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Validation of Serological Testing for Anti-Treponema pallidum from Postmortem Blood on the Siemens-BEP®-III Automatic System
Summary Background Infectious disease marker testing is obligatory for the release of human tissue for transplantation. Most CE-marked tests are not validated for postmortem blood. In a previous study we have validated the testing for anti-HIV-1/2, anti-HCV, HBsAg, and anti-HBc. Here, we present the validation of testing for antibodies against T. pallidum, which is the last marker obligatory for tissue release for transplantation. Methods 17 samples of postmortem sera and 10 samples of both pre-und postmortem sera were obtained from cornea donors and tested for anti-T. pallidum on the Siemens-BEP-III-System. These sera were spiked with anti-T. pallidum-positive standard sera in concentrations which give low- and high-positive results at the respective dilution. Results Two of the unspiked postmortem sera were false-positive most likely due to intense hemolysis (free hemoglobin > 50 mg/dl). Of the 25 negative postmortem sera, none of the spiked samples was false-negative after 0, 24 and 60 h. Conclusion There is no indication that postmortem samples give false-negative or false-positive results with the test system and test kits used in cases of low hemolysis. The procedure described might serve as a model for validating other test kits on postmortem samples.
Kalus, Ulrich; Wilkemeyer, Ina; Pruss, Axel; Caspari, Gregor
Development of a colloidal gold-immunochromatography assay to detect immunoglobulin G antibodies to Treponema pallidum with TPN17 and TPN47.
Syphilis remains a worldwide public health problem; it is necessary to develop a new diagnostic approach that is easier and faster than conventional tests. Here, we report a new testing method named colloidal gold-immunochromatography assay (GICA) to detect syphilis instead of fluorescent treponemal antibody-absorption (FTA-Abs). Syphilis-specific immunoglobulin G (IgG) antibody was detected with GICA established on syphilis-specific recombinant proteins, TPN17 and TPN47. FTA-Abs Treponema pallidum (TP)-IgG was set as the gold standard. A GICA test was performed to detect the serum of 14?967 subjects who took a serologic test for syphilis at the Xiamen Center of Clinical Laboratory, Fujian, China, from March 2009 to February 2010, among which 1326 cases were diagnosed as syphilitic. The results showed that the sensitivity, specificity, and positive predictive value were 99.38% (1279/1287), 99.96% (12,975/12,980), and 99.61% (1279/1284), respectively. The positive rate between the 2 test methods had no significant difference (?(2) = 0.003, P > 0.05). Detection on 500 interference specimens indicated that the biologic false-positive rate of the GICA test was extremely low and free from other biologic and chemical factors. The characteristics of GICA TP-IgG correspond to that of FTA-Abs TP-IgG (EUROIMMUN Medizinische Labordiagnostika, Germany). The GICA test is convenient, fast, and inexpensive, and it can be used both as a confirmatory test and a screening indicator, instead of FTA-Abs TP-IgG. PMID:20846810
Lin, Li-Rong; Fu, Zuo-Gen; Dan, Bing; Jing, Guang-Jun; Tong, Man-li; Chen, De-Teng; Yu, Yang; Zhang, Chang-Gong; Yang, Tian-Ci; Zhang, Zhong-Ying
Comparative Evaluation of Nine Different Enzyme-Linked Immunosorbent Assays for Determination of Antibodies against Treponema pallidum in Patients with Primary Syphilis
Nine different enzyme-linked immunosorbent assays (ELISAs) with a sonicate or recombinant proteins of Treponema pallidum as antigen have been evaluated comparatively by testing 52 highly selected sera from patients with primary syphilis, all negative in the microhemagglutination test for T. pallidum (MHA-TP). Eight tests exhibited greater sensitivity (48.5 to 76.9%) than the commonly used Venereal Disease Research Labo- ratory test
BRUNO L. SCHMIDT; MARZIEH EDJLALIPOUR; ANTON LUGER; Ludwig Boltzmann
Plasmonic ELISA for the ultrasensitive detection of Treponema pallidum.
In this report, we have developed a plasmonic ELISA strategy for the detection of syphilis. Plasmonic ELISA is an enzyme-linked immunoassay combined with enzyme-mediated surface plasmon resonance (SPR) of gold nanoparticles (AuNPs). Immune response of the Treponema pallidum (T. pallidum) antibodies triggers the acetylcholinesterase-catalyzed hydrolysis of acetylthiocholine to produce abundant thiocholine. The positive charged thiol, in turn, alters the surface charge distribution the AuNPs and leads to the agglomeration of the AuNPs. The induced strong localized SPR effect of the agglomerate AuNPs can, thus, allow the quantitative assay of T. pallidum antibodies due to the remarkable color and absorption spectral response changes of the reaction system. The plasmonic ELISA exhibited a quasilinear response to the logarithmic T. pallidum antibody concentrations in the range of 1pg/mL-10ng/mL with a detection limit of 0.98pg/mL. Such a low detection limit was 1000-fold improvements in sensitivity over a conventional ELISA. The results of plasmonic ELISA in syphilis assays of serum specimens from 60 patients agreed with those obtained using a conventional ELISA method. The plasmonic ELISA has characteristics (analyte specific, cost-effective, ease of automatic, low limit of detection) that provide potential for diagnosis and therapeutic monitoring of syphilis. PMID:24662060
Nie, Xin-Min; Huang, Rong; Dong, Cai-Xia; Tang, Li-Juan; Gui, Rong; Jiang, Jian-Hui
Usefulness in clinical practice of a point-of-care rapid test for simultaneous detection of nontreponemal and Treponema pallidum-specific antibodies in patients suffering from documented syphilis.
The usefulness of a point-of-care immunochromatographic dual test for the simultaneous detection of both nontreponemal and Treponema pallidum-specific antibodies (Chembio Diagnostics Systems Inc., Medford, NY, USA) was assessed in various situations related to syphilis, by reference to conventional syphilis serology. Thawed sera were obtained from 100 adults including 36 primary syphilis, 6 secondary syphilis, 6 re-infection, 9 recently-treated syphilis, and 43 old syphilis. Doubtful reactivities for the treponemal line were considered positive; doubtful reactivities for the nontreponemal line were considered positive only when the treponemal line was present. The sensitivity, the specificity, and its concordance to gold standard serology of treponemal line were high, around 90%. The sensitivity of nontreponemal line was 96.3%, its specificity 76.7%, and its concordance 83.4%. In conclusion, the dual rapid test from Chembio Diagnostics Systems Inc. is useful for rapid point-of-care diagnosis in the various situations encountered with patients suffering from syphilis. PMID:23999937
Guinard, Jérôme; Prazuck, Thierry; Péré, Hélčne; Poirier, Claire; LeGoff, Jérôme; Boedec, Erwan; Guigon, Aurélie; Day, Nesrine; Bélec, Laurent
Activation and Proteolytic Activity of the Treponema pallidum Metalloprotease, Pallilysin
Treponema pallidum is a highly invasive pathogen that undergoes rapid dissemination to establish widespread infection. Previous investigations identified the T. pallidum adhesin, pallilysin, as an HEXXH-containing metalloprotease that undergoes autocatalytic cleavage and degrades laminin and fibrinogen. In the current study we characterized pallilysin's active site, activation requirements, cellular location, and fibrin clot degradation capacity through both in vitro assays and heterologous treponemal expression and degradation studies. Site-directed mutagenesis showed the pallilysin HEXXH motif comprises at least part of the active site, as introduction of three independent mutations (AEXXH [H198A], HAXXH [E199A], and HEXXA [H202A]) abolished pallilysin-mediated fibrinogenolysis but did not adversely affect host component binding. Attainment of full pallilysin proteolytic activity was dependent upon autocatalytic cleavage of an N-terminal pro-domain, a process which could not occur in the HEXXH mutants. Pallilysin was shown to possess a thrombin cleavage site within its N-terminal pro-domain, and in vitro studies confirmed cleavage of pallilysin with thrombin generates a truncated pallilysin fragment that has enhanced proteolytic activity, suggesting pallilysin can also exploit the host coagulation process to facilitate protease activation. Opsonophagocytosis assays performed with viable T. pallidum demonstrated pallilysin is a target of opsonic antibodies, consistent with a host component-interacting, surface-exposed cellular location. Wild-type pallilysin, but not the HEXXA mutant, degraded fibrin clots, and similarly heterologous expression of pallilysin in the non-invasive spirochete Treponema phagedenis facilitated fibrin clot degradation. Collectively these results identify pallilysin as a surface-exposed metalloprotease within T. pallidum that possesses an HEXXH active site motif and requires autocatalytic or host-mediated cleavage of a pro-domain to attain full host component-directed proteolytic activity. Furthermore, our finding that expression of pallilysin confers upon T. phagedenis the capacity to degrade fibrin clots suggests this capability may contribute to the dissemination potential of T. pallidum.
Houston, Simon; Hof, Rebecca; Honeyman, Lisa; Hassler, Julia; Cameron, Caroline E.
Footprint of Positive Selection in Treponema pallidum subsp. pallidum Genome Sequences Suggests Adaptive Microevolution of the Syphilis Pathogen
In the rabbit model of syphilis, infection phenotypes associated with the Nichols and Chicago strains of Treponema pallidum (T. pallidum), though similar, are not identical. Between these strains, significant differences are found in expression of, and antibody responses to some candidate virulence factors, suggesting the existence of functional genetic differences between isolates. The Chicago strain genome was therefore sequenced and compared to the Nichols genome, available since 1998. Initial comparative analysis suggested the presence of 44 single nucleotide polymorphisms (SNPs), 103 small (?3 nucleotides) indels, and 1 large (1204 bp) insertion in the Chicago genome with respect to the Nichols genome. To confirm the above findings, Sanger sequencing was performed on most loci carrying differences using DNA from Chicago and the Nichols strain used in the original T. pallidum genome project. A majority of the previously identified differences were found to be due to errors in the published Nichols genome, while the accuracy of the Chicago genome was confirmed. However, 20 SNPs were confirmed between the two genomes, and 16 (80.0%) were found in coding regions, with all being of non-synonymous nature, strongly indicating action of positive selection. Sequencing of 16 genomic loci harboring SNPs in 12 additional T. pallidum strains, (SS14, Bal 3, Bal 7, Bal 9, Sea 81-3, Sea 81-8, Sea 86-1, Sea 87-1, Mexico A, UW231B, UW236B, and UW249C), was used to identify "Chicago-" or "Nichols -specific" differences. All but one of the 16 SNPs were "Nichols-specific", with Chicago having identical sequences at these positions to almost all of the additional strains examined. These mutations could reflect differential adaptation of the Nichols strain to the rabbit host or pathoadaptive mutations acquired during human infection. Our findings indicate that SNPs among T. pallidum strains emerge under positive selection and, therefore, are likely to be functional in nature.
Centurion-Lara, Arturo; Jeffrey, Brendan M.; Le, Hoavan T.; Molini, Barbara J.; Lukehart, Sheila A.; Sokurenko, Evgeni V.; Rockey, Daniel D.
Skin homing of Treponema pallidum in early syphilis: an immunohistochemical study.
The incidence of syphilis is increasing in most parts of the world including some major European cities. The lesions developing during secondary syphilis may be difficult to diagnose clinically. Similarly, the histopathologic changes do not always fulfill the typical diagnostic criteria. The objective of this study was to assess the contribution of immunohistochemistry for the identification and localization of Treponema pallidum on the skin. We retrieved from our files 12 paraffin-embedded biopsies of skin lesions, which had posed diagnostic problems in the past. Only a serologic test had proven that the patients had syphilis. Controls consisted of lichenoid dermatoses unrelated to syphilis and borreliosis. Immunohistochemistry using an antispirochete (T. pallidum and Borrelia) antibody was performed retrospectively. In all samples from primary and secondary syphilis, T. pallidum was highlighted, but none of the control lesions unrelated to syphilis showed positivity. Interestingly enough, T. pallidum present in the lower mid-part of the epidermis often outnumbered that in the dermis. This difference was more striking in secondary syphilis compared with primary syphilis. Immunohistochemistry for T. pallidum considerably increased the sensitivity and the specificity of the histologic diagnosis. The strong epidermal homing of T. pallidum is highlighted in early syphilis. PMID:18800002
Quatresooz, Pascale; Piérard, Gérald E
Studies on the action lysozyme in immune immobilization of Treponema pallidum (Nichols strain)
The action of antibodies and of the haemolytic complement system does not induce immune immobilization of Treponema pallidum, but only destroys superficial wall structures of these Gram-negative organisms. In sensitized and complement coated treponemes the immobilization is a result of the lysozyme interaction with the mucopeptide layer of the cell wall. In a lysozyme-free medium, immunologically damaged treponemes are able to repair the defects in the outer layers of their cell wall structures. Furthermore, it is shown that the long lag period in immune immobilization of T. pallidum can only partly be explained by the interaction of lysozyme with the treponemes.
Muller, F.; Feddersen, H.; Segerling, M.
TP0453, a Concealed Outer Membrane Protein of Treponema pallidum, Enhances Membrane Permeability
The outer membrane of Treponema pallidum, the noncultivable agent of venereal syphilis, contains a paucity of protein(s) which has yet to be definitively identified. In contrast, the outer membranes of gram-negative bacteria contain abundant immunogenic membrane-spanning ?-barrel proteins mainly involved in nutrient transport. The absence of orthologs of gram-negative porins and outer membrane nutrient-specific transporters in the T. pallidum genome predicts that nutrient transport across the outer membrane must differ fundamentally in T. pallidum and gram-negative bacteria. Here we describe a T. pallidum outer membrane protein (TP0453) that, in contrast to all integral outer membrane proteins of known structure, lacks extensive ?-sheet structure and does not traverse the outer membrane to become surface exposed. TP0453 is a lipoprotein with an amphiphilic polypeptide containing multiple membrane-inserting, amphipathic ?-helices. Insertion of the recombinant, nonlipidated protein into artificial membranes results in bilayer destabilization and enhanced permeability. Our findings lead us to hypothesize that TP0453 is a novel type of bacterial outer membrane protein which may render the T. pallidum outer membrane permeable to nutrients while remaining inaccessible to antibody.
Hazlett, Karsten R. O.; Cox, David L.; Decaffmeyer, Marc; Bennett, Michael P.; Desrosiers, Daniel C.; La Vake, Carson J.; La Vake, Morgan E.; Bourell, Kenneth W.; Robinson, Esther J.; Brasseur, Robert; Radolf, Justin D.
Complete Genome Sequence of the Treponema pallidum subsp. pallidum Sea81-4 Strain.
Using the rabbit model of syphilis, the Sea81-4 strain of Treponema pallidum subsp. pallidum has been found to be more likely than other strains to invade the central nervous system (CNS). To identify possible explanations for this important phenotype at the genomic level, we sequenced the Sea81-4 strain genome. PMID:24744342
Giacani, Lorenzo; Iverson-Cabral, Stefanie L; King, Jordon C K; Molini, Barbara J; Lukehart, Sheila A; Centurion-Lara, Arturo
Defining the Interaction of the Treponema pallidum Adhesin Tp0751 with Laminin
Various invasive pathogens attach to host tissues via the extracellular matrix component laminin, the major glycoprotein found within basement membranes. Previous investigations identified the laminin-binding adhesin Tp0751 within the spirochete bacterium Treponema pallidum. In the current study, Tp0751 was shown to attach to a variety of laminin isoforms that are widely distributed throughout the host, including laminins 1, 2, 4, 8, and 10. Such universal attachment is conducive for an adhesin present within a highly invasive pathogen that encounters a variety of tissue sites during the course of infection. Additional studies systematically identified the amino acid residues within Tp0751 that contribute to laminin binding using synthetic peptides designed from the mature protein sequence. The minimum laminin-binding region of the adhesin was localized to 10 amino acids; peptides containing these residues inhibited attachment of Tp0751 and T. pallidum to laminin. Further, Tp0751-specific antibodies inhibited attachment of T. pallidum to laminin. This study furthers our knowledge of the interaction of T. pallidum with laminin, an association that is proposed to facilitate bacterial traversal of basement membranes and subsequent entry into the circulation and tissue invasion. As such, these investigations will reveal new targets for possible prevention of bacterial dissemination and establishment of chronic infection.
Cameron, Caroline E.; Brouwer, Nathan L.; Tisch, Lisa M.; Kuroiwa, Janelle M. Y.
Respiration and Oxidative Phosphorylation in Treponema pallidum
Exogenous and endogenously generated reduced pyridine nucleotides caused marked stimulation of O2 uptake when added to treponemal cell-free extracts, which indicated that terminal electron transport was coupled to the consumption of O2. Oxidation of reduced nicotinamide adenine dinucleotide (NADH) was shown to correlate stoichiometrically with O2 reduction, suggesting that NADH was being oxidized through a mainstream respiratory chain dehydrogenase. Oxygen evolution in treponemal extracts was observed after the completion of O2 uptake which was stimulated by exogenous NADH and endogenously generated reduced NAD phosphate. Oxygen evolution was inhibited by both cyanide and pyruvate, which was consistent with O2 release from H2O2 by catalase. The addition of exogenous H2O2 to treponemal extracts caused rapid O2 evolution characteristic of a catalase reaction. A spectrophotometric assay was used to measure ATP formation in T. pallidum cell-free extracts that were stimulated with NADH. P/O ratios from 0.5 to 1.1 were calculated from the amounts of ATP formed versus NADH oxidized. Phosphorylating activity was dependent on Pi concentration and was sensitive to cyanide, N, N?-dicyclohexylcarbodiimide, and carbonyl cyanide m-chlorophenyl hydrazone. Adenine nucleotide pools of T. pallidum were measured by the firefly luciferin-luciferase assay. Shifts in adenine nucleotide levels upon the addition of NADH to cell-free extracts were impossible to evaluate due to the presence of NAD+ nucleosidase. However, when whole cells, previously incubated under an atmosphere of 95% N2-5% CO2, were sparged with air, ATP and ADP levels increased, while AMP levels decreased. The shift was attributed to both oxidative phosphorylation and to the presence of an adenylate kinase activity. T. pallidum was also found to possess an Mg2+ - and Ca2+ -stimulated ATPase activity which was sensitive to N, N? -dicyclohexylcarbodiimide. These data indicated a capability for oxidative phosphorylation by T. pallidum.
Lysko, Paul G.; Cox, C. D.
Treponema pallidum receptor binding proteins interact with fibronectin.
Analysis of plasma proteins avidly bound to T. pallidum surfaces revealed the ability of T. pallidum to acquire numerous host macromolecules. No acquisition was evident by the avirulent spirochete, T. phagedenis biotype Reiter. Western blotting technology using hyperimmune antifibronectin serum as a probe revealed the ability of virulent treponemes to avidly bind fibronectin from a complex medium such as plasma. The specificity of the tiplike adherence of motile T. pallidum to fibronectin-coated glass surfaces and to fibronectin on HEp-2 cells was reinforced by the observation that pretreatment of coverslips or cell monolayers with monospecific antiserum against fibronectin substantially reduced T. pallidum attachment. The stoichiometric binding of T. pallidum to fibronectin-coated coverslips and the inability of unlabeled or 35S-radiolabeled treponemes to interact with glass surfaces treated with other plasma proteins further established the specific nature of the interaction between virulent T. pallidum and fibronectin. The avid association between three outer envelope proteins of T. pallidum and fibronectin was also demonstrated. These treponemal surface proteins have been previously identified as putative receptor-binding proteins responsible for T. pallidum parasitism of host cells. The data suggest that surface fibronectin mediates tip-oriented attachment of T. pallidum to host cells via a receptor-ligand mechanism of recognition. PMID:6304227
Peterson, K M; Baseman, J B; Alderete, J F
Adjuvant Activity of Sargassum pallidum Polysaccharides against Combined Newcastle Disease, Infectious Bronchitis and Avian Influenza Inactivated Vaccines
This study evaluates the effects of Sargassum pallidum polysaccharides (SPP) on the immune responses in a chicken model. The adjuvanticity of Sargassum pallidum polysaccharides in Newcastle disease (ND), infectious bronchitis (IB) and avian influenza (AI) was investigated by examining the antibody titers and lymphocyte proliferation following immunization in chickens. The chickens were administrated combined ND, IB and AI inactivated vaccines containing SPP at 10, 30 and 50 mg/mL, using an oil adjuvant vaccine as a control. The ND, IB and AI antibody titers and the lymphocyte proliferation were enhanced at 30 mg/mL SPP. In conclusion, an appropriate dose of SPP may be a safe and efficacious immune stimulator candidate that is suitable for vaccines to produce early and persistent prophylaxis.
Li, Li-Jie; Li, Ming-Yi; Li, Yan-Tuan; Feng, Jing-Jing; Hao, Feng-Qiang; Zhang, Lun
Treponema pallidum Invades Intercellular Junctions of Endothelial Cell Monolayers
NASA Astrophysics Data System (ADS)
The pathogenesis of syphilis reflects invasive properties of Treponema pallidum, but the actual mode of tissue invasion is unknown. We have found two in vitro parallels of treponemal invasiveness. We tested whether motile T. pallidum could invade host cells by determining the fate of radiolabeled motile organisms added to a HeLa cell monolayer; 26% of treponemes associated with the monolayer in a trypsin-resistant niche, presumably between the monolayer and the surface to which it adhered, but did not attain intracellularity. Attachment of T. pallidum to cultured human and rabbit aortic and human umbilical vein endothelial cells was 2-fold greater than to HeLa cells. We added T. pallidum to aortic endothelial cells grown on membrane filters under conditions in which tight intercellular junctions had formed. T. pallidum was able to pass through the endothelial cell monolayers without altering tight junctions, as measured by electrical resistance. In contrast, heat-killed T. pallidum and the nonpathogen Treponema phagedenis biotype Reiter failed to penetrate the monolayer. Transmission electron micrographs of sections of the monolayer showed T. pallidum in intercellular junctions. Our in vitro observations suggest that these highly motile spirochetes may leave the circulation by invading the junctions between endothelial cells.
Thomas, D. Denee; Navab, Mahamad; Haake, David A.; Fogelman, Alan M.; Miller, James N.; Lovett, Michael A.
Capacity of virulent Treponema pallidum (Nichols) for deoxyribonucleic acid synthesis.
Treponema pallidum (Nichols) was extracted from infected rabbit tissue, and cell lysates were prepared for monitoring thymidine kinase and deoxyribonucleic acid polymerase activities. No thymidine kinase could be demonstrated in preparations of T. pallidum or the cultivable T. phagedenis biotype Reiter. Significant levels of deoxyribonucleic acid polymerase were detected in both treponemal samples. Interestingly, comparisons of polymerase activity among a spectrum of bacterial genera revealed a direct correlation between enzyme concentrations and estimated generation time. Incorporation of [3H]uridine and [3H]thymidine into macromolecules by intact T. pallidum and the Reiter treponeme was examined. Selective ribonuclease-deoxyribonuclease digestion and cesium chloride gradient banding demonstrated that T. pallidum, independent of the host, and T. phagedenis were capable of synthesizing deoxyribonucleic acid only from the [3H]-uridine precursor.
Baseman, J B; Nichols, J C; Mogerley, S
Serum lipoprotein binding by Treponema pallidum: possible role for proteoglycans.
Acquisition by the syphilis spirochaete, Treponema pallidum, of radioiodinated total human plasma lipoprotein and lipoprotein subfractions was examined. Time dependent and saturation binding kinetics were observed for total lipoproteins and subfractions, including high density lipoproteins, low density lipoproteins (LDL), and very low density lipoproteins. All subfractions competed equally well in binding iodinated total lipoproteins and individual subfractions, but apoproteins common to all subfractions were ineffective in inhibiting lipoprotein acquisition. The interaction of LDL with T pallidum was studied further and, interestingly, the presence of 17% sulphated dextran sulphate (DS) in the reaction mixture containing treponemes and LDL resulted in up to 172 times more LDL being bound by live treponemes. Biological variability was observed in the extent of increased LDL bound in the presence of 17% sulphated DS by preparations of T pallidum isolated from different infected rabbits. Saturation kinetics of iodinated LDL acquisition was obtained in the presence of 17% sulphated DS but not 1% sulphated DS. Other proteoglycan molecules, such as chondroitin sulphate, hyaluronic acid and heparin, and fibronectin, the extracellular matrix protein targeted by treponemes in parasitism of host cells and tissues neither diminished nor enhanced LDL binding by live treponemes. Only 5% and 10% of associated radioactivity was released from treponemal surfaces after T pallidum was incubated with iodinated LDL and 17% sulphated-DS for 15 and 30 minutes, respectively. These data show binding and possible internalisation of host lipoproteins by T pallidum, which may be mediated by sulphated proteoglycan. Sulphated proteoglycans accumulate during T pallidum infections of host cells.
Alderete, J F; Baseman, J B
Molecular characterization and cellular localization of TpLRR, a processed leucine-rich repeat protein of Treponema pallidum, the syphilis spirochete.
Automated Edman degradation was used to obtain N-terminal and internal amino acid sequences from a 26-kDa protein in isolated Treponema pallidum outer membranes (OMs). The resulting sequences enabled us to PCR amplify from T. pallidum DNA a 275-bp fragment of the corresponding gene. The complete nucleotide sequence of the gene was determined from fragments amplified by long-distance PCR. Primer extension verified the assigned translational start of the open reading frame (ORF) and putative upstream promoter elements. The ORF encoded a highly basic (pI 9.6) 26-kDa protein which contained an N-terminal 25-amino-acid leader peptide terminated by a signal peptidase I cleavage site. The mature protein contained seven tandemly spaced copies (as well as an eighth incomplete copy) of a leucine-rich repeat (LRR), a motif previously identified in a number of prokaryotic and eukaryotic proteins. Accordingly, the polypeptide was designated T. pallidum leucine-rich repeat protein (TpLRR). Although Triton X-114 phase partitioning showed that TpLRR was hydrophilic, cell localization studies showed that most of the antigen was associated with the peptidoglycan-cytoplasmic membrane complex rather than being freely soluble in the periplasmic space. Immunoblot studies showed that syphilis patients develop a weak antibody response to the antigen. Lastly, the lrr(T. pallidum) gene was mapped to a 60-kb SfiI-SpeI fragment of the T. pallidum chromosome which also contains the rrnA and flaA genes. The function(s) of TpLRR is currently unknown; however, protein-protein and/or protein-lipid interactions mediated by its LRR motifs may facilitate interactions between components of the T. pallidum cell envelope.
Shevchenko, D V; Akins, D R; Robinson, E; Li, M; Popova, T G; Cox, D L; Radolf, J D
An antibody is a protein produced by the body's immune system when it detects harmful substances, called antigens. Examples ... as bacteria, fungi, parasites, and viruses) and chemicals. Antibodies may be produced when the immune system mistakenly ...
NSDL National Science Digital Library
Antibody: A blood protein that is produced in response to and counteracts an antigen. Antibodies are produced in response to disease and help the body fight against the particular disease. In this way, antibodies help the body develop an immunity to disease.
BEGIN:VCARD VERSION:2.1 FN:Darryl Leja N:Leja;Darryl ORG:National Human Genome Research Institute REV:2005-04-04 END:VCARD
Genetic diversity in Treponema pallidum: implications for pathogenesis, evolution and molecular diagnostics of syphilis and yaws.
Pathogenic uncultivable treponemes, similar to syphilis-causing Treponema pallidum subspecies pallidum, include T. pallidum ssp. pertenue, T. pallidum ssp. endemicum and Treponema carateum, which cause yaws, bejel and pinta, respectively. Genetic analyses of these pathogens revealed striking similarity among these bacteria and also a high degree of similarity to the rabbit pathogen, Treponema paraluiscuniculi, a treponeme not infectious to humans. Genome comparisons between pallidum and non-pallidum treponemes revealed genes with potential involvement in human infectivity, whereas comparisons between pallidum and pertenue treponemes identified genes possibly involved in the high invasivity of syphilis treponemes. Genetic variability within syphilis strains is considered as the basis of syphilis molecular epidemiology with potential to detect more virulent strains, whereas genetic variability within a single strain is related to its ability to elude the immune system of the host. Genome analyses also shed light on treponemal evolution and on chromosomal targets for molecular diagnostics of treponemal infections. PMID:22198325
Smajs, David; Norris, Steven J; Weinstock, George M
Substance P in the ventral pallidum: Projection from the ventral striatum, and electrophysiological and behavioral cinsequences of pallidal substance P
The ventral pallidum of the basal forebrain contains a high concentration of substance P and receives a massive projection from the nucleus accumbens. The present study was designed to determine whether the accumbens serves as a source for substance P-containing fibers in the ventral pallidum and characterize the function of this tachykinin peptide within the ventral pallidum.By combining in situ
T. C. Napier; I. Mitrovic; L. Churchill; M. A. Klitenick; X.-Y. Lu; P. W. Kalivas
A redescripton of Lyrosoma pallidum (Eschscholtz) and distributional range extension of Lyrosoma Mannerheim (Coleoptera, Agyrtidae)
Abstract A redescription with illustrations of the species Lyrosoma pallidum and a key to the Korean species of the family Agyrtidae are provided. New distributional data, including a range extension, of the two Lyrosoma Mannerheim species are presented. Lyrosoma pallidum (Eschscholtz) is recorded for the first time in Korea.
Yoo, In-Seong; Sikes, Derek; Ahn, Kee-Jeong
Phagocytosis of Borrelia burgdorferi and Treponema pallidum Potentiates Innate Immune Activation and Induces Gamma Interferon Production
We examined the interactions of live and lysed spirochetes with innate immune cells. THP-1 monocytoid cells were activated to comparable extents by live Borrelia burgdorferi and by B. burgdorferi and Treponema pallidum lysates but were poorly activated by live T. pallidum. Because THP-1 cells poorly internalized live spirochetes, we turned to an ex vivo peripheral blood mononuclear cell system that
Meagan W. Moore; Adriana R. Cruz; Carson J. LaVake; Amanda L. Marzo; Christian H. Eggers; Juan C. Salazar; Justin D. Radolf
Involvement of the medial pallidum in focal myoclonic dystonia: A clinical and neurophysiological case study
We successfully treated a patient with familial myo- clonic dystonia (FMD), which primarily affected his neck muscles, with bilateral deep brain stimulation (DBS) to the medial pallidum, and investigated the role of the medial palli- dum in FMD. A patient with FMD underwent bilateral implan- tation of DBS electrodes during which field potentials (FPs) in the medial pallidum and electromyograms
Xuguang Liu; Ivan C. Griffin; Simon G. Parkin; R. Christopher Miall; Jeremy G. Rowe; Ralph P. Gregory; Richard B. Scott; Tipu Z. Aziz; John F. Stein
Effect of spectinomycin on T. pallidum in incubating experimental syphilis.
Animal experiments were performed to determine if a single-dose treatment for acute gonorrhoea with 2 g. spectinomycin could cure a simultaneously acquired syphilis at a very early stage of incubation. 300 treponemes (Nichols stain T. pallidum) were inoculated intratesticularly and 3 days later spectinomycin was administered in a dose which produced spectinomycin serum levels similar to those in patients who had received a single oral dose of 2 g. This dosage of spectinomycin did not prevent the development of syphilitic orchitis or reactivity to the FTA-ABS test, but it prolonged the subclinical incubation period.
Petzoldt, D
Evaluation of a PCR test for detection of treponema pallidum in swabs and blood.
Syphilis diagnosis is based on clinical observation, serological analysis, and dark-field microscopy (DFM) detection of Treponema pallidum subsp. pallidum, the etiological agent of syphilis, in skin ulcers. We performed a nested PCR (nPCR) assay specifically amplifying the tpp47 gene of T. pallidum from swab and blood specimens. We studied a cohort of 294 patients with suspected syphilis and 35 healthy volunteers. Eighty-seven of the 294 patients had primary syphilis, 103 had secondary syphilis, 40 had latent syphilis, and 64 were found not to have syphilis. The T. pallidum nPCR results for swab specimens were highly concordant with syphilis diagnosis, with a sensitivity of 82% and a specificity of 95%. Reasonable agreement was observed between the results obtained with the nPCR and DFM methods (kappa = 0.53). No agreement was found between the nPCR detection of T. pallidum in blood and the diagnosis of syphilis, with sensitivities of 29, 18, 14.7, and 24% and specificities of 96, 92, 93, and 97% for peripheral blood mononuclear cell (PBMC), plasma, serum, and whole-blood fractions, respectively. HIV status did not affect the frequency of T. pallidum detection in any of the specimens tested. Swab specimens from mucosal or skin lesions seemed to be more useful than blood for the efficient detection of the T. pallidum genome and, thus, for the diagnosis of syphilis. PMID:22219306
Grange, P A; Gressier, L; Dion, P L; Farhi, D; Benhaddou, N; Gerhardt, P; Morini, J P; Deleuze, J; Pantoja, C; Bianchi, A; Lassau, F; Avril, M F; Janier, M; Dupin, N
21 CFR 866.3830 - Treponema pallidum tre-ponemal test reagents.
Code of Federal Regulations, 2013 CFR
... Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3830 Treponema pallidum tre-ponemal test reagents....
Seroprevalence of Antibodies to Microorganisms Known To Cause Arterial and Myocardial Damage in Patients with or without Coronary Stenosis
Infections are assumed to play a role in coronary artery disease (CAD) and cardiomyopathies. It is unknown whether the seroprevalence of antibodies to these microorganisms is higher in patients with than without CAD. The seroprevalence of antibodies to Bartonella henselae, Borrelia burgdorferi, Chlamydia pneumoniae, Coxiella bur- netii, Helicobacter pylori, human granulocytic Ehrlichia, Leptospira, Rickettsia conorii, and Treponema pallidum was assessed
C. Stollberger; G. Molzer; J. Finsterer
Element analysis of the Polysphondylium pallidum gp64 promoter.
gp64 mRNA in Polysphondylium pallidum is expressed extensively during vegetative growth, and begins to rapidly decrease at the onset of development. To examine this unique regulation, 5' deletion analysis of the gp64 promoter was undertaken, and two growth-phase activated elements have been found: a food-dependent, upstream regulatory region (FUR, -222 to -170) and a vegetatively activated, downstream region (VAD, -110 to -63). Here we concentrate our analysis on an A1 and A2 sequences in the FUR region: A1 consists of a GATTTTTTTA sequence called a corresponding sequence and A2 consists of the direct repeat TTTGTTGTG. The cells carrying a combined construct of A1 and A2 acted synergistically in a reporter activity. A point mutation analysis in A1 indicates that a G residue is required for the activation of A1. From analyses of promoter regulation in a liquid or a solid medium, the promoter activity of the cells fed on bacteria in A-medium (axenic medium for Polysphondylium) or grown in A-medium alone was only one fourth of that of the cells fed on bacteria. By the gel retardation, we detected a protein bound to the A1 sequence. PMID:11997096
Takaoka, Naohisa; Fukuzawa, Masashi; Kato, Atsushi; Saito, Tamao; Ochiai, Hiroshi
Structural elucidation of polysaccharide fractions from brown seaweed Sargassum pallidum.
The structural characteristics of two purified fractions of polysaccharides from Sargassum pallidum (SPS) were investigated in the present study. As results, the molecular weights of the two polysaccharide fractions, SPS-3-1 and SPS-3-2, were determined to be 5.87 and 7.25 kDa, respectively. SPS-3-1 was composed of glucose, mannose and galactose in a molar ratio of 11.18:1.00:0.96, while SPS-3-2 was composed of fucose, xylose, mannose, glucose and galactose in a molar ratio of 2.53:0.61:1.00:0.46:0.92. Both SPS-3-1 and SPS-3-2 exhibited the characteristics of polysaccharide in the frequency range of 4000-400 cm(-1) based on their Fourier-transform infrared spectra. Furthermore, the results of periodic acid oxidation, Smith degradation, methylation analysis and nuclear magnetic resonance spectroscopic analysis suggested that SPS-3-2 was composed of (1?4)-linked fucopyranosyl backbone and (1?3)-linked galactopyranosyl, (1?3)-linked mannopyranosyl, (1?2)-linked xylopyranosyl and (1?6)-linked glucopyranosyl branch chains. PMID:23911498
Ye, Hong; Zhou, Chunhong; Li, Wei; Hu, Bing; Wang, Xiaoqing; Zeng, Xiaoxiong
Identification of the Treponema pallidum subsp. pallidum TP0092 (RpoE) Regulon and Its Implications for Pathogen Persistence in the Host and Syphilis Pathogenesis
Bacteria often respond to harmful environmental stimuli with the induction of extracytoplasmic function (ECF) sigma (?) factors that in turn direct RNA polymerase to transcribe specific groups of response genes (or regulons) to minimize cellular damage and favor adaptation to the changed extracellular milieu. In Treponema pallidum subsp. pallidum, the agent of syphilis, the TP0092 gene is predicted to code for the pathogen's only annotated ECF ? factor, homologous to RpoE, known in Escherichia coli to control a key transduction pathway for maintenance of envelope homeostasis in response to external stress and cell growth. Here we have shown that TP0092 is highly transcribed during experimental syphilis. Furthermore, TP0092 transcription levels significantly increase as infection progresses toward immune clearance of the pathogen, suggesting a role for TP0092 in helping T. pallidum respond to harmful stimuli in the host environment. To investigate this hypothesis, we determined the TP0092 regulon at two different time points during infection using chromatin immunoprecipitation followed by high-throughput sequencing. A total of 22 chromosomal regions, all containing putative TP0092-binding sites and corresponding to as many T. pallidum genes, were identified. Noteworthy among them are the genes encoding desulfoferrodoxin and thioredoxin, involved in detoxification of reactive oxygen species (ROS). Because T. pallidum does not possess other enzymes for ROS detoxification, such as superoxide dismutase, catalase, or glutathione peroxidase, our results suggest that the TP0092 regulon is important in protecting the syphilis spirochete from damage caused by ROS produced at the site of infection during the inflammatory response.
Denisenko, Oleg; Tompa, Martin; Centurion-Lara, Arturo
Length of guanosine homopolymeric repeats modulates promoter activity of subfamily II tpr genes of Treponema pallidum ssp. pallidum
In Treponema pallidum, homopolymeric guanosine repeats of varying length are present upstream of both Subfamily I (tprC, D, F and I) and II (tprE, G and J) tpr genes, a group of potential virulence factors, immediately upstream of the +1 nucleotide. To investigate the influence of these poly-G sequences on promoter activity, tprE, G, J, F and I promoter regions containing homopolymeric tracts with different numbers of Gs, the ribosomal binding site and start codon were cloned in frame with the green fluorescent protein reporter gene (GFP), and promoter activity was measured both as fluorescence emission from Escherichia coli cultures transformed with the different plasmid constructs and using quantitative RT-PCR. For tprJ, G and E-derived clones, fluorescence was significantly higher with constructs containing eight Gs or fewer, while plasmids containing the same promoters with none or more Gs gave modest or no signal above the background. In contrast, tprF/I-derived clones induced similar levels of fluorescence regardless of the number of Gs within the promoter. GFP mRNA quantification showed that all of the promoters induced measurable transcription of the GFP gene; however, only for Subfamily II promoters was message synthesis inversely correlated to the number of Gs in the construct.
Giacani, Lorenzo; Lukehart, Sheila; Centurion-Lara, Arturo
Perinatal Treponema pallidum: evidence based guidelines to reduce mother to child transmission.
Universal antenatal screening for T. pallidum is standard in Irish maternity units. The prevalence of adult syphilis has increased in Ireland. We audited the neonatal management of infants exposed to T. pallidum in utero. A cross sectional retrospective analysis of all pregnancies with confirmed positive serology for T. pallidum from January 2005 to December 2010 was conducted at the National Maternity Hospital, Holles St. Data were analysed using SPSS 14.0. Ethical approval was obtained. There were 55,058 live births during the study period. Fifty-eight women had positive serology and 41 met inclusion criteria. Infant evaluation and follow up was decided by allocation to an evidence based algorithm. Twenty-one infants (51%) were accurately allocated and assessed, 5 (12%) had a partial assessment and the algorithm was incorrectly applied in 15 (36%) of cases. Failure to adhere to evidence based neonatal guidelines is common and undermines efficacy of the screening program. PMID:24592640
Freyne, B; Stafford, A; Knowles, S; O'Hora, A; Molloy, E
Adoptive transfer of immunity to Treponema pallidum Nichols infection in inbred strain 2 and C4D guinea pigs.
T lymphocytes purified from lymph nodes and spleens of chancre-immune, inbred strain 2 guinea pigs, when infused into syngeneic guinea pigs, conferred protection against challenge with Treponema pallidum subsp. pallidum Nichols. No protection was conferred by similar injections of cell suspensions from normal guinea pigs or guinea pigs immunized with T. phagedenis biotype Reiter or T. pallidum-free testis supernatants from infected rabbits. Similar results were obtained with homozygous C4D guinea pigs. After several months of infection, 2 of 11 strain 2 and 1 of 8 strain C4D recipients of T. pallidum-immune cells developed an erythematous reaction of short duration at the injection site; 2 of these recipients were positive for T. pallidum. Throughout the experimental period the humoral response to treponemal antigens was substantially lower in the adoptively immune guinea pigs than in various unprotected control groups. Passive immunity to infection with T. pallidum, however, seems to be dose related, since asymptomatic infection persisted for as long as 3 months after challenge in strain 2 guinea pigs transfused with 10(8) T. pallidum-immune lymphocytes, but not in C4D recipients of twice as many immune cells. Images
Wicher, V; Wicher, K; Jakubowski, A; Nakeeb, S M
Treponema pallidum Infection in the Wild Baboons of East Africa: Distribution and Genetic Characterization of the Strains Responsible
It has been known for decades that wild baboons are naturally infected with Treponema pallidum, the bacterium that causes the diseases syphilis (subsp. pallidum), yaws (subsp. pertenue), and bejel (subsp. endemicum) in humans. Recently, a form of T. pallidum infection associated with severe genital lesions has been described in wild baboons at Lake Manyara National Park in Tanzania. In this study, we investigated ten additional sites in Tanzania and Kenya using a combination of macroscopic observation and serology, in order to determine whether the infection was present in each area. In addition, we obtained genetic sequence data from six polymorphic regions using T. pallidum strains collected from baboons at two different Tanzanian sites. We report that lesions consistent with T. pallidum infection were present at four of the five Tanzanian sites examined, and serology was used to confirm treponemal infection at three of these. By contrast, no signs of treponemal infection were observed at the six Kenyan sites, and serology indicated T. pallidum was present at only one of them. A survey of sexually mature baboons at Lake Manyara National Park in 2006 carried out as part of this study indicated that roughly ten percent displayed T. pallidum-associated lesions severe enough to cause major structural damage to the genitalia. Finally, we found that T. pallidum strains from Lake Manyara National Park and Serengeti National Park were genetically distinct, and a phylogeny suggested that baboon strains may have diverged prior to the clade containing human strains. We conclude that T. pallidum infection associated with genital lesions appears to be common in the wild baboons of the regions studied in Tanzania. Further study is needed to elucidate the infection's transmission mode, its associated morbidity and mortality, and the relationship between baboon and human strains.
Harper, Kristin N.; Fyumagwa, Robert D.; Hoare, Richard; Wambura, Philemon N.; Coppenhaver, Dorian H.; Sapolsky, Robert M.; Alberts, Susan C.; Tung, Jenny; Rogers, Jeffrey; Kilewo, Morris; Batamuzi, Emmanuel K.; Leendertz, Fabian H.; Armelagos, George J.; Knauf, Sascha
Evaluation of the Recombinant Protein TpF1 of Treponema pallidum for Serodiagnosis of Syphilis
Syphilis is a chronic infection caused by Treponema pallidum subsp. pallidum, and diagnosis with sensitive and specific methods is a challenging process that is important for its prevention and treatment. In the present study, we established a recombinant protein TpF1-based indirect immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) and a Western blot assay for human and rabbit sera. The 20-kDa recombinant protein TpF1 was detected by Western blotting performed with sera from rabbits immunized with recombinant TpF1 and infected with the T. pallidum Nichols strain and T. pallidum clinical isolates but was not detected by Western blotting with sera from uninfected rabbits. The sensitivity of the recombinant protein was determined by screening sera from individuals with primary, secondary, latent, and congenital syphilis (n = 82). The specificity of the recombinant protein was determined by screening sera from uninfected controls (n = 30) and individuals with potentially cross-reactive infections, including Lyme disease (n = 30) and leptospirosis (n = 5). The sensitivities of TpF1-based ELISAs were 93.3%, 100%, 100%, and 100% for primary, secondary, latent, and congenital syphilis, respectively, and the specificities were all 100% for sera from uninfected controls and individuals with potentially cross-reactive infections. In Western blot assays, the sensitivities and specificities of TpF1 for human sera were all 100%. The reactivities of TpF1 with syphilitic sera were proportional to the titers of the T. pallidum particle agglutination (TPPA) assay. These data indicate that the recombinant protein TpF1 is a highly immunogenic protein in human and rabbit infections and a promising marker for the screening of syphilis.
Jiang, Chuanhao; Zhao, Feijun; Xiao, Jinhong; Zeng, Tiebing; Yu, Jian; Ma, Xiaohua; Wu, Haiying
Antioxidant activities in vitro of ethanol extract from brown seaweed Sargassum pallidum
The crude extract (CE) was obtained by extracting the powder of Sargassum pallidum with a solution of 70% ethanol. Then, CE dissolved in distilled water was fractionated with chloroform (Cf), ethyl acetate\\u000a (EtOAc), and n-butanol (n-BuOH), respectively, affording four fractions of Cf, EtOAc, n-BuOH and aqueous. First, the contents of total polyphenols, vitamin C (VC) and vitamin E (VE) in
Hong Ye; Chunhong Zhou; Yi Sun; Xin Zhang; Jun Liu; Qiuhui Hu; Xiaoxiong Zeng
Purification, antitumor and antioxidant activities in vitro of polysaccharides from the brown seaweed Sargassum pallidum
Supercritical CO2 extraction, ultrasonic-aid extraction and membrane separation technology were applied to prepare Sargassum pallidum polysaccharides (SP). Three main fractions, SP-1, SP-2 and SP-3, were obtained by membranes of 1.0×10?4mm pore size and normal molecular-weight cut-off of 50kDa. The resulting three preparations were further purified by DEAE Cellulose-52 chromatography to afford seven polysaccharide fractions. Furthermore, the antitumor and antioxidant activities,
Hong Ye; Keqi Wang; Chunhong Zhou; Jun Liu; Xiaoxiong Zeng
Cryo-Electron Tomography Elucidates the Molecular Architecture of Treponema pallidum, the Syphilis Spirochete? †
Cryo-electron tomography (CET) was used to examine the native cellular organization of Treponema pallidum, the syphilis spirochete. T. pallidum cells appeared to form flat waves, did not contain an outer coat and, except for bulges over the basal bodies and widening in the vicinity of flagellar filaments, displayed a uniform periplasmic space. Although the outer membrane (OM) generally was smooth in contour, OM extrusions and blebs frequently were observed, highlighting the structure's fluidity and lack of attachment to underlying periplasmic constituents. Cytoplasmic filaments converged from their attachment points opposite the basal bodies to form arrays that ran roughly parallel to the flagellar filaments along the inner surface of the cytoplasmic membrane (CM). Motile treponemes stably attached to rabbit epithelial cells predominantly via their tips. CET revealed that T. pallidum cell ends have a complex morphology and assume at least four distinct morphotypes. Images of dividing treponemes and organisms shedding cell envelope-derived blebs provided evidence for the spirochete's complex membrane biology. In the regions without flagellar filaments, peptidoglycan (PG) was visualized as a thin layer that divided the periplasmic space into zones of higher and lower electron densities adjacent to the CM and OM, respectively. Flagellar filaments were observed overlying the PG layer, while image modeling placed the PG-basal body contact site in the vicinity of the stator-P-collar junction. Bioinformatics and homology modeling indicated that the MotB proteins of T. pallidum, Treponema denticola, and Borrelia burgdorferi have membrane topologies and PG binding sites highly similar to those of their well-characterized Escherichia coli and Helicobacter pylori orthologs. Collectively, our results help to clarify fundamental differences in cell envelope ultrastructure between spirochetes and gram-negative bacteria. They also confirm that PG stabilizes the flagellar motor and enable us to propose that in most spirochetes motility results from rotation of the flagellar filaments against the PG.
Izard, Jacques; Renken, Christian; Hsieh, Chyong-Ere; Desrosiers, Daniel C.; Dunham-Ems, Star; La Vake, Carson; Gebhardt, Linda L.; Limberger, Ronald J.; Cox, David L.; Marko, Michael; Radolf, Justin D.
Purification, crystallization and preliminary X-ray analysis of TP0435 (Tp17) from the syphilis spirochete Treponema pallidum
Syphilis, caused by the bacterial spirochete Treponema pallidum, remains a prominent sexually transmitted infection worldwide. Despite sequencing of the genome of this obligate human pathogen 15 years ago, the functions of a large number of the gene products of T. pallidum are still unknown, particularly with respect to those of the organism's periplasmic lipoproteins. To better understand their functions, a structural biology approach has been pursued. To this end, the soluble portion of the T. pallidum TP0435 lipoprotein (also known as Tp17) was cloned, hyper-expressed in Escherichia coli and purified to apparent homogeneity. The protein crystals obtained from this preparation diffracted to 2.4?Ĺ resolution and had the symmetry of space group R3. In the hexagonal setting, the unit-cell parameters were a = b = 85.7, c = 85.4?Ĺ.
Brautigam, Chad A.; Deka, Ranjit K.; Norgard, Michael V.
Evaluation of antioxidant and immunity-enhancing activities of Sargassum pallidum aqueous extract in gastric cancer rats.
The effect of Sargassum pallidum (brown seaweed) aqueous extract on the immunity function and antioxidant activities in was studied gastric cancer rats. Treatment with Sargassum pallidum aqueous extract at oral doses 400, 600 or 800 mg/kg body weight was found to provide a dose-dependent protection against N-methyl-N?-nitro-Nnitrosoguanidine (MNNG)-induced immunity damage and oxidative injury by enhancing serum interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-10 (IL-10) levels, decreasing interleukin-6 (IL-6), interleukin-1? (IL-1?), tumor necrosis factor-alpha (TNF-?) levels, preserving normal antioxidant enzymes activities, and by inhibiting lipid peroxidation in gastric mucosa. It can be concluded that Sargassum pallidum aqueous extract may enhance the immunity and antioxidant activities in gastric cancer rats. PMID:22785269
Zhang, Rui-Li; Luo, Wen-Da; Bi, Tie-Nan; Zhou, Shen-Kang
Novel mitochondrial gene content and gene arrangement indicate illegitimate inter-mtDNA recombination in the chigger mite, Leptotrombidium pallidum.
To better understand the evolution of mitochondrial (mt) genomes in the Acari (mites and ticks), we sequenced the mt genome of the chigger mite, Leptotrombidium pallidum (Arthropoda: Acari: Acariformes). This genome is highly rearranged relative to that of the hypothetical ancestor of the arthropods and the other species of Acari studied. The mt genome of L. pallidum has two genes for large subunit rRNA, a pseudogene for small subunit rRNA, and four nearly identical large noncoding regions. Nineteen of the 22 tRNAs encoded by this genome apparently lack either a T-arm or a D-arm. Further, the mt genome of L. pallidum has two distantly separated sections with identical sequences but opposite orientations of transcription. This arrangement cannot be accounted for by homologous recombination or by previously known mechanisms of mt gene rearrangement. The most plausible explanation for the origin of this arrangement is illegitimate inter-mtDNA recombination, which has not been reported previously in animals. In light of the evidence from previous experiments on recombination in nuclear and mt genomes of animals, we propose a model of illegitimate inter-mtDNA recombination to account for the novel gene content and gene arrangement in the mt genome of L. pallidum. PMID:15931495
Shao, Renfu; Mitani, Harumi; Barker, Stephen C; Takahashi, Mamoru; Fukunaga, Masahito
Evaluation of an Immunochromatographic Point-of-Care Test for the Simultaneous Detection of Nontreponemal and Treponemal Antibodies in Patients With Syphilis.
We described the evaluation of the Syphilis Screening & Confirm Assay for the simultaneous detection of nontreponemal and treponemal antibodies. A total of 248 samples were evaluated. The sensitivity of the tests was 98.8%, 99.5% and 98.9%, while specificity was 94.7%, 88.9% and 93.2%, respectively, as compared with the rapid plasma reagin, Treponema pallidum hemagglutination assay, and fluorescent treponemal antibody absorption tests. PMID:25013972
Castro, Rita; Lopes, Angela; da Luz Martins Pereira, Filomena
Role of D1 dopamine receptors of the ventral pallidum in inhibitory avoidance learning.
The mesolimbic dopaminergic system (MLDS) originating from the ventral tegmental area has important role in the regulation of motivation, learning and memory. The ventral pallidum (VP), innervated by the MLDS, is involved in the regulation of adaptive behavior, but its exact role is not known in inhibitory avoidance learning. The VP contains both D1 and D2 dopamine receptors, but the density of the former subtype is more excessive. Therefore, in our present experiments, the role of D1 dopamine receptors of the VP in one trial step-through inhibitory avoidance paradigm was investigated. In the conditioning trial, animals were shocked 3 times with 0.5mA current for 1s, and subsequently were microinjected bilaterally with D1 dopamine receptor agonist SKF38393 into the VP in three doses (0.1?g, 1.0?g or 5.0?g in 0.4?l saline). To clarify whether the agonist effect was specific, we also applied the D1 dopamine receptor antagonist SCH23390 (5.0?g in 0.4?l saline) 15min prior the agonist treatment. The D1 dopamine receptor agonist, in a dose-dependent manner, significantly increased the step-through latency during the test trials: retention was significant relative to the controls even after 2 weeks of conditioning. The D1 dopamine receptor antagonist SCH23390 pretreatment eliminated SKF38393 effects in the ventral pallidum. Our results show that D1 dopamine receptor mediated mechanisms in the VP facilitate learning and memory in inhibitory avoidance paradigm and this facilitation is specific because it can be eliminated by D1 dopamine receptor antagonist. PMID:24815313
Péczely, László; Ollmann, Tamás; László, Kristóf; Kovács, Anita; Gálosi, Rita; Szabó, Adám; Karádi, Zoltán; Lénárd, László
Antigenic variation in Treponema pallidum: TprK sequence diversity accumulates in response to immune pressure during experimental syphilis1
Pathogens that cause chronic infections often employ antigenic variation to evade the immune response and persist in the host. In Treponema pallidum (T. pallidum), the causative agent of syphilis, the TprK antigen undergoes variation of seven variable regions (V1-V7) by nonreciprocal recombination of silent donor cassettes with the tprK expression site. These V regions are the targets of the host humoral immune response during experimental infection. The present study addresses the causal role of the acquired immune response in the selection of TprK variants in two ways: 1) by investigating TprK variants arising in immunocompetent vs immunosuppressed hosts, and 2) by investigating the effect of prior specific immunization on selection of TprK variants during infection. V region diversity, particularly in V6, accumulates more rapidly in immunocompetent rabbits than in pharmacologically immunosuppressed rabbits (treated with weekly injections of methylprednisolone acetate). In a complementary experiment, rabbits pre-immunized with V6 region synthetic peptides had more rapid accumulation of V6 variant treponemes than control rabbits. These studies demonstrate that the host immune response selects against specific TprK epitopes expressed on T. pallidum, resulting in immune selection of new TprK variants during infection, confirming a role for antigenic variation in syphilis.
Giacani, Lorenzo; Molini, Barbara J.; Kim, Eric Y.; Godornes, B. Charmie; Leader, B. Troy; Tantalo, Lauren C.; Centurion-Lara, Arturo; Lukehart, Sheila A.
A role for the ventral pallidum in context-induced and primed reinstatement of alcohol seeking.
The ventral pallidum (VP) is a major target of projections from the nucleus accumbens, and has been implicated in the reinstatement of psychostimulant seeking as part of a cortical-striatal-pallidal 'final common pathway' for relapse. Here, we studied the role of the VP in context-induced and primed reinstatement of alcoholic beer seeking, using a combination of microinjections and tract tracing studies. In experiment 1, rats were trained to respond to alcoholic beer in one context (A), and then extinguished in a second context (B), prior to testing for reinstatement (ABA renewal) and extinction (ABB). VP microinjection of the ?-opioid receptor (MOR) antagonist CTAP prevented reinstatement. In experiment 2, VP microinjection of CTAP also prevented the primed reinstatement of alcoholic beer seeking after rats were trained, extinguished, and tested in the same context. In experiment 3, we employed retrograde neural tract tracing together with c-Fos immunohistochemistry to identify the VP afferents recruited during context-induced reinstatement of alcoholic beer seeking. There was evidence for the recruitment of accumbens core?VP, basolateral amygdala?VP and paraventricular thalamus?VP pathways during context-induced reinstatement. These results indicate that the VP MORs are critical for context-induced reinstatement, and that the VP receives inputs from a number of regions known to be important in reinstatement of drug seeking. PMID:23773238
Perry, Christina J; McNally, Gavan P
Promoter analysis of the membrane protein gp64 gene of the cellular slime mold Polysphondylium pallidum.
We cloned a genomic fragment of the membrane protein gp64 gene of the cellular slime mold Polysphondylium pallidum by inverse PCR. Primer extension analysis identified a major transcription start site 65 bp upstream of the translation start codon. The promoter region of the gp64 gene contains sequences homologous to a TATA box at position -47 to -37 and to an initiator (Inr, PyPyCAPyPyPyPy) at position -3 to +5 from the transcription start site. Successively truncated segments of the promoter were tested for their ability to drive expression of the beta-galactosidase reporter gene in transformed cells; also the difference in activity between growth conditions was compared. The results indicated that there are two positive vegetative regulatory elements extending between -187 and -62 bp from the transcription start site of the gp64 promoter; also their activity was two to three times higher in the cells grown with bacteria in shaken suspension than in the cells grown in an axenic medium. PMID:10542319
Takaoka, N; Fukuzawa, M; Saito, T; Sakaitani, T; Ochiai, H
The growth of the cellular slime mold, Polysphondylium pallidum, was studied on a semidefined medium in shaken suspension. When the medium contained large quantities of particulate material, growth was more rapid and the cellular size and protein content were smaller than when growth occurred on a medium containing less particulate material. The cellular levels of DNA, RNA, and protein; of lysosomal enzymes (acid phosphatase, acid proteinase); and of peroxisomal enzymes (catalase) were assayed during growth and the subsequent stationary phase that led eventually to encystment. Only DNA remained at a constant cellular level. Encystment of exponentially growing cells could also be initiated by washing them and introducing them into a soluble peptone medium. The rate of encystment was proportional to the osmolarity of this medium. The encystment process was followed with respect to the cellular levels of DNA, RNA, protein, carbohydrates, acid phosphatase, acid ?-N-Ac-glucosaminidase, and catalase. The most dramatic change occurred in the cellular cellulose content, which increased by at least an order of magnitude by the time encystment was morphologically complete. It was concluded that the encystment of this slime mold in suspension exhibits a number of biochemical similarities to the development of this and other cellular slime molds on a surface.
Githens, S.; Karnovsky, M. L.
Trifolium pallidum and Trifolium scabrum extracts in the protection of human plasma components.
Clovers (genus: Trifolium) have been used in traditional medicine by many cultures, but the biological activity of the most of these plants still remains unknown. The aim of our in vitro study was to assess the antioxidative action of phenolic extracts from aerial parts of Trifolium scabrum and Trifolium pallidum in human blood plasma, exposed to oxidative stress. In the present study we also demonstrate, for the first time the effects of the tested extracts on coagulative properties and fibrinolytic activity of blood plasma. The protective properties of the examined extracts (0.5-50 ?g/ml) against peroxynitrite-induced oxidative stress were estimated by the measurements of 3-nitrotyrosine, thiol groups and the thiobarbituric acid-reactive substances levels. The extracts considerably prevented the oxidative and nitrative damage to plasma proteins. Even the lowest doses of the Trifolium extracts (0.5 ?g/ml) were able to markedly reduce 3-nitrotyrosine formation (by about 50%) and to increase the level of -SH groups (by about 30%), in comparison to the plasma exposed to ONOO(-) in the absence of the extracts. The protective action of all the used concentrations of the Trifolium extracts in the prevention of lipid peroxidation was also found. The tested extracts influenced neither the coagulative properties nor fibrinolytic activity of plasma. Moreover, the extracts were able to significantly reduce the inhibitory effect of ONOO(-) on fibrinolytic activity of plasma (assessed with the use of a chromogenic substrate for plasmin). PMID:23335023
Kolodziejczyk-Czepas, Joanna; Olas, Beata; Malinowska, Joanna; Wachowicz, Barbara; Moniuszko-Szajwaj, Barbara; Kowalska, Iwona; Oleszek, Wieslaw; Stochmal, Anna
Evidence for a methyl-accepting chemotaxis protein gene (mcp1) that encodes a putative sensory transducer in virulent Treponema pallidum.
The clinical and histopathological manifestations of syphilis and the invasive behavior of Treponema pallidum in tissue culture systems reflect the propensity for treponemes to migrate through skin, hematogenously disseminate, and invade targeted tissues. Treponemal motility is believed to be essential to this process and thereby an important facet of syphilis pathogenesis. By analogy with other bacterial pathogens, it is plausible that treponemal motility and tissue invasion are modulated by sensory transduction events associated with chemotactic responses. Recent studies have demonstrated the existence in T. pallidum of accessory molecules typically associated with sensory transduction events involving methyl-accepting chemotaxis proteins (MCPs). Intrinsic radiolabeling of T. pallidum in vitro with L-[methyl-3H] methionine revealed one methylated treponemal polypeptide with an apparent molecular mass of 64 kDa. A degenerate oligonucleotide probe corresponding to a highly conserved C-terminal domain within Bacillus subtilis and Escherichia coli MCPs was used in Southern blotting of T. pallidum DNA to identify and subsequently clone a putative T. pallidum MCP gene (mcp1). Computer analyses predicted a near-consensus promoter upstream of mcp1, and primer extension analysis employing T. pallidum RNA revealed a transcriptional initiation site. T. pallidum mcp1 encoded a 579-amino-acid (64.6-kDa) polypeptide which was highly homologous to at least 69 other known or putative sensory transducer proteins, with the highest degrees of homology existing between the C terminus of mcp1 and the C-terminal (signaling) domains of the other bacterial MCPs. Other salient features of Mcp1 included (i) six potential membrane-spanning domains at the N terminus, (ii) two predicted alpha-helical coiled coil regions containing at least three putative methylation sites, and (iii) homologies with two ligand-binding domains (LI-1 and LI-2) of the E. coli MCPs Trg and Tar. This study is the first to provide both metabolic and genetic evidence for an MCP sensory transducer in T. pallidum. The combined findings prompt key questions regarding the relationship(s) among sensory transduction, regulation of endoflagellar rotation, and chemotactic responses (in particular, the role of glucose) during virulence expression by T. pallidum.
Hagman, K E; Porcella, S F; Popova, T G; Norgard, M V
Cocaine Dysregulates Opioid Gating of GABA Neurotransmission in the Ventral Pallidum
The ventral pallidum (VP) is a target of dense nucleus accumbens projections. Many of these projections coexpress GABA and the neuropeptide enkephalin, a ? and ? opioid receptor (MOR) ligand. Of these two, the MOR in the VP is known to be involved in reward-related behaviors, such as hedonic responses to palatable food, alcohol intake, and reinstatement of cocaine seeking. Stimulating MORs in the VP decreases extracellular GABA, indicating that the effects of MORs in the VP on cocaine seeking are via modulating GABA neurotransmission. Here, we use whole-cell patch-clamp on a rat model of withdrawal from cocaine self-administration to test the hypothesis that MORs presynaptically regulate GABA transmission in the VP and that cocaine withdrawal changes the interaction between MORs and GABA. We found that in cocaine-extinguished rats pharmacological activation of MORs no longer presynaptically inhibited GABA release, whereas blocking the MORs disinhibited GABA release. Moreover, MOR-dependent long-term depression of GABA neurotransmission in the VP was lost in cocaine-extinguished rats. Last, GABA neurotransmission was found to be tonically suppressed in cocaine-extinguished rats. These substantial synaptic changes indicated that cocaine was increasing tone on MOR receptors. Accordingly, increasing endogenous tone by blocking the enzymatic degradation of enkephalin inhibited GABA neurotransmission in yoked saline rats but not in cocaine-extinguished rats. In conclusion, our results indicate that following withdrawal from cocaine self-administration enkephalin levels in the VP are elevated and the opioid modulation of GABA neurotransmission is impaired. This may contribute to the difficulties withdrawn addicts experience when trying to resist relapse.
Scofield, Michael D.; Rice, Kenner C.; Cheng, Kejun; Roques, Bernard P.
An orexin hotspot in ventral pallidum amplifies hedonic 'liking' for sweetness.
Orexin (hypocretin) is implicated in stimulating appetite as well as arousal, and in both food reward and drug reward. The ventral pallidum (VP) receives orexin projections from lateral hypothalamus neurons (LH), and orexin terminals are especially dense in the posterior half of VP, which is also the location of an opioid hedonic hotspot. The VP hotspot is a roughly cubic-millimeter site where mu opioid stimulation can amplify the hedonic impact of sweetness, expressed as an increase in 'liking' reactions to sucrose taste. The anatomical overlap in posterior VP between opioid hotspot and orexin inputs raises the possibility that the hedonic hotspot might allow orexin to amplify 'liking' too. We examined whether microinjections of orexin-A into the VP hotspot enhance the hedonic impact of sucrose, as assessed via affective taste reactivity measures of 'liking' reactions, and additionally compared effects at nearby sites in adjacent LH and extended amygdala. Taste reactivity results indicated that orexin stimulation specifically in the VP hotspot nearly doubled the magnitude of positive 'liking' reactions elicited by the taste of sucrose. Mapping results for localization of function, aided by Fos plume measures of the local spread of orexin impact, suggested that hedonic enhancement was generated by essentially the same cubic-millimeter of posterior VP previously identified as the opioid hotspot. By contrast, microinjection sites in the anterior half of VP, or in LH or extended amygdala, generally failed to produce any hedonic enhancement. We conclude that an orexin hedonic hotspot exists in posterior VP, with similar boundaries to the opioid hotspot. An orexin hedonic hotspot may permit regulatory hypothalamic circuitry to make foods more 'liked' during hunger by acting through VP. Dysfunction in a VP orexin hotspot in addiction or mood disorders might also contribute to some types of affective psychopathology. PMID:23463152
Ho, Chao-Yi; Berridge, Kent C
US Patent & Trademark Office Database
The present invention relates to the production of antibody libraries. In particular, the present invention relates to the use of integrating retroviral vectors to generate libraries comprising a plurality of combinations of antibody light chains and heavy chains. The present invention thus provides improved methods of generating and screening antibody libraries comprising large numbers of unique antibodies.
Monoclonal antibodies and immobilized antibodies
Antibodies in both their free and immobilized state have been the object of considerable industrial and academic interest.\\u000a A variety of methods are used for preparing and immobilizing antibodies. Applications for monoclonal antibodies include the\\u000a preparation of therapeutics, diagnostics, and in affinity fractionation. Recent US patents on monoclonal and immobilized antibodies\\u000a and scientific literature on monoclonal antibodies are surveyed. A
Robert J. Linhardt; C. W. Abell; R. M. Denney; B. W. Altrock; R. Auerbach; S. D. Bernal; R. E. Canfield; P. H. Ehrlich; W. R. Moyle; T. S. Chan; T. W. Chang; N. T. Chang; J. A. Cidlowski; M. D. Viceps; R. J. Cote; D. M. Morrissey; A. N. Houghton; E. J. Beattie; H. F. Oettgen; L. J. Old; C. M. Croce; R. S. Cubicciotti; A. E. Karu; R. M. Krauss; J. S. Cullor; A. Deutsch; H. Brandwein; H. Platt; D. M. Hunter; A. Dubitsky; S. M. Durham; F. A. Dolbeare; J. W. Gray; G. R. Dreesman; C. E. Kendall; J. C. Egrie; A. R. Frackelton; H. N. Eisen; A. H. Ross; S. Gay; G. Geirnaert; J. E. Geltosky; E. H. Goldberg; E. Goldwasser; C. Kavinsky; T. L. Weiss; H. G. Gratzner; B. Hampar; M. Zweig; S. D. Showalter; H. H. Handley; M. C. Glassy; Y. Hagiwara; H. Hagiwara; C. M. Huang; S. N. Cohen; J. V. Hughes; E. M. Scolnick; J. E. Tomassini; R. Jefferis; J. Steensgaard; H. S. Kaplan; N. N. H. Teng; K. S. Earn; R. F. Calvo; L. Kass; J. R. Kettman; M. V. Norgard; M. B. Khazaeli; W. H. Beierwaltes; B. G. England; P. C. Kung; G. Goldstein; L. Lanier; J. Phillips; N. L. Warner; J. W. Larrick; A. R. Raubitschek; K. E. Truitt; H. Lazarus; J. F. Schwaber; J. Lewicki; C. Lewis; J. V. Olander; W. R. Tolbert; E. L. Milford; C. B. Carpenter; J. M. Paradysz; D. F. Mosher; J. L. Mulshine; J. D. Minna; K. A. Murray; D. M. Neville; R. J. Youle; M. Nicolson; I. Pastan; M. C. Willingham; D. J. Fitzgerald; A. Pucci; A. M. Smithyman; M. B. Slade; P. W. French; G. Wijffels; C. S. Pukel; K. O. Lloyd; L. R. Travassos; W. G. Dippold; R. P. Reckel; J. L. Harris; R. Wellerson; S. M. Shaw; P. M. Kaplan; E. L. Reinherz; S. F. Schlossman; S. C. Mener; J. Sakamoto; C. C. Cordon; E. Friedman; C. L. Finstad; W. E. Enker; M. R. Melamed; J. F. Oettgen; P. J. Scannon; L. E. Spitler; H. M. Lee; R. T. Kawahata; R. P. Mischak; J. Schlom; D. Colcher; M. Nuti; P. H. Hand; F. Austin; G. D. Shockman; D. E. Jackson; W. Wong; Z. Steplewski; H. Koprowski; M. Herlyn; M. Strand; I. S. Trowbridge; D. L. Urdal; C. J. March; S. K. Dower; J. R. Wands; V. R. Zurawski; C. A. White; R. Dulbecco; W. R. Allen; E. C. Arnold; M. Flasher; H. H. Freedman; T. D. Heath; P. Shek; D. Papahadjopoulos; M. Ikeda; S. Sakamoto; K. Suzuki; M. Kuboyama; Y. Harada; A. Kawashiri; E. Takahashi; H. S. Lee; S. Margel; R. C. Nowinski; A. S. Hoffman; J. W. Peterson; K. B. Platt; D. E. Reed; F. X. Real; M. J. Mattes; P. O. Livingston; A. Rembaum; R. C. K. Yen; R. Rosenstein; B. Schneider
Effects of electrolytic lesions of the lateral pallidum on motor coordination, spatial learning, and regional brain variations of cytochrome oxidase activity in rats
In view of recent theories suggesting a role for basal ganglia circuits in motor control and cognition, rats with bilateral electrolytic lesions of the lateral part of the globus pallidus were compared with control rats on motor coordination tasks and spatial learning in the Morris water maze. By comparison with sham-operated controls, rats with lesions of the lateral pallidum were
M. Jeljeli; C. Strazielle; J. Caston; R. Lalonde
New Tests for Syphilis: Rational Design of a PCR Method for Detection of Treponema pallidum in Clinical Specimens Using Unique Regions of the DNA Polymerase I Gene
A sensitive and specific PCR method to detect Treponema pallidum in clinical specimens was developed. PCR primers were designed based on two unique features of the DNA polymerase I gene (polA). The first distinctive characteristic is that the region codes for a high cysteine content and has low homology with similar regions of DNA polymerase I gene from known microorganisms.
HSI LIU; BERTA RODES; C.-Y. Chen; BRET STEINER
A Novel Lactamase Activity from a Penicillin-binding Protein of Treponema pallidum and Why Syphilis Is Still Treatable with Penicillin
Treponema pallidum, the causative agent of syphilis, is sensitive to penicillins. Yet, an abundant membrane- bound protein of this organism, Tp47, turns over peni- cillins. It is shown herein that the turnover process is a hydrolytic reaction that results in the corresponding penicilloates, products that have their -lactam bonds hydrolyzed. This is the reaction of -lactamases, bona fide resistance enzymes
J. Y. Cha; Akihiro Ishiwata; Shahriar Mobashery
Pharmacokinetic properties of arsenic species after oral administration of Sargassum pallidum extract in rats using an HPLC-HG-AFS method.
Sargassum pallidum is one of the Traditional Chinese Medicine widely used for phlegm elimination and detumescence. Arsenic is present in high concentration in seaweed belonging to the genus Sargassum. Therefore, the consumption of S. pallidum is a route of exposure to arsenic. Since the toxicity of arsenic is highly dependent on its chemical speciation, the determination of total arsenic is not adequate to assess the risks. Here, a high performance liquid chromatography-hydride generation-atomic fluorescence spectrometry (HPLC-HG-AFS) was developed for determination of the common arsenic species including arsenite [As(III)], dimethylarsinate (DMA), methylarsonate (MMA) and arsenate [As(V)] simultaneously. This method was applied to study the pharmacokinetic profile of these arsenic species in rats after oral administration of S. pallidum extract at different doses. The described assay was validated for limit of quantification, linearity, intra-day and inter-day precisions, accuracy, extraction recovery and stability according to the FDA validation guidelines. As(III) or MMA was not detected in any samples collected at all time points using the present HPLC-HG-AFS method. As(V) and DMA in the S. pallidum could be readily absorbed and eliminated in rats. A trend of dose-dependence was shown for DMA and As(V) in the drug concentration-time profiles. This study would be helpful for the apprehension of the action mechanism and clinical application of medicinal seaweeds. PMID:24763266
Cao, Yan; Duan, Jinao; Guo, Jianming; Li, Weixia; Tao, Weiwei
Estimation of the genome sizes of the chigger mites Leptotrombidium pallidum and Leptotrombidium scutellare based on quantitative PCR and k-mer analysis
Background Leptotrombidium pallidum and Leptotrombidium scutellare are the major vector mites for Orientia tsutsugamushi, the causative agent of scrub typhus. Before these organisms can be subjected to whole-genome sequencing, it is necessary to estimate their genome sizes to obtain basic information for establishing the strategies that should be used for genome sequencing and assembly. Method The genome sizes of L. pallidum and L. scutellare were estimated by a method based on quantitative real-time PCR. In addition, a k-mer analysis of the whole-genome sequences obtained through Illumina sequencing was conducted to verify the mutual compatibility and reliability of the results. Results The genome sizes estimated using qPCR were 191?±?7 Mb for L. pallidum and 262?±?13 Mb for L. scutellare. The k-mer analysis-based genome lengths were estimated to be 175 Mb for L. pallidum and 286 Mb for L. scutellare. The estimates from these two independent methods were mutually complementary and within a similar range to those of other Acariform mites. Conclusions The estimation method based on qPCR appears to be a useful alternative when the standard methods, such as flow cytometry, are impractical. The relatively small estimated genome sizes should facilitate whole-genome analysis, which could contribute to our understanding of Arachnida genome evolution and provide key information for scrub typhus prevention and mite vector competence.
Hemichorea in a thymoma patient without anti-CRMP-5 antibody.
We reported a 72-year-old man with thymoma who presented with hemichorea. Although his brain CT and MRI revealed no abnormality, regional cerebral blood flow changes, identified by single photon emission computed tomography, suggested that the mechanism underlying the chorea seemed to be a dysfunction of the subthalamic nucleus and pallidum. His hemichorea was completely resolved after thymectomy. Absence of serum anti-neural autoantibodies, including small-cell lung carcinoma-related chorea anti-CRMP-5 antibody, suggests that mechanisms different from cross-talk neural-targeted tumor immune response can be responsible for the thymoma-associated paraneoplastic chorea. PMID:24413817
Nakae, Yoshiharu; Ikeda, Shingo; Yamamoto, Ryoo; Tanaka, Fumiaki; Johkura, Ken
Seroprevalence survey of Egyptian tourism workers for hepatitis B virus, hepatitis C virus, human immunodeficiency virus, and Treponema pallidum infections: association of hepatitis C virus infections with specific regions of Egypt.
Blood samples from 740 Egyptian Nationals working in the tourism industry at two sites in the South Sinai governorate were screened for markers of infection with hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), and Treponema pallidum. Study subjects included 467 individuals from a rural seashore tourist village and 273 persons at two hotels in a well-established resort town. Subjects' ages ranged from 15 to 70 years; 99.3% were male. The prevalence of serologic markers for currently asymptomatic or past HBV infection alone was 20.7% (n = 153), of markers for past or chronic HCV infection alone was 7.4% (n = 55), and of markers for both HBV and HCV was 6.9% (n = 51). Of the 204 individuals positive for anti-HBV core antibody, 12 (5.9%) were also positive for hepatitis B surface antigen. Two individuals (0.3%) had a serologic market suggestive of an active syphilitic infection. No subject was found to be HIV-seropositive. History of prior injections and number of injections were associated with infection with HCV. Primary residence in the Nile delta and valley areas where schistosomiasis is highly endemic, was also a statistically significant risk factor for HCV, but not HBV infection. PMID:8780457
el-Sayed, N M; Gomatos, P J; Rodier, G R; Wierzba, T F; Darwish, A; Khashaba, S; Arthur, R R
ERIC Educational Resources Information Center
Monoclonal antibodies have provided an exciting addition to the "armory" of the molecular biologist and immunologist. This article discusses briefly the concept of, techniques available for, production of, and possible uses of monoclonal antibodies. (Author)
Killington, R. A.; Powell, K. L.
[Antiphospholipid antibodies].
The concept of antiphospholipid syndrome(APS) has been widely accepted. Antiphospholipid antibodies originally included anticardiolipin antibodies and lupus anticoagulants as serological marker of APS. However, recent advances have shown that most pathogenic antiphospholipid antibodies are directed to phospholipid binding proteins such as beta 2-glycoprotein I and prothrombin as well as phospholipids. The preliminary classification criteria for definite APS have been advocated as the "Sapporo criteria". Further prospective investigations are required to re-evaluate the clinical significance of so-called antiphospholipid antibodies. PMID:12924247
Kaburaki, Junichi; Kuwana, Masataka; Ikeda, Yasuo
Syphilis-causing strains belong to separate SS14-like or Nichols-like groups as defined by multilocus analysis of 19 Treponema pallidum strains.
Treponema pallidum strains are closely related at the genome level but cause distinct diseases. Subspecies pallidum (TPA) is the causative agent of syphilis, subspecies pertenue (TPE) causes yaws while subspecies endemicum (TEN) causes bejel (endemic syphilis). Compared to the majority of treponemal genomic regions, several chromosomal loci were found to be more diverse. To assess genetic variability in diverse genomic positions, we have selected (based on published genomic data) and sequenced five variable loci, TP0304, TP0346, TP0488, TP0515 and TP0558, in 19 reference Treponema pallidum strains including all T. pallidum subspecies (TPA, TPE and TEN). Results of this multilocus analysis divided syphilitic isolates into two groups: SS14-like and Nichols-like. The SS14-like group is comprised of SS14, Grady, Mexico A and Philadelphia 1 strains. The Nichols-like group consisted of strains Nichols, Bal 73-1, DAL-1, MN-3, Philadelphia 2, Haiti B and Madras. The TP0558 locus was selected for further studies because it clearly distinguished between the SS14- and Nichols-like groups and because the phylogenetic tree derived from the TP0558 locus showed the same clustering pattern as the tree constructed from whole genome sequences. In addition, TP0558 was shown as the only tested locus that evolved under negative selection within TPA strains. Sequencing of a short fragment (573bp) of the TP0558 locus in a set of 25 clinical isolates from 22 patients collected in the Czech Republic during 2012-2013 revealed that clinical isolates follow the SS14- and Nichols-like distribution. PMID:24841252
Nechvátal, Lukáš; P?trošová, Helena; Grillová, Linda; Pospíšilová, Petra; Mikalová, Lenka; Strnadel, Radim; Kuklová, Ivana; Kojanová, Martina; Kreidlová, Miluše; Va?ousová, Daniela; Procházka, P?emysl; Zákoucká, Hana; Krch?áková, Alena; Smajs, David
Production of proinflammatory cytokines in the human THP-1 monocyte cell line following induction by Tp0751, a recombinant protein of Treponema pallidum.
The tissue destruction characteristic of syphilis infection may be caused by inflammation due to Treponema pallidum and the ensuing immune responses to the pathogen. T. pallidum membrane proteins are thought to be potent inducers of inflammation during the early stages of infection. However, the actual membrane proteins that induce inflammatory cytokine production are not known, nor are the molecular mechanisms responsible for triggering and sustaining the inflammatory cascades. In the present study, Tp0751 recombinant protein from T. pallidum was found to induce the production of proinflammatory cytokines, including TNF-alpha, IL-1beta and IL-6, in a THP-1 human monocyte cell line. The signal transduction pathways involved in the production of these cytokines were then further investigated. No inhibition of TNF-a, IL-1beta, or IL-6 production was observed following treatment with the SAPK/JNK specific inhibitor SP600125 or with an ERK inhibitor PD98059. By contrast, anti-TLR2 mAb, anti-CD14 mAb, and the p38 inhibitor SB203580 significantly inhibited the production of all three cytokines. In addition, pyrrolidine dithiocarbamate (PDTC), a specific inhibitor of NF-kappaB, profoundly inhibited the production of these cytokines. Tp0751 treatment strongly activated NF-kappaB, as revealed by Western blotting. However, NF-kappaB translocation was significantly inhibited by treatment with PDTC. These results indicated that TLR2, CD14, MAPKs/p38, and NF-kappaB might be implicated in the inflammatory reaction caused by T. pallidum infection. PMID:20596832
Liu, ShuangQuan; Wang, ShiPing; Wu, YiMou; Zhao, FeiJun; Zeng, TieBing; Zhang, YueJun; Zhang, QiuGui; Gao, DongMei
Antithyroid microsomal antibody
Thyroid antimicrosomal antibody; Antimicrosomal antibody; Microsomal antibody; Thyroid peroxidase antibody; TPOAb ... test is done to confirm the cause of thyroid problems, including Hashimoto's thyroiditis . The test may also ...
[Laboratory diagnosis of Treponema pallidum infection in patients with early syphilis and neurosyphilis through a PCR-based test].
Syphilis is a sexually transmitted disease caused by Treponema pallidum. The diagnosis is based mainly in clinical presentation and non-specific assays. PCR-based diagnosis has been suggested as an attractive alternative method. The aim of this study was the validation of a PCR-based test for the diagnosis of early syphilis (ES) and neurosyphilis (NS). Clinical samples of mucocutaneous lesions and cerebrospinal fluid (CSF) specimens from patients previously diagnosed for ES and NS respectively using an enlarged gold standard, were tested by PCR. The reaction was done using primers targeting the tpN47 gene. Twenty out of 21 mucocutaneous samples from patients diagnosed with ES were positive by PCR, with a clinical sensitivity of 95%. Four out of 8 CSF samples from patients previously diagnosed with NS were positive by PCR, with a clinical sensitivity of 50%. The clinical specificity for both ES and NS was 100%. The PCR sensitivity and specificity for mucocutaneous samples allowed us to implement this assay in our laboratory for routine diagnosis. Although the sensitivity of the PCR in CSF was low, it may be useful to support clinical diagnosis. PMID:22052394
García, Patricia; Grassi, Bruno; Fich, Félix; Salvo, Aurelio; Araya, Luis; Abarzúa, Fernando; Soto, Julia; Poggi, Helena; Lagos, Marcela; Vásquez, Patricia; León, Eugenia P; Pérez, Carlos; Wozniak, Aniela
Europium Nanoparticle-Based High Performing Immunoassay for the Screening of Treponemal Antibodies
Treponema pallidum subspecies pallidum (Tp) is the causative agent of syphilis which mainly spreads through sexual contact, blood transfusion and perinatal route. In order to curtail the spread of the infection and to clinically manage the disease, timely, accurate and reliable diagnosis is very important. We have developed an immunoassay for the detection of treponemal antibodies in human serum or plasma samples. In vivo biotinylated and non-biotinylated versions of the recombinant antigen were designed by the fusion of three Tp-specific antigens namely Tp15, Tp17 and Tp47. These fusion antigens were expressed in E. coli and purified using single-step metal affinity chromatography. Biotinylated fusion antigen immobilized on streptavidin coated plate was used to capture the treponemal antibodies and the non-biotinylated antigen coated on europium nanoparticles was used as tracer. Assays with two different incubation times of 10 min and 1 h were developed, and following the incubation the europium fluorescence was measured using time-resolved fluorometry. The developed time-resolved fluorometric (TRF) immunoassays were evaluated with in-house and commercial serum/plasma sample panels. For well-established treponemal antibodies positive or negative samples, the sensitivity of TRF immunoassay with 10 min incubation time was 97.4%, and of TRF immunoassay with 1 h incubation time was 98.7%, and the specificities of both the TRF immunoassays were 99.2%. For the samples with discordant results with the reference assays, both the TRF immunoassays showed better specificity than the Enzygnost syphilis enzyme immunoassay as a screening test. The two different incubation times did not have any significant effect on the signal to cutoff (S/Co) ratios obtained with the two immunoassays (p?=?0.06). Our results indicate that the developed immunoassay with a short incubation time of 10 min has the potential to be used in clinical laboratories and in blood-bank settings as a screening test for treponemal antibodies.
Talha, Sheikh M.; Hytonen, Jukka; Westhorpe, Adam; Kumar, Sushil; Khanna, Navin; Pettersson, Kim
Europium nanoparticle-based high performing immunoassay for the screening of treponemal antibodies.
Treponema pallidum subspecies pallidum (Tp) is the causative agent of syphilis which mainly spreads through sexual contact, blood transfusion and perinatal route. In order to curtail the spread of the infection and to clinically manage the disease, timely, accurate and reliable diagnosis is very important. We have developed an immunoassay for the detection of treponemal antibodies in human serum or plasma samples. In vivo biotinylated and non-biotinylated versions of the recombinant antigen were designed by the fusion of three Tp-specific antigens namely Tp15, Tp17 and Tp47. These fusion antigens were expressed in E. coli and purified using single-step metal affinity chromatography. Biotinylated fusion antigen immobilized on streptavidin coated plate was used to capture the treponemal antibodies and the non-biotinylated antigen coated on europium nanoparticles was used as tracer. Assays with two different incubation times of 10 min and 1 h were developed, and following the incubation the europium fluorescence was measured using time-resolved fluorometry. The developed time-resolved fluorometric (TRF) immunoassays were evaluated with in-house and commercial serum/plasma sample panels. For well-established treponemal antibodies positive or negative samples, the sensitivity of TRF immunoassay with 10 min incubation time was 97.4%, and of TRF immunoassay with 1 h incubation time was 98.7%, and the specificities of both the TRF immunoassays were 99.2%. For the samples with discordant results with the reference assays, both the TRF immunoassays showed better specificity than the Enzygnost syphilis enzyme immunoassay as a screening test. The two different incubation times did not have any significant effect on the signal to cutoff (S/Co) ratios obtained with the two immunoassays (p=0.06). Our results indicate that the developed immunoassay with a short incubation time of 10 min has the potential to be used in clinical laboratories and in blood-bank settings as a screening test for treponemal antibodies. PMID:24386329
Talha, Sheikh M; Hytönen, Jukka; Westhorpe, Adam; Kumar, Sushil; Khanna, Navin; Pettersson, Kim
Prevalence survey of infection with Treponema pallidum among HIV-positive patients in Tehran
Objective To identify the frequency of syphilis among Iranian HIV-positive patients. Methods A cross-sectional study on the prevalence of syphilis and HIV co-infection among 450 patients diagnosed with HIV infection was conducted between 2004 and 2008 at Imam Khomeini hospital, Tehran, Iran. The lab tests including CD4 cell count, cerebrospinal fluid, veneral disease research laboratory (VDRL), fluorescent treponema antibody-absorption (FTA-Abs) and viral load were performed for all the patients. Data regarding medical history and their demographics were also collected. Results Of all 450 HIV-positive patients, 24 (5.3%) had a positive VDRL test and only two men had a FTA-Abs positive test which means 0.45% of them had a definite co-infection of syphilis. 65.3% of the HIV-positive patients were injection drug users that the co-infection prevalence of them was 0.7%. We did not find any patient with neurosyphilis. Conclusions Considering the increasing prevalence of HIV and also extensive use of highly active antiretroviral therapy in developing nations, the diagnosis of syphilis should be timely established using screening tests among such patients.
Badie; Yavari, Zeinab; Esmaeeli, Shooka; Paydary, Koosha; Emamzadeh-Fard, Sahra; SeyedAlinaghi, SeyedAhmad; Rasoulinejad, Mehrnaz
The antibody molecule is modular and separate domains can be extracted through biochemical or genetic means. It is clear from review of the literature that a wave of novel, antigen-specific molecular forms may soon enter clinical evaluation. This report examines the developmental histories of therapeutics derived from antigen-specific fragments of antibodies produced by recombinant processes. Three general types of fragments were observed, antigen-binding fragments (Fab), single chain variable fragments (scFv) and "third generation" (3G), each representing a successive wave of antibody fragment technology. In parallel, drug developers have explored multi-specificity and conjugation with exogenous functional moieties in all three fragment types. Despite high hopes and an active pipeline, enthusiasm for differentiating performance of fragments should, perhaps, be tempered as there are yet few data that suggest these molecules have distinct clinical properties due only to their size.
Tromp1, a putative rare outer membrane protein, is anchored by an uncleaved signal sequence to the Treponema pallidum cytoplasmic membrane.
Treponema pallidum rare outer membrane protein 1 (Tromp1) has extensive sequence homology with substrate-binding proteins of ATP-binding cassette transporters. Because such proteins typically are periplasmic or cytoplasmic membrane associated, experiments were conducted to clarify Tromp1's physicochemical properties and cellular location in T. pallidum. Comparison of the sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobilities of (i) native Tromp1 and Tromp1 synthesized by coupled in vitro transcription-translation and (ii) native Tromp1 and recombinant Tromp1 lacking the N-terminal signal sequence revealed that the native protein is not processed. Other studies demonstrated that recombinant Tromp1 lacks three basic porin-like properties: (i) the ability to form aqueous channels in liposomes which permit the influx of small hydrophilic solutes, (ii) an extensive beta-sheet secondary structure, and (iii) amphiphilicity. Subsurface localization of native Tromp1 was demonstrated by immunofluorescence analysis of treponemes encapsulated in gel microdroplets, while opsonization assays failed to detect surface-exposed Tromp1. Incubation of motile treponemes with 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)-diazarine, a photoactivatable, lipophilic probe, also did not result in the detection of Tromp1 within the outer membranes of intact treponemes but, instead, resulted in the labeling of a basic 30.5-kDa presumptive outer membrane protein. Finally, analysis of fractionated treponemes revealed that native Tromp1 is associated predominantly with cell cylinders. These findings comprise a body of evidence that Tromp1 actually is anchored by an uncleaved signal sequence to the periplasmic face of the T. pallidum cytoplasmic membrane, where it likely subserves a transport-related function.
Akins, D R; Robinson, E; Shevchenko, D; Elkins, C; Cox, D L; Radolf, J D
Manipulation of GABA in the ventral pallidum, but not the nucleus accumbens, induces intense, preferential, fat consumption in rats.
Injections of the GABAA antagonist bicuculline into the medial ventral pallidum (VPm) induce marked increases in food intake, but nothing is known about the way in which these injections alter the distribution of intake in a macronutrient selection situation. We investigated this topic by adapting rats to a diet containing independent sources of protein, carbohydrate and fat, and then examining the effects of intra-VPm bicuculline on diet selection. Under these conditions, bicuculline produced a massive, preferential increase in fat intake with subjects consuming a mean of 97% of their calories from fat. Furthermore, all treated subjects ate fat before any other macronutrient, suggesting that the animals' behavior was directed selectively toward this dietary component even before consumption had begun. Similar effects were not observed following food deprivation, which exerted its largest effect on carbohydrate intake. To compare the intra-VPm bicuculline response to that seen after activation of GABA receptors in the nucleus accumbens shell (AcbSh), a major source of projections to the VPm, we conducted similar experiments with intra-AcbSh injections of muscimol and baclofen. These injections also enhanced food intake, but did not reproduce the selective preference for fat seen after intra-VPm bicuculline. These experiments provide the first demonstration of preferential enhancement of fat intake following manipulations of a nonpeptide neurotransmitter. Since mean intakes of fat under baseline conditions and after deprivation tended to be lower than those of carbohydrates, it seems unlikely that the effects of intra-VPm bicuculline are related to the intrinsic "rewarding" properties of fat, but might rather reflect the induction of a state of "fat craving." PMID:24867334
Covelo, Ignacio R; Patel, Zaid I; Luviano, Jennifer A; Stratford, Thomas R; Wirtshafter, David
Delta-opioid receptor blockade in the ventral pallidum increases perceived palatability and consumption of saccharin solution in rats.
The ventral pallidum (VP) is involved in ingestive behaviour. It receives dense GABAergic projections from the nucleus accumbens. GABAergic terminals in the VP co-express enkephalin, an endogenous ligand of delta-opioid receptors. The role of the delta-opioid receptors in the VP in the context of ingestive behaviour remains unclear, in contrast to the well-understood involvement of the mu-opioid receptors. We used the single-bottle test to examine the effects of VP microinjections of the delta-opioid receptor antagonist naltrindole on consumption of a saccharin solution. Naltrindole injections significantly increased the intake of saccharin, but not water, during a 2-h test session. We also investigated perceived palatability of saccharin using a taste reactivity test. The drug treatments increased ingestive responses to intraorally infused saccharin. Further experimentation explored the role of VP delta-opioid receptors in behavioural responses to saccharin that were previously paired with malaise upon the retrieval of conditioned taste aversion (CTA). Naltrindole-injected rats exhibited longer latency for the first occurrence of aversive responses than vehicle-injected control rats. However, there was no between-group difference in total aversive responses. These results suggest that naltrindole injections into the VP induce an enhancement of perceived palatability of a normally preferred saccharin solution, and thereby facilitate consumption of the solution. On the other hand, delayed aversive responses to the conditioned aversive saccharin suggest that the delta-opioid receptors in the VP mediate the initiation of aversive taste reactivity responses to the conditioned stimulus upon CTA retrieval. PMID:24739358
Inui, Tadashi; Shimura, Tsuyoshi
NSDL National Science Digital Library
This site contains a thorough overview of the fundamentals of antibodies. The site starts with an introduction to antigens and antibodies, antibody production and titer including practical information.
Injection of a sufficient dose of bacteriophage ?X 174 into guinea pigs results in the formation of rapidly sedimenting antibody molecules (19S), and later, slowly sedimenting molecules (7S). Above a threshold dose of antigen, the relative rate of 19S formation is maximal and dose-independent; below this dose, slower relative rates are obtained. The time for doubling the serum 19S level is as short as 6 to 8 hours, suggesting that the absolute rate of antibody formation per cell is increasing in addition to proliferation of antibody-producing cells. Synthesis of 19S after injection of 1010 ?X virtually ceases at 10 days after which 19S antibody activity disappears from the circulation with a half-life of approximately 24 hours. A second injection of ?X on day 5 or 9 prolongs 19S synthesis, indicating that antigen not only can regulate the relative rate, but also is essential for continued synthesis of 19S. 19S synthesis is also prolonged in guinea pigs by injection of ?X with endotoxin or by 400 r whole body x-irradiation 24 hours after injection of phage into rabbits. The primary 7S response is not detected until approximately 1 week after immunization and relative rates are antigen-dependent. Primary 7S synthesis can continue for many months and leads to preparation for a secondary antibody response (immunological memory) during which only 7S is detected. In contrast, in animals that form precipitating 19S without detectable 7S, a second injection of phage 1 month later results in a second 19S response which closely resembles the first. These findings have led to the suggestion that formation of 19S does not lead to persisting immunological memory.
Uhr, Jonathan W.; Finkelstein, Martin S.
Reagents Data Portal AntibodiesNCI announces the release of monoclonal antipeptide antibodies from rabbit for distribution on the antibody portal. There are 60 recently added monoclonal antibodies, with 56 generated from mouse and 4 generated from rabbit. Print
The TP0796 Lipoprotein of Treponema pallidum Is a Bimetal-dependent FAD Pyrophosphatase with a Potential Role in Flavin Homeostasis*
Treponema pallidum, an obligate parasite of humans and the causative agent of syphilis, has evolved the capacity to exploit host-derived metabolites for its survival. Flavin-containing compounds are essential cofactors that are required for metabolic processes in all living organisms, and riboflavin is a direct precursor of the cofactors FMN and FAD. Unlike many pathogenic bacteria, Treponema pallidum cannot synthesize riboflavin; we recently described a flavin-uptake mechanism composed of an ABC-type transporter. However, there is a paucity of information about flavin utilization in bacterial periplasms. Using a discovery-driven approach, we have identified the TP0796 lipoprotein as a previously uncharacterized Mg2+-dependent FAD pyrophosphatase within the ApbE superfamily. TP0796 probably plays a central role in flavin turnover by hydrolyzing exogenously acquired FAD, yielding AMP and FMN. Biochemical and structural investigations revealed that the enzyme has a unique bimetal Mg2+ catalytic center. Furthermore, the pyrophosphatase activity is product-inhibited by AMP, indicating a possible role for this molecule in modulating FMN and FAD levels in the treponemal periplasm. The ApbE superfamily was previously thought to be involved in thiamine biosynthesis, but our characterization of TP0796 prompts a renaming of this superfamily as a periplasmic flavin-trafficking protein (Ftp). TP0796 is the first structurally and biochemically characterized FAD pyrophosphate enzyme in bacteria. This new paradigm for a bacterial flavin utilization pathway may prove to be useful for future inhibitor design.
Deka, Ranjit K.; Brautigam, Chad A.; Liu, Wei Z.; Tomchick, Diana R.; Norgard, Michael V.
The TP0796 lipoprotein of Treponema pallidum is a bimetal-dependent FAD pyrophosphatase with a potential role in flavin homeostasis.
Treponema pallidum, an obligate parasite of humans and the causative agent of syphilis, has evolved the capacity to exploit host-derived metabolites for its survival. Flavin-containing compounds are essential cofactors that are required for metabolic processes in all living organisms, and riboflavin is a direct precursor of the cofactors FMN and FAD. Unlike many pathogenic bacteria, Treponema pallidum cannot synthesize riboflavin; we recently described a flavin-uptake mechanism composed of an ABC-type transporter. However, there is a paucity of information about flavin utilization in bacterial periplasms. Using a discovery-driven approach, we have identified the TP0796 lipoprotein as a previously uncharacterized Mg(2+)-dependent FAD pyrophosphatase within the ApbE superfamily. TP0796 probably plays a central role in flavin turnover by hydrolyzing exogenously acquired FAD, yielding AMP and FMN. Biochemical and structural investigations revealed that the enzyme has a unique bimetal Mg(2+) catalytic center. Furthermore, the pyrophosphatase activity is product-inhibited by AMP, indicating a possible role for this molecule in modulating FMN and FAD levels in the treponemal periplasm. The ApbE superfamily was previously thought to be involved in thiamine biosynthesis, but our characterization of TP0796 prompts a renaming of this superfamily as a periplasmic flavin-trafficking protein (Ftp). TP0796 is the first structurally and biochemically characterized FAD pyrophosphate enzyme in bacteria. This new paradigm for a bacterial flavin utilization pathway may prove to be useful for future inhibitor design. PMID:23447540
Deka, Ranjit K; Brautigam, Chad A; Liu, Wei Z; Tomchick, Diana R; Norgard, Michael V
Newly available antibodies with practical applications in surgical pathology.
Selected antibodies that have become available in recent years and have applications in diagnostic pathology are discussed. They include antibodies that are organ-related, provide information on cellular differentiation or histogenetic type, have predictive value in tumors, and highlight infective agents. PAX8 (paired box gene 8) is a marker expressed in the lower female genital tract, thyroid, and kidney and their tumors. Napsin A is expressed in the lung and kidney and is an alternative marker for pulmonary adenocarcinoma. Arginase A is a sensitive and specific marker for liver tumors. ERG (Ets-related gene) is an excellent marker for endothelium and vascular tumors as well as prostatic cancer (about 50% of cases). SOX10 (SRY-related HMG box) is expressed predominantly in melanocytic and Schwann cells and the corresponding tumors. DOG1 (discovered on GIST 1) is an excellent marker for gastrointestinal stromal tumor (GIST) and acinic cell carcinoma. OCT3/4 is a pan-germ cell tumor marker, except yolk sac tumor. SALL4 is positive in various types of germ cell tumors, including yolk sac tumor. MUC4 (mucin-related antigen 4) is a sensitive and specific marker for low-grade fibromyxoid sarcoma. Langerin is a specific marker for Langerhans cells and their tumors. SOX11 is a sensitive marker for mantle cell lymphoma. New generation antibodies against anaplastic lymphoma kinase (ALK) are required to reliably demonstrate ALK gene translocation in pulmonary carcinomas. Lack of expression of succinate dehydrogenase B is seen in paragangliomas of the hereditary form and in the pediatric type of GIST. Antibodies against Trepenoma pallidum can facilitate the diagnosis of syphilis, whereas those against SV40 (simian virus 40) are helpful for diagnosis of BK virus infection and progressive multifocal leukoencephalopathy. PMID:24225578
Chan, John K C
Detection of the A2058G and A2059G 23S rRNA Gene Point Mutations Associated with Azithromycin Resistance in Treponema pallidum by Use of a TaqMan Real-Time Multiplex PCR Assay
Macrolide treatment failure in syphilis patients is associated with a single point mutation (either A2058G or A2059G) in both copies of the 23S rRNA gene in Treponema pallidum strains. The conventional method for the detection of both point mutations uses nested PCR combined with restriction enzyme digestions, which is laborious and time-consuming. We initially developed a TaqMan-based real-time duplex PCR assay for detection of the A2058G mutation, and upon discovery of the A2059G mutation, we modified the assay into a triplex format to simultaneously detect both mutations. The point mutations detected by the real-time triplex PCR were confirmed by pyrosequencing. A total of 129 specimens PCR positive for T. pallidum that were obtained from an azithromycin resistance surveillance study conducted in the United States were analyzed. Sixty-six (51.2%) of the 129 samples with the A2058G mutation were identified by both real-time PCR assays. Of the remaining 63 samples that were identified as having a macrolide-susceptible genotype by the duplex PCR assay, 17 (27%) were found to contain the A2059G mutation by the triplex PCR. The proportions of macrolide-susceptible versus -resistant genotypes harboring either the A2058G or the A2059G mutation among the T. pallidum strains were 35.6, 51.2, and 13.2%, respectively. None of the T. pallidum strains examined had both point mutations. The TaqMan-based real-time triplex PCR assay offers an alternative to conventional nested PCR and restriction fragment length polymorphism analyses for the rapid detection of both point mutations associated with macrolide resistance in T. pallidum.
Chi, Kai-Hua; Pillay, Allan; Nachamkin, Eli; Su, John R.; Ballard, Ronald C.
[Prevalence, risk factors and genetic characterization of human T-cell lymphotropic virus types 1 and 2 in patients infected with human immunodeficiency virus type 1 in the cities of Ribeirăo Preto and Săo Paulo].
The aim of this study was to define the prevalence of human T cell lymphotropic virus types 1 and 2 in patients who were positive for human immunodeficiency virus type 1 in the State of Săo Paulo, Brazil. We evaluated 319 individuals infected with HIV type 1 who were attended at specialized clinics in two cities (Ribeirăo Preto and Săo Paulo). The patients were interviewed and tested for antibodies against HTLV types 1 and 2 (Orthoâ HTLV-1/HTLV-2 Ab-Capture enzyme immunoassay). Direct DNA sequencing of polymerase chain reaction products from the tax region of HTLV type 2 and the long terminal repeat region of HTLV types 1 and 2 were performed to differentiate and determine the subtypes. The overall prevalence of anti-HTLV type 1 and 2 antibodies was 7.5% (24/319; 95% CI: 5.2-11.5). HTLV type 1 and 2 infection was associated with a history of injected drug use and with antibodies for hepatitis C virus (p < 0.001), but not with age (p = 0.2), sex (p = 0.9), sexual behavior or serological markers for sexually transmitted diseases (anti-Treponema pallidum, anti-human herpesvirus type 8 or anti-hepatitis B virus antibodies) (p > 0.05). HTLV DNA was detected in 13 out of 24 samples, of which 12 were characterized as HTLV subtype 2c and one as HTLV subtype 1a. Among the 12 HTLV type 2 samples, seven were from injected drug users, thus indicating that this route is an important risk factor for HTLV type 2 transmission among our population infected with HIV type 1. PMID:19684973
Kleine Neto, Walter; Sanabani, Sabri Saeed; Jamal, Leda Fátima; Sabino, Ester Cerdeira
Proposal of a new halobacterial genus Natrinema gen. nov., with two species Natrinema pellirubrum nom. nov. and Natrinema pallidum nom. nov.
A phylogenetic analysis of 69 halobacterial 16S rRNA gene sequences has been carried out, integrating data from new isolates, previously described halobacteria and cloned sequences from uncultivated halobacteria. Halobacterium halobium NCIMB 777, Halobacterium trapanicum NCIMB 784 and Halobacterium salinarium NCIMB 786, together with several other strains (strains T5.7, L11 and Halobacterium trapanicum NCIMB 767) constitute a distinct lineage with at least 98.2% sequence similarity. These strains have been incorrectly assigned to the genus Halobacterium. Therefore, based on a variety of taxonomic criteria, it is proposed that Halobacterium salinarium NCIMB 786 is renamed as Natrinema pellirubrum nom. nov., the type species of the new genus Natrinema gen. nov., and that Halobacterium halobium NCIMB 777 and Halobacterium trapanicum NCIMB 784 are renamed as a single species, Natrinema pallidum nom. nov. It was notable that halobacteria closely related to the proposed new genus have been isolated from relatively low-salt environments. PMID:9828420
McGenity, T J; Gemmell, R T; Grant, W D
Reagents Data Portal Antibodies
NCI announces the release of monoclonal antipeptide antibodies from rabbit for distribution on the antibody portal. There are 60 recently added monoclonal antibodies, with 56 generated from mouse and 4 generated from rabbit.
National Technical Information Service (NTIS)
The invention relates, in general, to antibodies to p40 (both polyclonal and monoclonal). Additionally, the invention relates to hybridomas which produce monoclonal antibodies to p40 and diagnostic kits comprising antibodies to p40.
T. G. Fanning
Antibody Phage Display Technology
In the past decade, the drive to develop completely human antibodies for human therapy has led to the develop- ment of phage display technology. This technology is able to deliver the ultimate in antibody engineering, that is, high-affinity fully human antibodies to any antigen of choice. Here, this application of phage display technology is reviewed, and the many other antibody
Patrick Chames; Hennie R Hoogenboom
Antibodies with infinite affinity
Here we report an approach to the design and production of antibody\\/ligand pairs, to achieve functional affinity far greater than avidin\\/biotin. Using fundamental chemical principles, we have developed antibody\\/ligand pairs that retain the binding specificity of the antibody, but do not dissociate. Choosing a structurally characterized antibody\\/ligand pair as an example, we engineered complementary reactive groups in the antibody binding
Albert J. Chmura; Molly S. Orton; Claude F. Meares
... CeliacDisease.net People with celiac disease who eat gluten have higher than normal levels of certain antibodies ... rye and barley that are generically known as "gluten." Find Out For Sure Antibody tests are only ...
Identification and sequences of the Treponema pallidum fliM', fliY, fliP, fliQ, fliR and flhB' genes.
Information regarding the biology and virulence attributes of Treponema pallidum (Tp) is limited due to the lack of genetic exchange mechanisms and the inability to continuously cultivate this spirochete. We have utilized TnphoA mutagenesis of a Tp genomic DNA library in Escherichia coli (Ec) to identify genes encoding exported proteins, a subset of which are likely to be important in treponemal pathogenesis. We report here the identification and nucleotide (nt) sequence of a 5-kb treponemal DNA insert that contains seven open reading frames (ORFs). The proteins encoded by six of these ORFs have homology with members of a newly described protein family involved in the biogenesis/assembly of flagella and the control of flagellar rotation in Ec, Salmonella typhimurium (St) and Bacillus subtilis (Bs). Certain members of this family are also involved in the export of virulence factors in Yersinia (Yr) spp., St and Shigella flexneri (Sf). We have named these six ORFs fliM', fliY, fliP, fliQ, fliR and flhB'. The operon containing these ORFs has been designated as the fla operon. We hypothesize that the protein products of these genes are involved in the biogenesis/assembly of flagella and the control of flagellar rotation in Tp. PMID:8529894
Hardham, J M; Frye, J G; Stamm, L V
Prevalence of Treponema pallidum seropositivity and herpes simplex virus type 2 infection in a cohort of men who have sex with men, Bangkok, Thailand, 2006-2010.
We report prevalence of Treponema pallidum (TP) seropositivity and herpes simplex virus type 2 (HSV-2) infection and risk factors associated with their prevalence in a cohort of men who have sex with men (MSM) in Bangkok, Thailand. Between April 2006 and March 2010 we enrolled Thai MSM into a cohort study based at the Silom Community Clinic, with baseline behavioural data and laboratory testing for sexually transmitted infections (STIs). Logistic regression was used to analyse risk factors associated with the prevalence of TP seropositivity and HSV-2 infection. From a total of 1544 enrolled men (mean age 26 years) TP, HSV-2 and HIV seropositive rates were 4.4%, 20.7% and 21.6%, respectively. After multivariable analysis, participating in group sex, reporting paying for sex, reporting sex with a casual partner in a park and being HSV-2 seropositive were associated with TP prevalence. Age ?30 years, having less than a high school education, past use of recreational drugs, meeting casual sexual partners at a public venue (sauna) and TP seropositivity were associated with HSV-2 infection. The significant baseline prevalence of TP seropositivity and HSV-2 infection in this cohort demonstrates the need for screening and treatment of these STIs and targeted prevention interventions in Thai MSM in Bangkok. PMID:22807537
Holtz, T H; Thienkrua, W; McNicholl, J M; Wimonsate, W; Chaikummao, S; Chonwattana, W; Wasinrapee, P; Varangrat, A; Mock, P A; Sirivongrangson, P; van Griensven, F
Assignment of disulfide bonds in gp64, a putative cell-cell adhesion protein of Polysphondylium pallidum. Presence of Sushi domains in the cellular slime mold protein.
The 64-kDa membrane-bound glycoprotein of the cellular slime mold Polysphondylium pallidum (referred to as gp64), seems to be implicated in cell-cell adhesion. Previously we have isolated a full-length gp64 cDNA, determined its nucleotide sequence, and found that all cysteine residues in the protein are involved in the formation of disulfide bonds. The disulfide arrangement of the 36 cysteines in gp64 was established by analysis of proteolytically cleaved protein and sequence analysis of cystine-containing fragments. Since gp64 has 36 Cys residues, 18 disulfide bonds must exist and the positions of 15 of them were determined. The 15 disulfide bonds in gp64 constitute five characteristic, so-called Sushi domains. In a Sushi domain, the first Cys in a sequence is connected to the third one and the second Cys to the fourth one. This is the first report describing the presence of Sushi domains in a cellular slime mold protein. From these data, gp64 appears to be distinct from all other previously described cell-adhesion proteins. PMID:7961835
Saito, T; Kumazaki, T; Ochiai, H
Effects of electrical lesions of the medial preoptic area and the ventral pallidum on mate-dependent paternal behavior in mice.
In laboratory animals, less is known about the neural circuits that mediate paternal behavior than those that influence maternal behavior. In mice, we recently reported that when sires are separated with their mate dams from their pups, ultrasound and pheromonal signals from the dams can evoke and initiate maternal-like retrieval behavior in the sires upon reunion with the offspring; this is termed mate-dependent paternal care. We used electrolytic brain lesion (EBL) methods to identify the potential roles of the medial preoptic area (mPOA) and ventral pallidum (VP) regions in regulating paternal care, areas known to be critical for the expression of maternal behavior. Electrolytic lesions of the mPOA or VP disrupted mate-dependent paternal care; latencies to initiate pup retrieval, grooming and crouching were longer in the EBL-treated sires relative to the sham-operated mice. The number of grooming episodes and duration of crouching were also lower in sires with the EBL in both areas. These results indicate that the mPOA and VP regions are essential for mate-dependent paternal care in mice. PMID:24721669
Akther, Shirin; Fakhrul, Azam A K M; Higashida, Haruhiro
Intracellular antibodies for proteomics
The intracellular antibody technology has many applications for proteomics studies.The potential of intracellular antibodies for the systematic study of the proteome has been made possible by the development of new experimental strategies that allow the selection of antibodies under conditions of intracellular expression. The Intracellular Antibody Capture Technology (IACT) is an in vivo two-hybrid-based method originally developed for the selection
Michela Visintin; Giovanni Antonio Meli; Isabella Cannistraci; Antonino Cattaneo
The use of monoclonal antibodies (mAbs) for cancer therapy has achieved considerable success in recent years. Antibody–drug conjugates are powerful new treatment options for lymphomas and solid tumours, and immunomodulatory antibodies have also recently achieved remarkable clinical success. The development of therapeutic antibodies requires a deep understanding of cancer serology, protein-engineering techniques, mechanisms of action and resistance, and the interplay
Jedd D. Wolchok; Lloyd J. Old; Andrew M. Scott
ERIC Educational Resources Information Center
Understanding antibody structure and function is difficult for many students. The rearrangement of constant and variable regions during antibody differentiation can be effectively simulated using a paper model. Describes a hands-on laboratory exercise which allows students to model antibody diversity using readily available resources. (PVD)
Baker, William P.; Moore, Cathy Ronstadt
US Patent & Trademark Office Database
Antibodies that interact with osteoprotegerin ligand (OPGL) are described. Methods of treating osteopenic disorders by administering a pharmaceutically effective amount of antibodies to OPGL are described. Methods of detecting the amount of OPGL in a sample using antibodies to OPGL are described.
[Bispecific antibodies: what future?].
Monoclonal antibodies have emerged as a very successful class of therapeutic agents. In their native format, monoclonal antibodies are monospecific in that they recognize only one epitope, but their Fc domain also binds to FcfR-expressing cells. Attempts to improve the cytotoxicity of antibodies, particularly in the cancer field, have led to the design of bispecific antibodies: this can occur through various strategies, such as quadroma, thioether-linked Fab' gamma fragments or genetic engineering. Such bispecific antibodies have been developped to enhance immunotherapy, by bridging tumor cells and T cells, or radioimmunotherapy by combining bispecific antibodies and radiolabeled bivalent haptens that bind cooperatively to target cells. Multiple further applications can be envisaged such as targeting two different antigens on the same cell, or two epitopes of the same antigen. Although progresses have been slowed by technical constraints, there is little doubt that this class of novel antibodies derivatives will experience a promising development. PMID:20035697
Pčlegrin, André; Robert, Bruno
Activation of human monocytic cells by Treponema pallidum and Borrelia burgdorferi lipoproteins and synthetic lipopeptides proceeds via a pathway distinct from that of lipopolysaccharide but involves the transcriptional activator NF-kappa B.
There is increasing evidence that lipoproteins of Treponema pallidum and Borrelia burgdorferi are key inflammatory mediators during syphilis and Lyme disease. A principal objective of the present study was to identify more precisely similarities and divergences among lipopolysaccharide (LPS)- and lipoprotein-lipopeptide-induced immune cell signaling events. Like LPS, purified native B. burgdorferi OspA and synthetic analogs of OspA, OspB, and two T. pallidum lipoproteins (Tpp47 and Tpp17) all induced NF-kappa B translocation in THP-1 human monocytoid cells. Acylation of OspA and the synthetic peptides was requisite for cell activation. Polymyxin B abrogated only the response to LPS. By using 70Z/3-derived pre-B-cell lines either lacking or expressing human CD14 (the LPS receptor), it was observed that expression of human CD14 imparted responsiveness to LPS but not to OspA or spirochetal lipopeptides (assessed by induction of NF-kappa B and expression of surface immunoglobulin M). Finally, the biological relevance of the observation that T. pallidum lipoproteins-lipopeptides induce both NF-kappa B and cytokine production in monocytes was supported by the ability of the synthetic analogs to promote human immunodeficiency virus replication in chronically infected U1 monocytoid cells; these observations also suggest a potential mechanism whereby a syphilitic chancre can serve as a cofactor for human immunodeficiency virus transmission. The combined data lend additional support to the proposal that spirochetal lipoproteins and LPS initiate monocyte activation via different cell surface events but that the signaling pathways ultimately converge to produce qualitatively similar cellular responses.
Norgard, M V; Arndt, L L; Akins, D R; Curetty, L L; Harrich, D A; Radolf, J D
Optogenetic activation of GABAergic neurons in the nucleus accumbens decreases the activity of the ventral pallidum and the expression of cocaine-context-associated memory.
GABAergic medium-sized spiny neurons (MSNs) in the nucleus accumbens (NAc) differentially express D1 and D2 dopamine receptors. Both D2- and D1-MSNs in the NAc form projections into the ventral pallidum, whereas only D1-MSNs directly project into midbrain neurons. They are critical in rewarding and aversive learning, and understanding the function of these NAc efferents and the alteration of their targeted brain regions in responding to a reward-associated context is important. In this study, we activated the GABAergic neurons in the NAc of mice expressing channelrhodopsin-2 under the control of the vesicular GABA transporter promoter by an optogenetic approach, and examined its effects on the expression of cocaine-context-associated memory. In vivo optogenetic activation of the NAc GABAergic neurons inhibited the expression of cocaine-conditioned place preference (CPP). When tested 24 h later, these mice exhibited normal cocaine-induced CPP, indicating that the inhibitory effect on the expression of CPP was transient and reversible. Activation of the NAc GABAergic neurons also attenuated the learning of cocaine-induced reinforcement, as indicated by the results of behavioural sensitization. To explore how the cocaine-context-associated information was processed and integrated, we assessed the activity of NAc MSN-targeted brain nuclei and found that the activation of NAc GABAergic neurons during CPP expression resulted in a decrease of c-Fos+ cells in the ventral palladium. Our data suggested that the NAc GABAergic efferents inhibit the ventral palladium activity and negatively regulate the expression of motivational effects induced by cocaine-context-associated cues. PMID:24456857
Wang, Li; Shen, Minjie; Yu, Yongchun; Tao, Yezheng; Zheng, Ping; Wang, Feifei; Ma, Lan
Changes in Accumbal and Pallidal pCREB and ?FosB in Morphine-Sensitized Rats: Correlations with Receptor-Evoked Electrophysiological Measures in the Ventral Pallidum
Activation of ?-opioid receptors in the ventral pallidum (VP) is important for the induction of behavioral sensitization to morphine in rats. The present study was designed to ascertain if neurons within the VP demonstrate sensitization at a time when morphine-induced behavioral sensitization occurred (ie 3 or 14 days after five once-daily injections of 10 mg/kg i.p. morphine) in rats. Western blotting was used to evaluate transcription factors altered by opiates, CREB and ?FosB. CREB levels did not change in the VP, but there was a significant decrease in levels of its active, phosphorylated form (pCREB) at both 3- and 14-days withdrawal. ?FosB levels were elevated following a 3-day withdrawal, but returned to normal by 14 days. This profile also was obtained from nucleus accumbens tissue. In a separate group of similarly treated rats, in vivo electrophysiological recordings of VP neuronal responses to microiontophoretically applied ligands were carried out after 14-days withdrawal. The firing rate effects of local applications of morphine were diminished in rats withdrawn from i.p. morphine. Repeated i.p. morphine did not alter GABA-mediated suppression of firing, or the rate enhancing effects of the D1 dopamine receptor agonist SKF82958 or glutamate. However, VP neurons from rats withdrawn from repeated i.p. morphine showed a higher propensity to enter a state of depolarization inactivation to locally applied glutamate. Overall, these findings reveal that decreased pCREB in brain regions such as the VP accompanies persistent behavioral sensitization to morphine and that this biochemical alteration may influence the excitability of neurons in this brain region.
McDaid, John; Dallimore, Jeanine E; Mackie, Alexander R; Napier, T Celeste
[Antiganglioside complex antibody].
Antiganglioside antibodies are known to play a pathogenic role in development of Guillain-Barré syndrome (GBS) and Fisher syndrome (FS). Recently, antibodies to ganglioside complexes (GSCs) consisting of two different gangliosides were found in some patients with GBS and FS. Some anti-GSC antibodies such as anti-GD1a/GD1b, anti-GM1/GQ1b or anti-GM1/GalNAc-GD1a antibodies correlate with specific clinical features. Anti-GSCs antibodies can be found also in chronic inflammatory demyelinating polyneuropathy and neuropathy associated with IgM monoclonal gammopathy. Latest studies have revealed molecular basis of anti-GSCs antibody-mediated nerve injury. The concept of GSCs will provide new vistas on understanding of pathogenesis of GBS and related autoimmune diseases. PMID:23777091
Hongo, Yu; Kaida, Kenichi
Biobarcodes: Antibodies and Nanosensors
NSDL National Science Digital Library
In this activity/demo, learners investigate biobarcodes, a nanomedical technology that allows for massively parallel testing that can assist with disease diagnosis. Learners define antibodies and learn how each antibody binds to a unique protein. Learners also discover how biobarcoding uses nanoparticles, antibodies, DNA and magnetism to detect diseases earlier than we could detect before. Learners assemble a jigsaw puzzle that models how biobarcodes work.
Network, Nanoscale I.; Industry, Oregon M.
Cholesterol-dependent complement activation has been proposed as a factor that might influence the pathogenesis of atherosclerosis. Although antibodies to cholesterol conjugates have been reported, cholesterol is widely regarded as a poorly immunogenic substance. Monoclonal IgM complement-fixing antibodies to cholesterol were obtained in the present study after immunizing mice with liposomes containing high amounts of cholesterol (71 mol % relative to phosphatidylcholine) and lipid A as an adjuvant. Clones were selected for the ability of secreted antibodies to react with liposomes containing 71% cholesterol but not with liposomes containing 43% cholesterol. The antibodies also reacted with crystalline cholesterol in a solid-phase enzyme-linked immunosorbent assay. Binding of monoclonal antibodies to the surface of crystalline cholesterol was demonstrated by electron microscopy by utilizing a second antibody (anti-IgM) labeled with colloidal gold. The immunization period required to induce monoclonal antibodies was very short (3 days) and a high fraction of the hybrid cells (at least 70%) were secreting detectable antibodies to cholesterol. The results demonstrate that cholesterol can be a highly immunogenic molecule and that complement-fixing antibodies to cholesterol can be readily obtained. Images
Swartz, G M; Gentry, M K; Amende, L M; Blanchette-Mackie, E J; Alving, C R
Serum antibodies against genitourinary infectious agents in prostate cancer and benign prostate hyperplasia patients: a case-control study
Background Infection plays a role in the pathogenesis of many human malignancies. Whether prostate cancer (PCa) - an important health issue in the aging male population in the Western world - belongs to these conditions has been a matter of research since the 1970 s. Persistent serum antibodies are a proof of present or past infection. The aim of this study was to compare serum antibodies against genitourinary infectious agents between PCa patients and controls with benign prostate hyperplasia (BPH). We hypothesized that elevated serum antibody levels or higher seroprevalence in PCa patients would suggest an association of genitourinary infection in patient history and elevated PCa risk. Methods A total of 434 males who had undergone open prostate surgery in a single institution were included in the study: 329 PCa patients and 105 controls with BPH. The subjects' serum samples were analysed by means of enzyme-linked immunosorbent assay, complement fixation test and indirect immunofluorescence for the presence of antibodies against common genitourinary infectious agents: human papillomavirus (HPV) 6, 11, 16, 18, 31 and 33, herpes simplex virus (HSV) 1 and 2, human cytomegalovirus (CMV), Chlamydia trachomatis, Mycoplasma hominis, Ureaplasma urealyticum, Neisseria gonorrhoeae and Treponema pallidum. Antibody seroprevalence and mean serum antibody levels were compared between cases and controls. Tumour grade and stage were correlated with serological findings. Results PCa patients were more likely to harbour antibodies against Ureaplasma urealyticum (odds ratio (OR) 2.06; 95% confidence interval (CI) 1.08-4.28). Men with BPH were more often seropositive for HPV 18 and Chlamydia trachomatis (OR 0.23; 95% CI 0.09-0.61 and OR 0.45; 95% CI 0.21-0.99, respectively) and had higher mean serum CMV antibody levels than PCa patients (p = 0.0004). Among PCa patients, antibodies against HPV 6 were associated with a higher Gleason score (p = 0.0305). Conclusions Antibody seropositivity against the analyzed pathogens with the exception of Ureaplasma does not seem to be a risk factor for PCa pathogenesis. The presence or higher levels of serum antibodies against the genitourinary pathogens studied were not consistently associated with PCa. Serostatus was not a predictor of disease stage in the studied population.
In vitro selection has been an essential tool in the development of recombinant antibodies against various antigen targets. Deep sequencing has recently been gaining ground as an alternative and valuable method to analyze such antibody selections. The analysis provides a novel and extremely detailed view of selected antibody populations, and allows the identification of specific antibodies using only sequencing data, potentially eliminating the need for expensive and laborious low-throughput screening methods such as enzyme-linked immunosorbant assay. The high cost and the need for bioinformatics experts and powerful computer clusters, however, have limited the general use of deep sequencing in antibody selections. Here, we describe the AbMining ToolBox, an open source software package for the straightforward analysis of antibody libraries sequenced by the three main next generation sequencing platforms (454, Ion Torrent, MiSeq). The ToolBox is able to identify heavy chain CDR3s as effectively as more computationally intense software, and can be easily adapted to analyze other portions of antibody variable genes, as well as the selection outputs of libraries based on different scaffolds. The software runs on all common operating systems (Microsoft Windows, Mac OS X, Linux), on standard personal computers, and sequence analysis of 1–2 million reads can be accomplished in 10–15 min, a fraction of the time of competing software. Use of the ToolBox will allow the average researcher to incorporate deep sequence analysis into routine selections from antibody display libraries.
D'Angelo, Sara; Glanville, Jacob; Ferrara, Fortunato; Naranjo, Leslie; Gleasner, Cheryl D; Shen, Xiaohong; Bradbury, Andrew RM; Kiss, Csaba
Antibodies are critical in protection against extracellular microbial pathogens. Although antibodies also play a role in transplant/tumor rejection and in autoimmune disease, this paper focuses on defense against bovine infections. Effector mechanisms of different bovine isotypes, subisotypes and allotypes are discussed. The importance of antigen specificity is also stressed. PMID:12072231
Corbeil, L B
Expression of Recombinant Antibodies
Recombinant antibodies are highly specific detection probes in research, diagnostics, and have emerged over the last two decades as the fastest growing class of therapeutic proteins. Antibody generation has been dramatically accelerated by in vitro selection systems, particularly phage display. An increasing variety of recombinant production systems have been developed, ranging from Gram-negative and positive bacteria, yeasts and filamentous fungi, insect cell lines, mammalian cells to transgenic plants and animals. Currently, almost all therapeutic antibodies are still produced in mammalian cell lines in order to reduce the risk of immunogenicity due to altered, non-human glycosylation patterns. However, recent developments of glycosylation-engineered yeast, insect cell lines, and transgenic plants are promising to obtain antibodies with "human-like" post-translational modifications. Furthermore, smaller antibody fragments including bispecific antibodies without any glycosylation are successfully produced in bacteria and have advanced to clinical testing. The first therapeutic antibody products from a non-mammalian source can be expected in coming next years. In this review, we focus on current antibody production systems including their usability for different applications.
Frenzel, Andre; Hust, Michael; Schirrmann, Thomas
Antibodies with infinite affinity.
Here we report an approach to the design and production of antibody/ligand pairs, to achieve functional affinity far greater than avidin/biotin. Using fundamental chemical principles, we have developed antibody/ligand pairs that retain the binding specificity of the antibody, but do not dissociate. Choosing a structurally characterized antibody/ligand pair as an example, we engineered complementary reactive groups in the antibody binding pocket and the ligand, so that they would be in close proximity in the antibody/ligand complex. Cross-reactions with other molecules in the medium are averted because of the low reactivity of these groups; however, in the antibody/ligand complex the effective local concentrations of the complementary reactive groups are very large, allowing a covalent reaction to link the two together. By eliminating the dissociation of the ligand from the antibody, we have made the affinity functionally infinite. This chemical manipulation of affinity is applicable to other biological binding pairs. PMID:11447282
Chmura, A J; Orton, M S; Meares, C F
Potential bioweapons are biological agents (bacteria, viruses and toxins) at risk of intentional dissemination. Biodefense, defined as development of therapeutics and vaccines against these agents, has seen an increase, particularly in the US, following the 2001 anthrax attack. This review focuses on recombinant antibodies and polyclonal antibodies for biodefense that have been accepted for clinical use. These antibodies aim to protect against primary potential bioweapons or category A agents as defined by the Centers for Disease Control and Prevention (Bacillus anthracis, Yersinia pestis, Francisella tularensis, botulinum neurotoxins, smallpox virus and certain others causing viral hemorrhagic fevers) and certain category B agents. Potential for prophylactic use is presented, as well as frequent use of oligoclonal antibodies or synergistic effect with other molecules. Capacities and limitations of antibodies for use in biodefense are discussed, and are generally applicable to the field of infectious diseases.
Froude, Jeffrey W; Stiles, Bradley; Pelat, Thibaut
[Humanized antibodies as therapeutics].
Since 1997, nine humanized antibodies received the approval of the FDA to be used as drugs for the treatment of various diseases including transplant rejections, metastatic breast and colon cancers, leukaemia, non-Hodgkin lymphomas, allergic conditions or multiple sclerosis. This review describes techniques used to engineer these antibodies and presents the recent evolutions of these techniques : SDRs grafting or < abbreviated > CDRs grafting. Based on the illustrative examples of several antibodies, Mylotarg, Herceptin or Xolair, the therapeutic effectiveness of humanized antibodies are underlined and, with the example of Tysabri, the sometimes dramatic adverse effects associated with their clinical use is stressed. In a second part, this review presents some future and realistic avenues to improve the effectiveness of the humanized antibodies, to decrease their immunogenicity and to reduce their cost. PMID:16324646
Bellet, Dominique; Dangles-Marie, Virginie
Red Blood Cell Antibody Identification
... Antibody ID, RBC; RBC Ab ID Formal name: Red Blood Cell Antibody Identification Related tests: Direct Antiglobulin ... None The Test Sample What is being tested? Red blood cell antibodies are proteins produced by the ...
Antibody discovery: sourcing of monoclonal antibody variable domains.
Historically, antibody variable domains for therapeutic antibodies have been sourced primarily from the mouse IgG repertoire, and typically either chimerized or humanized. More recently, human antibodies from transgenic mice producing human IgG, phage display libraries, and directly from human B lymphocytes have been used more broadly as sources of antibody variable domains for therapeutic antibodies. Of the total 36 antibodies approved by major maket regulatory agencies, the variable domain sequences of 26 originate from the mouse. Of these, four are marketed as murine antibodies (of which one is a mouse-rat hybrid IgG antibody), six are mouse-human chimeric antibodies, and 16 are humanized. Ten marketed antibodies have originated from human antibody genes, three isolated from phage libraries of human antibody genes and seven from transgenic mice producing human antibodies. Five antibodies currently in clinical trials have been sourced from camelids, as well as two from non-human primates, one from rat, and one from rabbit. Additional sources of antibody variable domains that may soon find their way into the clinic are potential antibodies from sharks and chickens. Finally, the various methods for retrieval of antibodies from humans, mouse and other sources, including various display technologies and amplification directly from B cells, are described. PMID:24168292
Strohl, William R
To study the mechanism of antigenic stimulation in the secondary immune response, primed lymphoid cells were stimulated with antigen in various ways in vitro. The effect of antigenic stimulation was assessed by the antibody titres obtained after in vivo culture of primed cells in X-irradiated recipients. Primed cells were much more effectively stimulated by antigen—antibody (Ag—Ab) complex than by free antigen. Primed cells could also be stimulated by spleen or lymph node cells from normal mice which had been exposed to free antigen or Ag—Ab complex in vitro or in vivo and thoroughly washed. Under these conditions, Ag—Ab complex was again much more effective than free antigen. When the cells were incubated with Ag—Ab complex, the dose of antigen bound to the cells was somewhat increased. But this increased binding of antigen could not solely account for the increase in immunogenicity. It is suggested that the ingestion of antigen by macrophages is facilitated by the presence of antibody and that the macrophages mediate the effective immune stimulus to memory cells. The effect of antibody in increasing the immunogenicity of antigen was lost completely when antibody was digested with pepsin. Thus, the Fc portion of antibody seemed to be important for this effect. However, it was demonstrated that antibody does not operate by becoming attached to macrophages as cytophilic antibody, and that complement is not involved in this process. The augmenting mechanism of antibody on the antigenic stimulation mediated by macrophages was discussed. ImagesFIG. 2
Hamaoka, T.; Kitagawa, M.
Eight antigenically unique immunoglobulins have been identified in purified equine anti-p-azophenyl-?-lactoside (Lac) antibody isolated from a single horse. The Fc fragments of the ?Ga-, ?Gb-, ?Gc-, and -?A-globulins have been shown to possess unique antigenic determinants. Common ?G- and ?A-Fc fragment antigenic determinants, which were absent from the 10S?1- and ?M-globulins, have also been observed. All antibody populations share two antigenically distinct light (B, L) chain variants. The association of anti-Lac antibody with the hapten p-(p-dimethylamino-benzeneazo)-phenyl-?-lactoside has been measured by equilibrium dialysis and by fluorescence quenching. A variation in the affinity of anti-Lac antibody for hapten has been observed. The affinity of antibody was unaltered by enzymatic removal of the Fc fragments by peptic digestion or dissociation of the two combining sites on the papain 3.5S Fab fragments, indicating that the observed heterogeneity of affinities was not a direct function of the heterogeneity in structure of the Fc fragments. Isolated heavy (A, H) chains of ?A-anti-Lac antibody have been shown to have retamed affinity for Lac dye by equilibrium dialysis and by analytical ultracentrifugation, employing a combination of schlieren and absorption optics. The heavy (A, H) chains from two physically separable, antigenically distinct antibody populations, isolated from the same animal and having affinity for the same haptenic determinant, have been found to differ in their amino acid composition. Anti-Lac antibody light (B, L) chains have also been shown to be chemically heterogeneous, and contained populations of polypeptide chains possessing, and populations lacking methionine. The relevance of the observed structural heterogeneity of equine anti-Lac antibody to the problem of defining the mechanism of acquisition of immunological specificity is briefly discussed.
Rockey, John H.
Monoclonal antibody "gold rush".
The market, sales and regulatory approval of new human medicines, during the past few years, indicates increasing number and share of new biologics and emergence of new multibillion dollar molecules. The global sale of monoclonal antibodies in 2006 were $20.6 billion. Remicade had annual sales gain of $1 billion during the past 3 years and five brands had similar increase in 2006. Rituxan with 2006 sales of $4.7 billion was the best selling monoclonal antibody and biological product and the 6th among the top selling medicinal brand. It may be the first biologic and monoclonal antibody to reach $10 billion annual sales in the near future. The strong demand from cancer and arthritis patients has surpassed almost all commercial market research reports and sales forecast. Seven monoclonal antibody brands in 2006 had sales exceeding $1 billion. Humanized or fully human monoclonal antibodies with low immunogenicity, enhanced antigen binding and reduced cellular toxicity provide better clinical efficacy. The higher technical and clinical success rate, overcoming of technical hurdles in large scale manufacturing, low cost of market entry and IND filing, use of fully human and humanized monoclonal antibodies has attracted funds and resources towards R&D. Review of industry research pipeline and sales data during the past 3 years indicate a real paradigm shift in industrial R&D from pharmaceutical to biologics and monoclonal antibodies. The antibody bandwagon has been joined by 200 companies with hundreds of new projects and targets and has attracted billions of dollars in R&D investment, acquisitions and licensing deals leading to the current Monoclonal Antibody Gold Rush. PMID:17691940
Maggon, Krishan
Designing antibodies for oncology.
The past five years have witnessed the emergence of monoclonal antibodies as important therapeutics for cancer treatment. Lower toxicity for antibodies versus small molecules, the potential for increased efficacy by conjugation to radioisotopes and cellular toxins, or the ability to exploit immune cell functions have led to clinical performances on par or superior to conventional drug therapies. This review outlines the various immunoglobulin design strategies currently available, techniques used to reduce Ig antigenicity and toxicity, and points to consider during the manufacture of antibodies for use in clinical oncology. PMID:16408163
Tanner, Jerome E
Lyme disease serology; ELISA for Lyme disease; Western blot for Lyme disease ... see: Venipuncture . A laboratory specialist will look for Lyme disease antibodies in the blood sample using the ELISA ...
Future of antibody purification.
Antibody purification seems to be safely ensconced in a platform, now well-established by way of multiple commercialized antibody processes. However, natural evolution compels us to peer into the future. This is driven not only by a large, projected increase in the number of antibody therapies, but also by dramatic improvements in upstream productivity, and process economics. Although disruptive technologies have yet escaped downstream processes, evolution of the so-called platform is already evident in antibody processes in late-stage development. Here we perform a wide survey of technologies that are competing to be part of that platform, and provide our [inherently dangerous] assessment of those that have the most promise. PMID:17134947
Low, Duncan; O'Leary, Rhona; Pujar, Narahari S
Designing antibodies for oncology
Summary The past five years have witnessed the emergence of monoclonal antibodies as important therapeutics for cancer treatment.\\u000a Lower toxicity for antibodies versus small molecules, the potential for increased efficacy by conjugation to radioisotopes\\u000a and cellular toxins, or the ability to exploit immune cell functions have led to clinical performances on par or superior\\u000a to conventional drug therapies. This review outlines
Jerome E. Tanner
Intracellular antibody immunity.
Antibodies allow the immune system to target pathogens despite their tremendous diversity and rapid evolution. Once bound to a pathogen, antibodies induce a broad range of effector mechanisms, including phagocytosis and complement. However, these mechanisms are all initiated in the extracellular space, meaning that pathogens like viruses evade them upon infection of their target cells. Recently, it has been shown that, in addition to mediating extracellular immune responses, antibodies also activate immunity inside infected cells. Antibodies that are bound to the surface of non-enveloped viruses or bacteria are carried into the cell during pathogen entry. Once inside the cell, these pathogen-attached antibodies are recognised by a highly conserved, high affinity cytosolic antibody receptor called TRIM21. TRIM21 initiates both sensor and effector responses that reduce viral replication and induce an antiviral state. These responses are an important part of antiviral immunity and the removal of TRIM21 results in uncontrolled viraemia and death in a mouse model of infection. PMID:24722852
Watkinson, Ruth E; McEwan, William A; James, Leo C
Human antibodies to blood group A substance were purified by absorption on columns of insoluble polyleucyl hog blood group A + H substance and eluted first with N-acetylgalactosamine and then with an A active reduced pentasaccharide ARL0.52. The ?M and ?G antibodies in these eluates were separated by density gradient centrifugation. The antibodies were studied for their relative capacities to be inhibited by various blood group A active oligosaccharides. Antibodies eluted by the N-acetylgalactosamine could be inhibited by N-acetylgalactosamine, as well as by lower concentrations of A active tri- and pentasaccharides, while those eluted by the pentasaccharide ARL0.52 could only be inhibited by the two oligosaccharides, but not by N-acetylgalactosamine, indicating that the N-acetylgalactosamine eluate had more antibodies with smaller size combining sites than the ARL0.52 eluate. Measurements by equilibrium dialysis gave values ranging from 2 x 103 to 1 x 105 M–1 and the values obtained with the ARL0.52 eluate were somewhat higher than those with the GalNAc eluate. Only one of three anti-A sera had ?M anti-A in the ARL0.52 eluate, while all three had ?M in the N-acetylgalactosamine eluate. Data on the precipitating, hemagglutinating, complement fixing, hemolytic properties of the eluted antibodies, and of their content of ? and ? light chains are given.
Moreno, Carlos; Kabat, Elvin A.
Monoclonal antibodies and cancer therapy
These proceedings collect papers on the subject of monoclonal antibodies. Topics include: Monoclonal antibody, biochemical effects and cancer therapeutic potential of tunicamycin, use of monoclonal antibodies for detection of lymph node metastases, active specific immunotherapy, and applications of monoclonal antibodies to investigations of growth factors.
Reisfeld, R.A.; Sell, S.
Phage Display Derived Therapeutic Antibodies
This article gives an overview about the development of human therapeutic antibodies generated by phage dis- play. After an introduction to the method, the focus is on approved antibodies and those currently in clinical trials, 14 of which are described in detail. 1. ANTIBODY PHAGE DISPLAY In the mid 1980s, the development of recombinant tech- nologies for antibody chimerization and
Holger Thie; Torsten Meyer; Thomas Schirrmann; Michael Hust; Stefan Dubel
US Patent & Trademark Office Database
Problem to Be Solved It is intended to provide an antibody having an inhibitory activity against cell malignant transformation and/or tumor cell growth, etc. Solution The present invention provides an antibody which recognizes an epitope recognized by an antibody produced by a hybridoma SH348-1 (FERM BP-10836) or a hybridoma SH357-1 (FERM BP-10837), an antibody produced by the hybridoma SH348-1 or the hybridoma SH357-1, an antibody obtained by humanizing the antibody produced by the hybridoma SH348-1 or the hybridoma SH357-1, a pharmaceutical agent comprising the antibody as an active ingredient, etc.
Neurokinin-1 (NK1) receptors in cholinergic neurons of the rat ventral pallidum have a predominantly dendritic distribution that is affected by apomorphine when combined with startle-evoking auditory stimulation
Cholinergic neurons of the basal forebrain are implicated in startle reflex inhibition by a prior weak stimulus often referred to as prepulse inhibition (PPI) and used as an index of sensorimotor gating deficits in schizophrenia. Gating deficits can be produced in rodent models by acute systemic administration of apomorphine, a non-selective dopamine D1 and D2 receptor agonist that also affects trafficking of neurokinin-1 (NK1) receptors induced by startle evoking auditory stimulation (AS) in midbrain neurons. We used electron microscopic immunolabeling of NK1 receptors and the vesicular acetylcholine transporter (VAchT) to test the hypothesis that the subcellular distributions of these receptors in cholinergic neurons of the rat ventral pallidum are subject to a similar regulation. In vehicle controls, NK1 immunogold was often seen near cytoplasmic endomembranes in somata and large dendrites, but was more equally distributed in cytoplasmic and plasmalemmal compartments of medium dendrites, and principally located on the plasma membrane of small dendrites. These labeling patterns appeared to be largely independent of whether the NK1 receptor was co-expressed with VAchT, however only the medium and small VAchT-labeled dendrites showed significant treatment-specific differences in NK1 immunogold distributions. The NK1 receptor immunogold particle density on the plasma membrane of medium cholinergic dendrites was significantly enhanced by combined apomorphine and AS, while neither alone affected either the plasmalemmal density or the equality of the plasmalemmal and cytoplasmic distributions of NK1 receptors in these dendrites. Small cholinergic dendrites showed a significant AS-induced increase in both the plasmalemmal and cytoplasmic density of NK1 gold particles, and an apomorphine-induced disruption of the preferential plasmalemmal targeting of the NK1 receptors. These results provide ultrastructural evidence that NK1 receptors in cholinergic neurons of the ventral pallidum have subcellular locations and plasticity conducive to active involvement in dopamine-dependent sensorimotor processing.
Mengual, Elisa; Chan, June; Lane, Diane; Palenzuela, Marta San Luciano; Hara, Yuko; Lessard, Andree; Pickel, Virginia M.
Search for the Prevalence of Infection with the Human Immunodeficiency Virus, the Hepatitis B Virus and Treponema Pallidum among Different Groups and Areas of the Philippines.
National Technical Information Service (NTIS)
Information on what is now known as Acquired Immunodeficiency Syndrome has been circulated in the Philippines as early as 1983. It was only in 1985, however, that the first kit for AIDS antibody detection was marketed. Then people kept asking, 'Do we have...
V. Basaca-Sevilla C. G. Hayes G. Espinosa A. B. Andrada P. Mejia
[Production of antibodies to aflatoxins].
A panel of ten monoclonal antibodies to aflatoxins B1, B2, and G2 was produced and comprehensively characterized. The affinity and cross reactivity of these antibodies were determined using the methods of direct, indirect, and competitive ELISA. The structures of monoclonal antibody genes were comprehensively studied and the variable and constant domains of the antibody genes were cloned and sequenced. Sequencing analysis confirmed the results of isotyping the light and heavy antibody chains obtained by ELISA. Variable and constant fragments of the antibody genes were cloned into a bicistron expression vector for the recombinant Fab' fragment for one of the antibodies expressed in Escherichia coli and purified. Thus, data were obtained that can be useful for the development of an aflatoxin detection system on the basis of the described monoclonal antibodies and the creation of recombinant antibodies with changed parameters of specificity using protein engineering methods. PMID:20386586
Kalinichenko, A A; Toporova, V A; Panina, A A; Aliev, T K; Kriukova, E A; Shemchukova, O B; Solopova, O N; Pozdniakova, L P; Sveshnikov, P G; Dolgikh, D A; Kirpichnikov, M P
NSDL National Science Digital Library
An antibody molecule. (A) Schematic drawing of a typical antibody molecule. As indicated, this protein is Y-shaped and has two identical binding sites for its antigen, one on either arm of the 3Y.2 The protein is composed of four polypeptide chains (two identical heavy chains and two identical and smaller light chains) held together by disulfide bonds. Each chain is made up of several different domains, here shaded either blue or gray. The antigen-binding site is formed where a heavy chain variable domain (VH) and a light chain variable domain (VL) come close together. These are the domains that differ most in their sequence and structure in different antibodies. (B) Ribbon drawing of a light chain showing the parts of the VL domain most closely involved in binding to the antigen in red; these contribute half of the fingerlike loops that fold around each of the antigen molecules in (A).
BEGIN:VCARD VERSION:2.1 FN:Martin Raff N:Raff;Martin REV:2005-04-15 END:VCARD; BEGIN:VCARD VERSION:2.1 FN:Julian Lewis N:Lewis;Julian REV:2005-04-15 END:VCARD; BEGIN:VCARD VERSION:2.1 FN:Alexander Johnson N:Johnson;Alexander REV:2005-04-15 END:VCARD; BEGIN:VCARD VERSION:2.1 FN:Dennis Bray N:Bray;Dennis REV:2005-04-15 END:VCARD; BEGIN:VCARD VERSION:2.1 FN:Bruce Alberts N:Alberts;Bruce REV:2005-04-15 END:VCARD; BEGIN:VCARD VERSION:2.1 FN:Keith Roberts N:Roberts;Keith REV:2005-04-15 END:VCARD
Radiolabeled antibody fragments
This patent describes an antibody conjugate of the formula: wherein Ab is the residue of a Fab fragment of a monoclonal antibody. The antibody being one which retains antigen-binding activity following enzymatic removal for the F/sub c/ fragment and reductive cleavage of the disulfide bond joining the heavy chains; -S- is the residue of the free sulfhydryl group of the Fab' fragment; R is a divalent organic linker; and R' is a nontoxic, weakly acidic chelating group capable of forming a chelate with a radionuclide metal ion that is thermodynamically stable under physiological conditions and has a higher stability constant than a chelate of the radionuclide with transferrin in vivo.
Nicolotti, R.A.
Glycoproteomic Analysis of Antibodies*
Antibody glycosylation has been shown to change with various processes. This review presents mass spectrometric approaches for antibody glycosylation analysis at the level of released glycans, glycopeptides, and intact protein. With regard to IgG fragment crystallizable glycosylation, mass spectrometry has shown its potential for subclass-specific, high-throughput analysis. In contrast, because of the vast heterogeneity of peptide moieties, fragment antigen binding glycosylation analysis of polyclonal IgG relies entirely on glycan release. Next to IgG, IgA has gained some attention, and studies of its O- and N-glycosylation have revealed disease-associated glycosylation changes. Glycoproteomic analyses of IgM and IgE are lagging behind but should complete our picture of glycosylation's influence on antibody function.
Zauner, Gerhild; Selman, Maurice H. J.; Bondt, Albert; Rombouts, Yoann; Blank, Dennis; Deelder, Andre M.; Wuhrer, Manfred
During the past decade, new insights into the mechanisms by which T-cell activation and proliferation are regulated have led to the identification of checkpoint proteins that either up- or down-modulate T-cell reactivity. In the presence of active malignancy, pathophysiologic inhibition of T-cell activity may predominate over stimulation. A number of antibodies have been generated that can block inhibitory checkpoint proteins or promote the activity of activating molecules. In murine models, their use alone or with a vaccine strategy has resulted in regression of poorly immunogenic tumors and cures of established tumors. The prototypical immune regulatory antibodies are those directed against cytotoxic T-lymphocyte antigen-4, a molecule present on activated T cells. In this review, the preclinical rationale and clinical experience with 2 anticytotoxic T-lymphocyte antigen-4 antibodies are extensively discussed, demonstrating that abrogation of an immune inhibitory molecule can result in significant regression of tumors and long-lasting responses. The unique kinetics of antitumor response and the characteristic immune-related side effects of ipilimumab are also discussed. This clinical efficacy of this promising antitumor agent has been evaluated in 2 randomized phase III trials, whose results are eagerly awaited. Programmed death (PD)-1 is another immune inhibitory molecule against which an abrogating human antibody has been prepared. Initial preclinical testing with anti–PD-1 and anti–PD-L1 has shown encouraging results. Stimulatory molecules such as CD40, 41-BB, and OX-40 are also targets for antibody binding and activation, not blockade, and early dose ranging trials with antibodies against all 3 have shown that they can mediate regression of tumors, albeit with their own spectrum of side effects that are different from those that occur with abrogation of immune inhibition.
Wolchok, Jedd D.; Yang, Arvin S.; Weber, Jeffrey S.
NSDL National Science Digital Library
The experimental protocol in this Web site is just one of many microbiology resources provided by the University of Leicester. The procedure guides students in finding the antibody concentration of a test antiserum and the number of antibody binding sites on an antigen molecule. A results graph and correct answers to the required calculations are given, providing the option of performing a virtual experiment in lieu of an actual one. This activity is probably most appropriate for high school and undergraduate level biology labs.
Cann, Alan.
Therapeutic antibodies against cancer.
Antibody-based therapeutics against cancer are highly successful and currently enjoy unprecedented recognition of their potential; 13 monoclonal antibodies (mAbs) have been approved for clinical use in the European Union and in the United States. Bevacizumab, rituximab, and trastuzumab had sales in 2010 of more than $5 billion each. Hundreds of mAbs, including bispecific mAbs and multispecific fusion proteins, mAbs conjugated with small-molecule drugs, and mAbs with optimized pharmacokinetics, are in clinical trials. However, deeper understanding of mechanisms is needed to overcome major problems including resistance to therapy, access to targets, complexity of biological systems, and individual variations. PMID:22520975
Adler, Mark J; Dimitrov, Dimiter S
Selection of recombinant antibodies from antibody gene libraries.
Antibodies are indispensable detection reagents for research and diagnostics and represent the biggest class of biological therapeutics on the market. In vitro antibody selection systems offer many advantages over animal-based technologies because the whole selection process is independent of the in vivo immune response. In the last two decades antibody phage display has evolved to the most robust and widely used method and has already yielded thousands of antibodies. The selection of binders by phage display is also referred to as "panning" and based on the specific molecular interaction of antibody phage with an immobilized antigen thus allowing the enrichment and isolation of antigen-specific monoclonal binders from very large antibody gene libraries. Here, we give detailed protocols for the selection of recombinant antibody fragments from antibody gene libraries in microtiter plates. PMID:24233787
Hust, Michael; Frenzel, André; Schirrmann, Thomas; Dübel, Stefan
Sandwich antibody arrays using recombinant antibody-binding protein L.
Antibody arrays are a useful for detecting antigens and other antibodies. This technique typically requires a uniform and well-defined orientation of antibodies attached to a surface for optimal performance. A uniform orientation can be achieved by modification of antibodies to include a single site for attachment. Thus, uniformly oriented antibody arrays require a bioengineered modification for the antibodies directly immobilization on the solid surface. In this study, we describe a "sandwich-type" antibody array where unmodified antibodies are oriented through binding with regioselectively immobilized recombinant antibody-binding protein L. Recombinant proL-CVIA bearing C-terminal CVIA motif is post-translationally modified with an alkyne group by protein farnesyltransferase (PFTase) at the cysteine residue in the CVIA sequence to give proL-CVIApf, which is covalently attached to an azido-modified glass slide by a Huisgen [3 + 2] cycloaddition reaction. Slides bearing antibodies bound to slides coated with regioselectively immobilized proL-CVIApf gave stronger fluorescence outputs and those where the antibody-binding protein was immobilized in random orientations on an epoxy-modified slide. Properly selected capture and detection antibodies did not cross-react with immobilized proL-CVIApf in sandwich arrays, and the proL-CVIApf slides can be used for multiple cycles of detected over a period of several months. PMID:24841983
Seo, Jin-Soo; Poulter, C Dale
Double Antibody ELISA for HEV Antigen and Antibody.
National Technical Information Service (NTIS)
The objects of the invention are: to provide a more sensitive and specific assay for HEV antibody and antigen than the previously described single-antibody ELISA; to provide a facile and effective means for determining titers of maternal antibody to HEV i...
K. Nazerian L. F. Lee
Monoclonal antibodies in haematopathology
This book contains over 40 selections. Some of the titles are: Oncogene (c-myc, c-myb) amplification in acute myelogenous leukaemia; Ultrastructural characterization of leukaemic cells with monoloclonal antibodies; Origin of B-cell malignancies; Immunohistology of gut lymphomas; and Spurious evidence of lineage infidelity in monocytic leukaemia.
Grignani, F.; Martelli, M.F.; Mason, D.Y.
Summary Unlike humans or mice, some species have limited genome encoded combinatorial diversity potential, yet mount a robust antibody response. Cows are unusual in having exceptionally long CDR H3 loops and few V-regions, but the mechanism for creating diversity is not understood. Deep sequencing revealed that ultralong CDR H3s contain a remarkable complexity of cysteines, suggesting that disulfide-bonded mini-domains may arise during repertoire development. Indeed, crystal structures of two cow antibodies reveal that these CDR H3s form a very unusual architecture composed of a ?-strand "stalk" that supports a structurally diverse, disulfide-bonded, "knob" domain. Sequence analysis suggests that diversity arises from somatic hypermutation of an ultralong DH with a severe codon bias towards mutation to cysteine. These unusual antibodies can be elicited to recognize defined antigens through the knob domain. Thus, the bovine immune system produces an antibody repertoire composed of CDR H3s of unprecedented length that fold into a diversity of mini-domains generated through combinations of somatically generated disulfides.
Wang, Feng; Ekiert, Damian C.; Ahmad, Insha; Yu, Wenli; Zhang, Yong; Bazirgan, Omar; Torkamani, Ali; Raudsepp, Terje; Mwangi, Waithaka; Criscitiello, Michael F.; Wilson, Ian A.; Schultz, Peter G.; Smider, Vaughn V.
Therapeutic antibody engineering
It is an important event in any knowledge area when an authority in the field decides that it is time to share all accumulated knowledge and learnings by writing a text book. This does not occur often in the biopharmaceutical industry, likely due to both the highly dynamic environment with tight timelines and policies and procedures at many pharmaceutical companies that hamper knowledge sharing. To take on a task like this successfully, a strong drive combined with a desire and talent to teach, but also an accommodating and stimulating environment is required. Luckily for those interested in therapeutic monoclonal antibodies, Dr. William R. Strohl decided about two years ago that the time was right to write a book about the past, present and future of these fascinating molecules. Dr. Strohl's great expertise and passion for biotechnology is evident from his life story and his strong academic and industry track record. Dr. Strohl pioneered natural product biotechnology, first in academia as a full professor of microbiology and biochemistry at Ohio State University in Columbus, Ohio and later in industry while at Merck. Despite his notable advances in recombinant natural products, industry interest in this area waned and in 2001 Dr. Strohl sought new opportunities by entering the field of antibody therapeutics. He initiated antibody discovery through phage display at Merck, and then moved to Centocor Research and Development Inc. (now Janssen Biotech, Inc.) in 2008 to head Biologics Research, where he now directs the discovery of innovative therapeutic antibody candidates.
Parren, Paul W.H.I.; Lugovskoy, Alexey A.
Abstract For most expression studies focusing on the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein, sensitive and specific antibodies (Abs) are critically needed. Several Abs have been produced commercially,or by research laboratories for CFTR detection in both cell lines with heterologous or endogenous expression and native cells\\/tissues. Here, we review the applicability of most Abs currently in use in CF
Filipa Mendes; Carlos M. Farinha; M Onica Roxo-rosa; Pascale Fanen; Aleksander Edelman; Robert Dormer; Margaret Mcpherson; Heather Davidson; Edith Puchelle; Hugo De Jonge; Ghanshyam D. Heda; Martina Gentzsch; Gergely L. Lukacs; Deborah Penque; Margarida D. Amaral
For most expression studies focusing on the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein, sensitive and specific antibodies (Abs) are critically needed. Several Abs have been produced commercially or by research laboratories for CFTR detection in both cell lines with heterologous or endogenous expression and native cells\\/tissues. Here, we review the applicability of most Abs currently in use in CF
Filipa Mendesa; Carlos M. Farinhaa; Pascale Fanenc; Robert Dormere; Ghanshyam D. Hedai; Gergely L. Lukacsk; Margarida D. Amarala; Ricardo Jorge; S. C. Johnson
For most expression studies focusing on the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein, sensitive and specific antibodies (Abs) are critically needed. Several Abs have been produced commercially or by research laboratories for CFTR detection in both cell lines with heterologous or endogenous expression and native cells\\/tissues. Here, we review the applicability of most Abs currently in use in CF
Filipa Mendes; Carlos M. Farinha; Mónica Roxo-Rosa; Pascale Fanen; Aleksander Edelman; Robert Dormer; Margaret McPherson; Heather Davidson; Edith Puchelle; Hugo De Jonge; Ghanshyam D. Heda; Martina Gentzsch; Gergely Lukacs; Deborah Penque; Margarida D. Amaral
Humanized Antibodies for Antiviral Therapy
NASA Astrophysics Data System (ADS)
Antibody therapy holds great promise for the treatment of cancer, autoimmune disorders, and viral infections. Murine monoclonal antibodies are relatively easy to produce but are severely restricted for therapeutic use by their immunogenicity in humans. Production of human monoclonal antibodies has been problematic. Humanized antibodies can be generated by introducing the six hypervariable regions from the heavy and light chains of a murine antibody into a human framework sequence and combining it with human constant regions. We humanized, with the aid of computer modeling, two murine monoclonal antibodies against herpes simplex virus gB and gD glycoproteins. The binding, virus neutralization, and cell protection results all indicate that both humanized antibodies have retained the binding activities and the biological properties of the murine monoclonal antibodies.
Co, Man Sung; Deschamps, Marguerite; Whitley, Richard J.; Queen, Cary
ERIC Educational Resources Information Center
Provides background information for teachers on the nature and production of antibodies. Points out that the production of monoclonal antibodies blends the malignant with the beneficial to create a medical tool of exciting potential. (JN)
Headon, Denis R.
Trifunctional antibody ertumaxomab
Background: The trifunctional antibody ertumaxomab bivalently targets the human epidermal growth factor receptor 2 (Her2) on epithelial (tumor) cells and the T cell specific CD3 antigen, and its Fc region is selectively recognized by Fc? type I/III receptor-positive immune cells. As a trifunctional immunoglobulin, ertumaxomab therefore not only targets Her2 on cancer cells, but also triggers immunological effector mechanisms mediated by T and accessory cells (e.g., macrophages, dendritic cells, natural killer cells). Whether molecular effects, however, might contribute to the cellular antitumor efficiency of ertumaxomab are largely unknown. Methods: Potential molecular effects of ertumaxomab on Her2-overexpressing BT474 and SK-BR-3 breast cancer cells were evaluated. The dissociation constant Kd of ertumaxomab was calculated from titration curves that were recorded by flow cytometry. Treatment-induced changes in Her2 homodimerization were determined by flow cytometric fluorescence resonance energy transfer measurements on a cell-by-cell basis. Potential activation / deactivation of Her2, ERK1/2, AKT and STAT3 were analyzed by western blotting, Immunochemistry and immunofluorescent cell staining. Results: The Kd of ertumaxomab for Her2-binding was determined at 265 nM and the ertumaxomab binding epitope was found to not overlap with that of the therapeutic anti-Her2 monoclonal antibodies trastuzumab and pertuzumab. Ertumaxomab caused an increase in Her2 phosphorylation at higher antibody concentrations, but changed neither the rate of Her2-homodimerization /-phosphorylation nor the activation state of key downstream signaling proteins analyzed. Conclusions: The unique mode of action of ertumaxomab, which relies more on activation of immune-mediated mechanisms against tumor cells compared with currently available therapeutic antibodies for breast cancer treatment, suggests that modular or sequential treatment with the trifunctional bivalent antibody might complement the therapeutic activity of other anti-Her2/anti-ErbB receptor reagents.
Diermeier-Daucher, Simone; Ortmann, Olaf; Buchholz, Stefan; Brockhoff, Gero
Calculation of Monoclonal Antibody Affinity Constants Directly from Antibody Dilution Curves.
National Technical Information Service (NTIS)
A method for estimating the affinity constants of monoclonal antibody directly from antibody dilution curves is described. Antibody affinity is a major determinant of the interaction of antigen with antibody. Knowledge of antibody affinity, as well as spe...
W. R. Griswold D. P. Nelson
Monoclonal Antibody Pharmacokinetics and Pharmacodynamics
More than 20 monoclonal antibodies have been approved as therapeutic drugs by the US Food and Drug Administration, and it is quite likely that the number of approved antibodies will double in the next 7–10 years. Antibody drugs show several desirable characteristics, including good solubility and stability, long persistence in the body, high selectivity and specificity, and low risk for
W Wang; EQ Wang; JP Balthasar
Antibody-gold cluster conjugates
Antibody- or antibody fragment-gold cluster conjugates are shown wherein the conjugate size can be about 5.0 nm. Methods and reagents are disclosed in which antibodies or Fab' fragments thereof are covalently bound to a stable cluster of gold atoms. 2 figs.
Hainfeld, J.F.
From whole monoclonal antibodies to single domain antibodies: think small.
The development of therapeutic monoclonal antibodies over the last 35 years has led to the emergence of a new class of useful therapeutic molecules. These "first generation" antibodies have been obtained thanks to the conjugated and huge efforts of both academic and biotech researchers. About 30 monoclonal antibodies are currently approved for therapeutic use in Europe, USA, and China. Strikingly, only a restricted number of these antibodies are immunoglobulin fragments, single variable domains, or multiunit formats based on the engineering of immunoglobulin variable domains. In the present chapter, we will review the major steps of the therapeutic antibodies history and we will highlight the enormous potential of antibody fragments, either used as multiunits such as bispecific antibodies, single units, or as cell modifiers such as intrabodies or cell surface-expressed molecules. PMID:22886242
Teillaud, Jean-Luc
Antibody Engineering and Therapeutics Conference
The Antibody Engineering and Therapeutics conference, which serves as the annual meeting of The Antibody Society, will be held in Huntington Beach, CA from Sunday December 8 through Thursday December 12, 2013. The scientific program will cover the full spectrum of challenges in antibody research and development, and provide updates on recent progress in areas from basic science through approval of antibody therapeutics. Keynote presentations will be given by Leroy Hood (Institute of System Biology), who will discuss a systems approach for studying disease that is enabled by emerging technology; Douglas Lauffenburger (Massachusetts Institute of Technology), who will discuss systems analysis of cell communication network dynamics for therapeutic biologics design; David Baker (University of Washington), who will describe computer-based design of smart protein therapeutics; and William Schief (The Scripps Research Institute), who will discuss epitope-focused immunogen design. In this preview of the conference, the workshop and session chairs share their thoughts on what conference participants may learn in sessions on: (1) three-dimensional structure antibody modeling; (2) identifying clonal lineages from next-generation data sets of expressed VH gene sequences; (3) antibodies in cardiometabolic medicine; (4) the effects of antibody gene variation and usage on the antibody response; (5) directed evolution; (6) antibody pharmacokinetics, distribution and off-target toxicity; (7) use of knowledge-based design to guide development of complementarity-determining regions and epitopes to engineer or elicit the desired antibody; (8) optimizing antibody formats for immunotherapy; (9) antibodies in a complex environment; (10) polyclonal, oligoclonal and bispecific antibodies; (11) antibodies to watch in 2014; and (12) polyreactive antibodies and polyspecificity.
Almagro, Juan Carlos; Gilliland, Gary L; Scott, Jamie; Larrick, James W; Pluckthun, Andreas; Veldman, Trudi; Adams, Gregory P; Parren, Paul WHI; Chester, Kerry A; Bradbury, Andrew; Reichert, Janice M; Huston, James S
Monoclonal antibodies in oncology.
Molecular biology has made tremendous strides over the last five years. The new biology allows us to prepare monoclonal antibodies to defined antigens; to detect, isolate and clone specific genes; and to insert these genes into defined sites in different cells giving new functions to old organisms. These revolutionary developments have been followed closely by researchers, businessmen, politicians and philosophers, as well as by those involved in the clinical care of patients. Although our understanding of human molecular biology is increasing rapidly, it is the development of monoclonal antibodies that has the most immediate application in the clinic. There have been several reports of their use in the diagnosis, localisation and treatment of human malignant disease. This review describes developments that are likely to have direct relevance to patient care in the near future.
Sikora, K
Therapeutic antibodies against cancer
Antibody-based therapeutics against cancer are highly successful in clinic and currently enjoy unprecedented recognition of their potential; 13 monoclonal antibodies (mAbs) have been approved for clinical use in the European Union and in the United States (one, mylotarg, was withdrawn from market in 2010). Three of the mAbs (bevacizumab, rituximab, trastuzumab) are in the top six selling protein therapeutics with sales in 2010 of more than $5 bln each. Hundreds of mAbs including bispecific mAbs and multispecific fusion proteins, mAbs conjugated with small molecule drugs and mAbs with optimized pharmacokinetics are in clinical trials. However, challenges remain and it appears that deeper understanding of mechanisms is needed to overcome major problems including resistance to therapy, access to targets, complexity of biological systems and individual variations.
Adler, Mark J.; Dimitrov, Dimiter S.
Antibody Therapy for Histoplasmosis
The endemic human pathogenic fungus Histoplasma capsulatum is a major fungal pathogen with a broad variety of clinical presentations, ranging from mild, focal pulmonary disease to life-threatening systemic infections. Although azoles, such as itraconazole and voriconazole, and amphotericin B have significant activity against H. capsulatum, about 1 in 10 patients hospitalized due to histoplasmosis die. Hence, new approaches for managing disease are being sought. Over the past 10?years, studies have demonstrated that monoclonal antibodies (mAbs) can modify the pathogenesis of histoplasmosis. Disease has been shown to be impacted by mAbs targeting either fungal cell surface proteins or host co-stimulatory molecules. This review will detail our current knowledge regarding the impact of antibody therapy on histoplasmosis.
Nosanchuk, Joshua D.; Zancope-Oliveira, Rosely M.; Hamilton, Andrew J.; Guimaraes, Allan J.
Chemically programmed antibodies.
Due to their unlimited chemical diversity, small molecules can rival monoclonal antibodies (mAbs) with respect to specificity and affinity for target molecules. However, key pharmacological properties of mAbs remain unmatched by small molecules. Chemical programming strategies have been developed for site-specific and covalent conjugation of small molecules to mAbs with unique reactivity centers. In addition to blending favorable features of small molecules and mAbs, chemically programmed antibodies (cpAbs) are economically attractive because they utilize the same mAb for an almost unlimited number of target molecule specificities, reducing manufacturing costs and shortening drug discovery and development time. Preclinical studies and clinical trials have begun to demonstrate the broad utility of cpAbs for the treatment and prevention of human diseases. PMID:24630478
Rader, Christoph
Engineering antibodies by yeast display.
Since its first application to antibody engineering 15 years ago, yeast display technology has been developed into a highly potent tool for both affinity maturing lead molecules and isolating novel antibodies and antibody-like species. Robust approaches to the creation of diversity, construction of yeast libraries, and library screening or selection have been elaborated, improving the quality of engineered molecules and certainty of success in an antibody engineering campaign and positioning yeast display as one of the premier antibody engineering technologies currently in use. Here, we summarize the history of antibody engineering by yeast surface display, approaches used in its application, and a number of examples highlighting the utility of this method for antibody engineering. PMID:22450168
Boder, Eric T; Raeeszadeh-Sarmazdeh, Maryam; Price, J Vincent
Infertility and Antiphospholipid Antibodies
Whether antiphospholipid antibodies (aPL) play a pathogenic role in infertility is highly controversial. aPL have been suggested to represent one potential etiology of infertility, specifically in patients with unexplained implantation failure following in vitro fertilization (IVF). The rationale is appealing, as it represents a logical extension of the demonstrated pathogenicity of aPL in contributing to recurrent spontaneous abortion, where mechanisms
Lisa R. Sammaritano
Antibody therapy for pediatric leukemia.
Despite increasing cure rates for pediatric leukemia, relapsed disease still carries a poor prognosis with significant morbidity and mortality. Novel targeted therapies are currently being investigated in an attempt to reduce adverse events and improve survival outcomes. Antibody therapies represent a form of targeted therapy that offers a new treatment paradigm. Monoclonal antibodies are active in pediatric acute lymphoblastic leukemia (ALL) and are currently in Phase III trials. Antibody-drug conjugates (ADCs) are the next generation of antibodies where a highly potent cytotoxic agent is bound to an antibody by a linker, resulting in selective targeting of leukemia cells. ADCs are currently being tested in clinical trials for pediatric acute myeloid leukemia and ALL. Bispecific T cell engager (BiTE) antibodies are a construct whereby each antibody contains two binding sites, with one designed to engage the patient's own immune system and the other to target malignant cells. BiTE antibodies show great promise as a novel and effective therapy for childhood leukemia. This review will outline recent developments in targeted agents for pediatric leukemia including monoclonal antibodies, ADCs, and BiTE antibodies. PMID:24795859
Vedi, Aditi; Ziegler, David S
Antibody Therapy for Pediatric Leukemia
Despite increasing cure rates for pediatric leukemia, relapsed disease still carries a poor prognosis with significant morbidity and mortality. Novel targeted therapies are currently being investigated in an attempt to reduce adverse events and improve survival outcomes. Antibody therapies represent a form of targeted therapy that offers a new treatment paradigm. Monoclonal antibodies are active in pediatric acute lymphoblastic leukemia (ALL) and are currently in Phase III trials. Antibody-drug conjugates (ADCs) are the next generation of antibodies where a highly potent cytotoxic agent is bound to an antibody by a linker, resulting in selective targeting of leukemia cells. ADCs are currently being tested in clinical trials for pediatric acute myeloid leukemia and ALL. Bispecific T cell engager (BiTE) antibodies are a construct whereby each antibody contains two binding sites, with one designed to engage the patient's own immune system and the other to target malignant cells. BiTE antibodies show great promise as a novel and effective therapy for childhood leukemia. This review will outline recent developments in targeted agents for pediatric leukemia including monoclonal antibodies, ADCs, and BiTE antibodies.
Vedi, Aditi; Ziegler, David S.
The future of monoclonal antibody technology
With the rapid growth of monoclonal antibody-based products, new technologies have emerged for creating modified forms of antibodies, including fragments, conjugates and multi-specific antibodies. We created a database of 450 therapeutic antibodies in development to determine which technologies and indications will constitute the "next generation" of antibody products. We conclude that the antibodies of the future will closely resemble the antibodies that have already been approved for commercial sale.
Zider, Alexander
US Patent & Trademark Office Database
A monoclonal or polyclonal antibody directed against urokinase plasminogen activator receptor (u-PAR), or a subsequence, analogue or glycosylation variant thereof. Antibodies are disclosed which react with free u-PAR or with complexes between u-PA and u-PAR and which are capable of 1) catching u-PAR in ELISA, or 2) detecting u-PAR, e.g. in blotting, or 3) in radioimmunoprecipitation assay precipitate purified u-PAR in intact or fragment form, or 4) is useful for immunohistochemical detection of u-PAR, e.g. in immunostaining of cancer cells, such as in tissue sections at the invasive front, or 5) inhibits the binding of pro-u-PA and active u-PA and thereby inhibits cell surface plasminogen activation. Methods are disclosed 1) for detecting or quantifying u-PAR, 2) for targeting a diagnostic to a cell containing a u-PAR on the surface, 3) for preventing or counteracting proteolytic activity in a mammal. Methods for for selecting a substance suitable for inhibiting u-PA/u-PAR interaction, for preventing or counteracting localized proteolytical activity in a mammal, for inhibiting the invasion and/or metastasis comprise the use of the antibodies and of nude mice inoculated with human cancer cells which are known to invade and/or metastasize in mice and having a distinct color, f.x. obtained by means of an enzyme and a chromogenic substrate for the enzyme, the color being different from the cells of the mouse.
Antibody biodistribution coefficients
Tissue vs. plasma concentration profiles have been generated from a physiologically-based pharmacokinetic model of monoclonal antibody (mAb). Based on the profiles, we hypothesized that a linear relationship between the plasma and tissue concentrations of non-binding mAbs could exist; and that the relationship may be generally constant irrespective of the absolute mAb concentration, time, and animal species being analyzed. The hypothesis was verified for various tissues in mice, rat, monkey, and human using mAb or antibody-drug conjugate tissue distribution data collected from diverse literature. The relationship between the plasma and various tissue concentrations was mathematically characterized using the antibody biodistribution coefficient (ABC). Estimated ABC values suggest that typically the concentration of mAb in lung is 14.9%, heart 10.2%, kidney 13.7%, muscle 3.97%, skin 15.7%, small intestine 5.22%, large intestine 5.03%, spleen 12.8%, liver 12.1%, bone 7.27%, stomach 4.98%, lymph node 8.46%, adipose 4.78%, brain 0.351%, pancreas 6.4%, testes 5.88%, thyroid 67.5% and thymus is 6.62% of the plasma concentration. The validity of using the ABC to predict mAb concentrations in different tissues of mouse, rat, monkey, and human species was evaluated by generating validation data sets, which demonstrated that predicted concentrations were within 2-fold of the observed concentrations. The use of ABC to infer tissue concentrations of mAbs and related molecules provides a valuable tool for investigating preclinical or clinical disposition of these molecules. It can also help eliminate or optimize biodistribution studies, and interpret efficacy or toxicity of the drug in a particular tissue.
Shah, Dhaval K.; Betts, Alison M.
Affinity chromatography for antibody purification.
The availability of purified antibodies is prerequisite for many applications and the appropriate choice(s) of antibody-purification steps is crucial. Numerous methods have been developed for the purification of antibodies; however, affinity chromatography-based methods are the most extensively utilized. These methods are based on highly specific and reversible biological interactions between two molecules (e.g., between receptor and ligand or antibody and antigen). Affinity chromatography offers very high selectivity, involving minimal steps, providing simplicity of approach and rapidity. Implementing an effective protocol often requires meticulous planning and testing in order to achieve high purity and yields of desired antibody types/subtypes. This chapter describes the basic techniques for purification of monoclonal, polyclonal, and recombinant antibodies employing affinity chromatography. PMID:24648096
Arora, Sushrut; Ayyar, B Vijayalakshmi; O'Kennedy, Richard
Production of precipitating and complement-fixing antibody in rabbits and other animals was induced by immunization with live yeast-phase cells of Histoplasma capsulatum. Results of studies of polysaccharide antigens from three strains of H. capsulatum, by quantitative complement-fixation with human and rabbit antisera, strongly suggest the presence of type specificity. The variations of titer during 11 weeks in one patient with histoplasmosis and the variations of titer among a group of patients with histoplasmosis were studied by use of quantitative complement-fixation tests.
Markowitz, Harold
Human antibodies from transgenic animals
Laboratory mice provide a ready source of diverse, high-affinity and high-specificity monoclonal antibodies (mAbs). However, development of rodent antibodies as therapeutic agents has been impaired by the inherent immunogenicity of these molecules. One technology that has been explored to generate low immunogenicity mAbs for in vivo therapy involves the use of transgenic mice expressing repertoires of human antibody gene sequences.
Nils Lonberg
Phosphorylcholine antibodies in pulmonary infection
Phosphorylcholine (PC) antibodies in serum from patients with pulmonary infection, and from normal individuals, were studied. Anti-PC antibodies were detectable in the serum from normal individuals at mean concentrations of 320 µg\\/ ml for the IgG class and 110 µg\\/ml for the IgM class. Concentrations of anti-PC antibodies which were higher than normal for both the IgG and IgM classes
S. Nishinarita; S. Sawada; T. Horie
Function-first antibody discovery
Therapeutic antibodies may mediate antineoplastic effects by altering the biological functions of their target, by directly stimulating the demise of cancer cells or by activating antibody-dependent immune effector mechanisms. We have recently provided in vivo proof-of-concept for a "function-first" target and drug discovery platform in which antibodies against a multitude of tumor-associated antigens are screened for biological effects in a target-unbiased manner.
Frendeus, Bjorn
Human Antibodies by Guided Selection
\\u000a Guided selection is a method for humanization of pre-existing non-human (for example, mouse) antibodies, based on chain shuffling\\u000a of V-genes by using phage display technology. Mouse VH and VL domains are used to guide the selection of a human antibody\\u000a partner and are replaced sequentially or in parallel with human VH and VL domains, respectively to derive human antibody.\\u000a The
Sang Jick Kim; Insoo Park; Hyo Jeong Hong
Monoclonal antibodies for treating cancer
The purpose of this study is to assess the current status of in-vivo use of monoclonal antibodies for treating cancer. Publications appearing between 1980 and 1988 were identified by computer searches using MEDLINE and CANCERLIT, by reviewing the table of contents of recently published journals, and by searching bibliographies of identified books and articles. More than 700 articles, including peer-reviewed articles and book chapters, were identified and selected for analysis. The literature was reviewed and 235 articles were selected as relevant and representative of the current issues and future applications for in-vivo monoclonal antibodies for cancer therapy and of the toxicity and efficacy which has been associated with clinical trials. Approaches include using antibody alone (interacting with complement or effector cells or binding directly with certain cell receptors) and immunoconjugates (antibody coupled to radioisotopes, drugs, toxins, or other biologicals). Most experience has been with murine antibodies. Trials of antibody alone and radiolabeled antibodies have confirmed the feasibility of this approach and the in-vivo trafficking of antibodies to tumor cells. However, tumor cell heterogeneity, lack of cytotoxicity, and the development of human antimouse antibodies have limited clinical efficacy. Although the immunoconjugates are very promising, heterogeneity and the antimouse immune response have hampered this approach as has the additional challenge of chemically or genetically coupling antibody to cytotoxic agents. As a therapeutic modality, monoclonal antibodies are still promising but their general use will be delayed for several years. New approaches using human antibodies and reducing the human antiglobulin response should facilitate treatment. 235 references.
Dillman, R.O. (Hoag Cancer Center, Newport Beach, CA (USA))
Donor Specific Antibodies after Transplantation
The detection of donor specific antibodies after organ transplantation might provide an incisive way to monitor allo-specific immunity and predict graft outcome. Still, the availability of new assays for these antibodies prompts us to pose some questions about results that might be observed. These questions include whether the antibodies detected in the blood are a sensitive measure of alloimmunity, whether the detected antibodies are truly specific for the donor and whether they are noxious for the graft. Here we explain why answers to these questions might interest the basic scientist and clinician.
Platt, Jeffrey L.; Cascalho, Marilia
Taxonomic study of the genera Halogeometricum and Halosarcina: transfer of Halosarcina limi and Halosarcina pallida to the genus Halogeometricum as Halogeometricum limi comb. nov. and Halogeometricum pallidum comb. nov., respectively
Members of the haloarchaeal genera Halosarcina and Halogeometricum (family Halobacteriaceae) are closely related to each other and show 96.6–98?% 16S rRNA gene sequence similarity. This is higher than the accepted threshold value (95?%) to separate two genera, and a taxonomic study using a polyphasic approach of all four members of the two genera was conducted to clarify their relationships. Polar lipid profiles indicated that Halogeometricum rufum RO1-4T, Halosarcina pallida BZ256T and Halosarcina limi RO1-6T are related more to each other than to Halogeometricum borinquense CGMCC 1.6168T. Phylogenetic analyses using the sequences of three different genes (16S rRNA gene, rpoB? and EF-2) strongly supported the monophyly of these four species, showing that they formed a distinct clade, separate from the related genera Halopelagius, Halobellus, Haloquadratum, Haloferax and Halogranum. The results indicate that the four species should be assigned to the same genus, and it is proposed that Halosarcina pallida and Halosarcina limi be transferred to the genus Halogeometricum as Halogeometricum pallidum comb. nov. (type strain, BZ256T?=?KCTC 4017T?=?JCM 14848T) and Halogeometricum limi comb. nov. (type strain, RO1-6T?=?CGMCC 1.8711T?=?JCM 16054T).
Qiu, Xing-Xing; Zhao, Mei-Lin; Han, Dong; Zhang, Wen-Jiao; Dyall-Smith, Mike L.
Antibodies were elicited to FAD by using the hapten N-6-(6-aminohexyl)-FAD conjugated to the immunogenic carrier protein bovine serum albumin. Cross-reactivity was determined by Ouchterlony double-diffusion analysis with N-6-(6-aminohexyl)-FAD coupled to rabbit serum albumin. Anti-FAD IgG was partially purified by (NH4)2SO4 precipitation followed by DEAE-cellulose/CM-cellulose and bovine serum albumin-agarose chromatography. The partially purified anti-FAD IgG fraction failed to inhibit the catalytic activities of the flavin-containing enzymes nitrate reductase, xanthine oxidase and succinate dehydrogenase, whereas enzyme activity could be inhibited by addition of antibodies elicited against the native proteins. However, the partially purified anti-FAD IgG fraction could be used as a highly sensitive and specific probe to detect proteins containing only covalently bound flavin, such as succinate dehydrogenase, p-cresol methylhydroxylase and monoamine oxidase, by immuno-blotting techniques. Detection limits were estimated to be of the order of femtomolar concentrations of FAD with increased sensitivity for the 8 alpha-N(3)-histidyl linkage compared with 8 alpha-O-tyrosyl substitution. Images Fig. 1. Fig. 3. Fig. 4. Fig. 5. Fig. 6.
Barber, M J; Eichler, D C; Solomonson, L P; Ackrell, B A
When lymph node fragments from previously immunized rabbits were stimulated in vitro to produce a secondary response, the continuous presence of 50 µg/ml (0.15 mM) of chloramphenicol in the medium during the entire incubation period of 15 to 21 days produced nearly complete suppression of the response. Concentrations as low as 5 µg/ml (0.015 mM) produced approximately 80 per cent suppression of the response. When 50 µg/ml of chloramphenicol was present during only the first 6 days of culture, the secondary response was reduced 90 per cent. When it was absent for the first 6 days but present for the next 9 to 15 days, the response was reduced only 40 per cent. Since over 95 per cent of the antibody of the secondary response in most experiments appeared in the medium after the 6th day, chloramphenicol apparently inhibits antibody production by interfering with some early phase of the response. It is suggested that this interference involves messenger RNA and that animal cells have appeared resistant to this drug only because their complement of messenger RNA present when the drug has been added is stable over the short periods during which protein synthesis has usually been studied.
Ambrose, Charles T.; Coons, Albert H.
Antibodies: Protective, destructive and regulatory role
This book contains papers under 10 subject headings. The headings are: Production and Function of Antibodies, Protective Role of Antibodies, Antibodies to Foreign and Neoplastic Cells, Autoantibodies, Regulatory Mechanisms, Allergy, Immune Complexes, Antibodies in Pregnancy and Aging, Administration of Antibodies for Prevention and Therapy, and Abstracts of Poster Presentations.
Milgrom, F.; Abeyounis, C.J.; Albini, B.
Monoclonal antibodies: versatile platforms for cancer immunotherapy
Antibodies are important therapeutic agents for cancer. Recently, it has become clear that antibodies possess several clinically relevant mechanisms of action. Many clinically useful antibodies can manipulate tumour-related signalling. In addition, antibodies exhibit various immunomodulatory properties and, by directly activating or inhibiting molecules of the immune system, antibodies can promote the induction of antitumour immune responses. These immunomodulatory properties can
Rishi Surana; Shangzi Wang; Louis M. Weiner
Monoclonal antibodies in human cancer.
Mouse, chimeric, humanized and human monoclonal antibodies (MABs) are all in use for treatment of human cancer. Unconjugated antibodies have a complex mechanism of action, dependent on the nature of the target structure. Antibodies can activate the immune system (antibody-dependent cellular cytotoxicity [ADCC], complement-dependent cytotoxicity [CDC], induction of tumor immunity [idiotype network]). ADCC appears to be one of the most important immune effector functions. Antibodies may also induce apoptosis, cell cycle arrest, inhibition of cell proliferation as well as angiogenesis and metastatic spread. For most antibodies there is no clear dose-response relationship in vivo. The effect of antibodies can be enhanced by combination with chemotherapy and/or by agents which activate the immune system. The best therapeutic effect may be obtained if MABs are used early in the course of the disease. Rituximab (anti-CD20) was the first registered MAB for the therapy of follicular lymphoma. Impressive results have been seen in combination with CHOP chemotherapy (cyclophosphamide, doxorubicin, vincristine and prednisone) in follicular and high-grade lymphomas. In other non-Hodgkin's lymphoma subtypes, promising results are also seen in combination with chemotherapy. Trastuzumab (anti-Her2) is a breakthrough in the treatment of breast cancer in combination with chemotherapeutic agents. This antibody is also in clinical testing for adjuvant treatment. Alemtuzumab (anti-CD52) has shown impressive results both in refractory chronic lymphocytic leukemia and as up-front therapy. There are many other antibodies in late stages of testing for registration. Interesting MABs include cetuximab (anti-epidermal growth factor receptor [EGFR]), especially in combination with radiotherapy in head and neck cancer; ABX-EGF (anti-EGFR) in renal carcinoma; bevacizumab (anti-vascular endothelial growth factor) in several solid tumors. Antiepithelial cell adhesion molecule antibodies show promise in combination with chemotherapy in the adjuvant setting of colorectal carcinoma. It is estimated that about 20 antibodies will be in clinical use by the year 2010. PMID:14988743
Mellstedt, H
Norovirus Monoclonal Antibodies and Peptides.
National Technical Information Service (NTIS)
The present invention is drawn to monoclonal antibodies that bind to a Norovirus, peptides that inhibit monoclonal antibody binding to a Norovirus, and peptides that inhibit binding of a Norovirus to a cell. The compositions of the invention find use as N...
M. Hardy
Bispecific antibodies for cancer therapy
With 23 approvals in the US and other countries and four approvals outside US, antibodies are now widely recognized as therapeutic molecules. The therapeutic and commercial successes met by rituximab, trastuzumab, cetuximab and other mAbs have inspired antibody engineers to improve the efficacy of these molecules. Consequently, a new wave of antibodies with engineered Fc leading to much higher effector functions such as antibody-dependent cell-mediated cytotoxicity or complement-dependent cytotoxicity is being evaluated in the clinic, and several approvals are expected soon. In addition, research on a different class of antibody therapeutics, bispecific antibodies, has recently led to outstanding clinical results, and the first approval of the bispecific antibody catumaxomab, a T cell retargeting agent that was approved in the European Union in April 2009. This review describes the most recent advances and clinical study results in the field of bispecific antibodies, a new class of molecules that might outshine conventional mAbs as cancer immunotherapeutics in a near future.
Baty, Daniel
A revival of bispecific antibodies
Bispecific antibodies usually do not occur in nature but are constructed by recombinant DNA or cell-fusion technologies. Most are designed to recruit cytotoxic effector cells of the immune system effectively against pathogenic target cells. This complex task explains why, after more than 15 years of extensive research, many different formats of bispecific antibodies have been developed but only a few
Peter Kufer; Ralf Lutterbüse; Patrick A. Baeuerle
Micromechanical antibody sensor
A sensor apparatus is provided using a microcantilevered spring element having a coating of a detector molecule such as an antibody or antigen. A sample containing a target molecule or substrate is provided to the coating. The spring element bends in response to the stress induced by the binding which occurs between the detector and target molecules. Deflections of the cantilever are detected by a variety of detection techniques. The microcantilever may be approximately 1 to 200 .mu.m long, approximately 1 to 50 .mu.m wide, and approximately 0.3 to 3.0 .mu.m thick. A sensitivity for detection of deflections is in the range of 0.01 nanometers.
Thundat, Thomas G. (Knoxville, TN) [Knoxville, TN; Jacobson, K. Bruce (Oak Ridge, TN) [Oak Ridge, TN; Doktycz, Mitchel J. (Knoxville, TN) [Knoxville, TN; Kennel, Stephen J. (Oak Ridge, TN) [Oak Ridge, TN; Warmack, Robert J. (Knoxville, TN) [Knoxville, TN
Therapeutic antibodies against viral hepatitis.
Antibodies have the potential to be immunotherapeutic agents, used either as stand-alone therapy or as an adjunct for managing chronic viral infection. In addition, antibodies may be used prophylactically in individuals who have been accidentally exposed to hepatitis B virus (HBV) or hepatitis C virus (HCV), or to prevent re-infection of the liver in patients who have undergone liver transplantation. Human monoclonal antibodies to HBV and HCV were generated and their ability to reduce viral load was tested in different animal model systems, the Trimera mouse model and HBV-carrier chimpanzees. These antibodies were further developed and are currently being studied in clinical trials for chronic HBV or HCV and in liver transplant patients. The antibodies were shown to be safe, tolerable and could significantly reduce viral load. Their ability to inhibit HCV re-infection in the transplanted liver is being evaluated. PMID:12772504
Dagan, Shlomo; Eren, Rachel
Endogenous antibodies for tumor detection.
The study of cancer immunology has provided diagnostic and therapeutic instruments through serum autoantibody biomarkers and exogenous monoclonal antibodies. While some endogenous antibodies are found within or surrounding transformed tissue, the extent to which this exists has not been entirely characterized. We find that in transgenic and xenograft mouse models of cancer, endogenous gamma immunoglobulin (IgG) is present at higher concentration in malignantly transformed organs compared to non-transformed organs in the same mouse or organs of cognate wild-type mice. The enrichment of endogenous antibodies within the malignant tissue provides a potential means of identifying and tracking malignant cells in vivo as they mutate and diversify. Exploiting these antibodies for diagnostic and therapeutic purposes is possible through the use of agents that bind endogenous antibodies. PMID:24875800
Rich, Barrie S; Honeyman, Joshua N; Darcy, David G; Smith, Peter T; Williams, Andrew R; Lim, Irene Isabel P; Johnson, Linda K; Gönen, Mithat; Simon, Joel S; LaQuaglia, Michael P; Simon, Sanford M
Endogenous Antibodies for Tumor Detection
The study of cancer immunology has provided diagnostic and therapeutic instruments through serum autoantibody biomarkers and exogenous monoclonal antibodies. While some endogenous antibodies are found within or surrounding transformed tissue, the extent to which this exists has not been entirely characterized. We find that in transgenic and xenograft mouse models of cancer, endogenous gamma immunoglobulin (IgG) is present at higher concentration in malignantly transformed organs compared to non-transformed organs in the same mouse or organs of cognate wild-type mice. The enrichment of endogenous antibodies within the malignant tissue provides a potential means of identifying and tracking malignant cells in vivo as they mutate and diversify. Exploiting these antibodies for diagnostic and therapeutic purposes is possible through the use of agents that bind endogenous antibodies.
Rich, Barrie S.; Honeyman, Joshua N.; Darcy, David G.; Smith, Peter T.; Williams, Andrew R.; Lim, Irene Isabel P.; Johnson, Linda K.; Gonen, Mithat; Simon, Joel S.; LaQuaglia, Michael P.; Simon, Sanford M.
Computer-aided antibody design
Recent clinical trials using antibodies with low toxicity and high efficiency have raised expectations for the development of next-generation protein therapeutics. However, the process of obtaining therapeutic antibodies remains time consuming and empirical. This review summarizes recent progresses in the field of computer-aided antibody development mainly focusing on antibody modeling, which is divided essentially into two parts: (i) modeling the antigen-binding site, also called the complementarity determining regions (CDRs), and (ii) predicting the relative orientations of the variable heavy (VH) and light (VL) chains. Among the six CDR loops, the greatest challenge is predicting the conformation of CDR-H3, which is the most important in antigen recognition. Further computational methods could be used in drug development based on crystal structures or homology models, including antibody–antigen dockings and energy calculations with approximate potential functions. These methods should guide experimental studies to improve the affinities and physicochemical properties of antibodies. Finally, several successful examples of in silico structure-based antibody designs are reviewed. We also briefly review structure-based antigen or immunogen design, with application to rational vaccine development.
Kuroda, Daisuke; Shirai, Hiroki; Jacobson, Matthew P.; Nakamura, Haruki
Antimyenteric neuronal antibodies in scleroderma.
The pathogenesis of gastrointestinal (GI) dysmotility in scleroderma is incompletely understood, although previous studies have proposed a neuropathic mechanism. We studied patients with scleroderma as compared with other connective tissue disease patients and normal controls for the presence of circulating antibodies to myenteric neurons. Serial dilutions of sera were overlaid on rat intestine, double-labeled with antineurofilament antibody as a myenteric plexus marker, and imaged using indirect immunofluorescence techniques. High titer sera (> or = 1:50) from 19 out of 41 scleroderma patients stained myenteric neurons, whereas none of 22 normals or 5 patients with idiopathic GI dysmotility were positive. Although 6 out of 20 SLE and 6 out of 10 mixed connective tissue disease patients' sera stained myenteric plexus neurons, when positive sera were absorbed with calf thymus extract to remove antinuclear antibody, 15 scleroderma sera, 0 SLE, and 2 mixed connective tissue disease patients retained positive staining of myenteric neurons. Western blotting using actin and neuronal intermediate filament preparations failed to show immunoreactivity with scleroderma sera containing antimyenteric neuronal antibodies. Paraneoplastic sera associated with GI dysmotility stained myenteric neurons in a different pattern than seen with scleroderma sera. A positive correlation between the presence of Raynaud's phenomenon and antimyenteric neuronal antibodies was observed in scleroderma patients. Our results indicate that IgG antibodies reacting with myenteric neurons are present in many patients with scleroderma. Although the neuronal antigen has not yet been identified, the presence of myenteric neuronal antibodies in patients with GI dysmotility and scleroderma suggests a neuropathic process. PMID:8040331
Howe, S; Eaker, E Y; Sallustio, J E; Peebles, C; Tan, E M; Williams, R C
Reducing heterophilic antibody interference in immunoassays using single chain antibodies
Sandwich ELISA microarrays have the potential to simultaneously quantify the levels of multiple diagnostic targets in a biological sample. However, as seen with traditional ELISA diagnostics, heterophilic antibodies (HA) in patient sera have the potential to cause interference in these assays. We demonstrate here that reducing the diagnostic capture antibody to its minimal functional unit, the variable heavy and light domains artificially connected with a short polypeptide linker (scFv), is an effective strategy for reducing the HA assay interference.
Baird, Cheryl L.; Tan, Ruimin; Fischer, Christopher J.; Victry, Kristin D.; Zangar, Richard C.; Rodland, Karin D.
Immunotoxicity of monoclonal antibodies
Monoclonal antibodies (mAbs) are large molecules intended to bind to specific targets often expressed on the immune system, and to treat various immunopathological conditions. Therefore, mAbs can be considered to have a high potential for immunotoxicity, which is reflected in the clinical experience accumulated on mAbs-induced adverse effects related to immunosuppression, immunostimulation and hypersensitivity (immunogenicity). So far, non clinical immunotoxicity studies have been inadequate to address all safety issues in relation to the possible immunotoxicity of mAbs, because they are fraught with limitations and pitfalls primarily related to the lack of relevant animal species. In addition, clinical studies rarely include validated end-points dedicated to the prediction of immunotoxicity. With the ongoing development of mAbs as novel therapeutic strategies for a wide variety of diseases, efforts should be paid to improve our understanding of mAbs-induced immunotoxic effects and design dedicated strategies to assess their immunological safety, both non clinically and clinically.
Marketed therapeutic antibodies compendium
Therapeutic monoclonal antibodies (mAbs) are currently being approved for marketing in Europe and the United States, as well as other countries, on a regular basis. As more mAbs become available to physicians and patients, keeping track of the number, types, production cell lines, antigenic targets, and dates and locations of approvals has become challenging. Data are presented here for 34 mAbs that were approved in either Europe or the United States (US) as of March 2012, and nimotuzumab, which is marketed outside Europe and the US. Of the 34 mAbs, 28 (abciximab, rituximab, basiliximab, palivizumab, infliximab, trastuzumab, alemtuzumab, adalimumab, tositumomab-I131, cetuximab, ibrituximab tiuxetan, omalizumab, bevacizumab, natalizumab, ranibizumab, panitumumab, eculizumab, certolizumab pegol, golimumab, canakinumab, catumaxomab, ustekinumab, tocilizumab, ofatumumab, denosumab, belimumab, ipilimumab, brentuximab) are currently marketed in Europe or the US. Data for six therapeutic mAbs (muromonab-CD3, nebacumab, edrecolomab, daclizumab, gemtuzumab ozogamicin, efalizumab) that were approved but have been withdrawn or discontinued from marketing in Europe or the US are also included.
Reichert, Janice M.
Surface chemistries for antibody microarrays
Enzyme-linked immunosorbent assay (ELISA) microarrays promise to be a powerful tool for the detection of disease biomarkers. The original technology for printing ELISA microarray chips and capturing antibodies on slides was derived from the DNA microarray field. However, due to the need to maintain antibody structure and function when immobilized, surface chemistries used for DNA microarrays are not always appropriate for ELISA microarrays. In order to identify better surface chemistries for antibody capture, a number of commercial companies and academic research groups have developed new slide types that could improve antibody function in microarray applications. In this review we compare and contrast the commercially available slide chemistries, as well as highlight some promising recent advances in the field.
Seurynck-Servoss, Shannon L.; Baird, Cheryl L.; Rodland, Karin D.; Zangar, Richard C.
Human Monoclonal Antibody Against Mesothelin
Mesothelin is a cell surface protein that is naturally expressed at very low levels, but that is significantly increased in aggressive tumors such as mesotheliomas, and pancreatic and ovarian tumors. Therefore, mesothelin is an excellent candidate for tumor-targeted immunotherapeutics. However, the only antibodies against mesothelin that are currently available for clinical trials are of murine origin. The use of these antibodies may be limited by their potential to elicit adverse immune responses in patients with repeated doses.
Engineering recombinant antibodies for immunotherapy
Recombinant antibody fragments binding with high affinity to their target can be obtained either from hybridomas or directly\\u000a from antibody libraries on filamentous phage. These fragments are devoid of any activity other than antigen binding, and have\\u000a to be processed and functionalized in order to be suitable for clinical applications. This article presents the authors' view\\u000a on the procedures and
Dario Neri; Heike Petrul; Gabrio Roncucci
Bispecific Antibodies from Hybrid Hybridoma
\\u000a Hybrid hybridomas (also termed quadromas or tetradomas) are man-made cell lines that secrete bispecific antibodies (bsAb)\\u000a with two different specificities being able to crosslink two distinct molecules. Such antibodies do not occur in nature and\\u000a have been originally developed to improve immunohistochemical staining procedures and immunoassays (Milstein and Cuello 1983;\\u000a Suresh et al. 1986). Interestingly, the fusion of two immunoglobulin-producing
Gerhard Moldenhauer
Radioimmunoguided surgery using monoclonal antibody
The potential proficiency of radioimmunoguided surgery in the intraoperative detection of tumors was assessed using labeled monoclonal antibody B72.3 in 66 patients with tissue-proved tumor. Monoclonal antibody B72.3 was injected 5 to 42 days preoperatively, and the hand-held gamma-detecting probe was used intraoperatively to detect the presence of tumor. Intraoperative probe counts of less than 20 every 2 seconds, or tumor-to-adjacent normal tissue ratios less than 2:1 were considered negative (system failure). Positive probe counts were detected in 5 of 6 patients with primary colon cancer (83 percent), in 31 of 39 patients with recurrent colon cancer (79 percent), in 4 of 5 patients with gastric cancer (80 percent), in 3 of 8 patients with breast cancer (37.5 percent), and in 4 of 8 patients with ovarian cancer (50 percent) undergoing second-look procedures. Additional patients in each group were scored as borderline positive. Overall, radioimmunoguided surgery using B72.3 identified tumors in 47 patients (71.2 percent), bordered on positive in 6 patients (9.1 percent), and failed to identify tumor in 13 patients (19.7 percent). Improved selection of patients for antigen-positive tumors, the use of higher affinity second-generation antibodies, alternate routes of antibody administration, alternate radionuclides, and more sophisticatedly bioengineered antibodies and antibody combinations should all lead to improvements in radioimmunoguided surgery.
Martin, E.W. Jr.; Mojzisik, C.M.; Hinkle, G.H. Jr.; Sampsel, J.; Siddiqi, M.A.; Tuttle, S.E.; Sickle-Santanello, B.; Colcher, D.; Thurston, M.O.; Bell, J.G.
Neutralising antibodies against ricin toxin.
The Centers for Disease Control and Prevention have listed the potential bioweapon ricin as a Category B Agent. Ricin is a so-called A/B toxin produced by plants and is one of the deadliest molecules known. It is easy to prepare and no curative treatment is available. An immunotherapeutic approach could be of interest to attenuate or neutralise the effects of the toxin. We sought to characterise neutralising monoclonal antibodies against ricin and to develop an effective therapy. For this purpose, mouse monoclonal antibodies (mAbs) were produced against the two chains of ricin toxin (RTA and RTB). Seven mAbs were selected for their capacity to neutralise the cytotoxic effects of ricin in vitro. Three of these, two anti-RTB (RB34 and RB37) and one anti-RTA (RA36), when used in combination improved neutralising capacity in vitro with an IC(50) of 31 ng/ml. Passive administration of association of these three mixed mAbs (4.7 µg) protected mice from intranasal challenges with ricin (5 LD(50)). Among those three antibodies, anti-RTB antibodies protected mice more efficiently than the anti-RTA antibody. The combination of the three antibodies protected mice up to 7.5 hours after ricin challenge. The strong in vivo neutralising capacity of this three mAbs combination makes it potentially useful for immunotherapeutic purposes in the case of ricin poisoning or possibly for prevention. PMID:21633505
Prigent, Julie; Panigai, Laetitia; Lamourette, Patricia; Sauvaire, Didier; Devilliers, Karine; Plaisance, Marc; Volland, Hervé; Créminon, Christophe; Simon, Stéphanie
Since 2010, mAbs has documented the biopharmaceutical industry's progress in transitioning antibody therapeutics to first Phase 3 clinical studies and regulatory review, and its success at gaining first marketing approvals for antibody-based products. This installment of the "Antibodies to watch" series outlines events anticipated to occur between December 2013 and the end of 2014, including first regulatory actions on marketing applications for vedolizumab, siltuximab, and ramucirumab, as well as the Fc fusion proteins Factor IX-Fc and Factor VIII-Fc; and the submission of first marketing applications for up to five therapeutics (secukinumab, ch14.18, onartuzumab, necitumumab, gevokizumab). Antibody therapeutics in Phase 3 studies are described, with an emphasis on those with study completion dates in 2014, including antibodies targeting interleukin-17a or the interleukin-17a receptor (secukinumab, ixekizumab, brodalumab), proprotein convertase subtilisin/kexin type 9 (alirocumab, evolocumab, bococizumab), and programmed death 1 receptor (lambrolizumab, nivolumab). Five antibodies with US Food and Drug Administration's Breakthrough Therapy designation (obinutuzumab, ofatumumab, lambrolizumab, bimagrumab, daratumumab) are also discussed. PMID:24284914
Reichert, Janice M
Avian Diagnostic and Therapeutic Antibodies
A number of infectious agents have the potential of causing significant clinical symptomology and even death, but dispite this, the number of incidence remain below the level that supports producing a vaccine. Therapeutic antibodies provide a viable treatment option for many of these diseases. We proposed that antibodies derived from West Nile Virus (WNV) immunized geese would be able to treat WNV infection in mammals and potential humans. We demonstrated that WNV specific goose antibodies are indeed successful in treating WNV infection both prophylactically and therapeutically in a golden hamster model. We demonstrated that the goose derived antibodies are non-reactogenic, i.e. do not cause an inflammatory response with multiple exposures in mammals. We also developed both a specific pathogen free facility to house the geese during the antibody production phase and a patent-pending purification process to purify the antibodies to greater than 99% purity. Therefore, the success of these study will allow a cost effective rapidly producible therapeutic toward clinical testing with the necessary infrastructure and processes developed and in place.
Bradley, David Sherman [UND SMHS] [UND SMHS
Selecting and screening recombinant antibody libraries
During the past decade several display methods and other library screening techniques have been developed for isolating monoclonal antibodies (mAbs) from large collections of recombinant antibody fragments. These technologies are now widely exploited to build human antibodies with high affinity and specificity. Clever antibody library designs and selection concepts are now able to identify mAb leads with virtually any specificity.
Hennie R Hoogenboom
Colorectal carcinoma antigens detected by hybridoma antibodies
Hybridoma cells which secrete colorectal carcinoma-specific antibodies have been produced and used to study the antigenic structure of these tumor cells. Nineteen antibodies have been studied in detail, and 15 of these are colorectal carcinoma specific. Only two antibodies reactive with carcinoembryonic antigen (CEA) have been discovered and five other antibodies that react with distinct epitopes on the cell surface
Hilary Koprowski; Zenon Steplewski; Kenneth Mitchell; Meenhard Herlyn; Dorothee Herlyn; Peter Fuhrer
Fragmentation of monoclonal antibodies.
Fragmentation is a degradation pathway ubiquitously observed in proteins despite the remarkable stability of peptide bond; proteins differ only by how much and where cleavage occurs. The goal of this review is to summarize reports regarding the non-enzymatic fragmentation of the peptide backbone of monoclonal antibodies (mAbs). The sites in the polypeptide chain susceptible to fragmentation are determined by a multitude of factors. Insights are provided on the intimate chemical mechanisms that can make some bonds prone to cleavage due to the presence of specific side-chains. In addition to primary structure, the secondary, tertiary and quaternary structures have a significant impact in modulating the distribution of cleavage sites by altering local flexibility, accessibility to solvent or bringing in close proximity side chains that are remote in sequence. This review focuses on cleavage sites observed in the constant regions of mAbs, with special emphasis on hinge fragmentation. The mechanisms responsible for backbone cleavage are strongly dependent on pH and can be catalyzed by metals or radicals. The distribution of cleavage sites are different under acidic compared to basic conditions, with fragmentation rates exhibiting a minimum in the pH range 5 to 6; therefore, the overall fragmentation pattern observed for a mAb is a complex result of structural and solvent conditions. A critical review of the techniques used to monitor fragmentation is also presented; usually a compromise has to be made between a highly sensitive method with good fragment separation and the capability to identify the cleavage site. The effect of fragmentation on the function of a mAb must be evaluated on a case-by-case basis depending on whether cleavage sites are observed in the variable or constant regions, and on the mechanism of action of the molecule. PMID:21487244
Vlasak, Josef; Ionescu, Roxana
Comparison of physical chemical properties of llama V HH antibody fragments and mouse monoclonal antibodies
Antigen specific llama VHH antibody fragments were compared to antigen specific mouse monoclonal antibodies with respect to specificity, affinity and stability. The llama VHH antibody fragments and the mouse monoclonal antibodies investigated were shown to be highly specific for the protein antigen hCG or the hapten antigen RR-6. The affinity of the interaction between monovalent llama VHH antibody fragments and
R. H. J. van der Linden; L. G. J. Frenken; B. de Geus; M. M. Harmsen; R. C. Ruuls; W. Stok; L. de Ron; S. Wilson; P. Davis; C. T. Verrips
Anti-actin antibodies revealed by counter-immunoelectrophoresis. Relation to smooth muscle antibodies and bile canalicular antibodies.
In investigations by counter-immunoelectrophoresis, anti-actin antibodies were found in 59% of patients with chronic hepatitis and in 8% of patients with non-hepatic diseases and normal blood donors. Anti-actin antibodies were found more frequently in patients with hepatitis and IgG smooth muscle antibodies than in other groups of diseases and normal subjects with IgG smooth muscle antibodies. Anti-actin antibodies showed no correlation with bile canalicular antibodies. Images Fig. 1 Fig. 2
Diederichsen, H; Riisom, K
Uses of monoclonal antibody 8H9
This invention provides a composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a suitable carrier. This invention provides a pharmaceutical composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a pharmaceutically acceptable carrier. This invention also provides an antibody other than the monoclonal antibody 8H9 comprising the complementary determining regions of monoclonal antibody 8H9 or a derivative thereof, capable of binding to the same antigen as the monoclonal antibody 8H9. This invention provides a substance capable of competitively inhibiting the binding of monoclonal antibody 8H9. This invention also provides an isolated scFv of monoclonal antibody 8H9 or a derivative thereof. This invention also provides the 8H9 antigen. This invention also provides different uses of the monoclonal antibody 8H9 or its derivative.
Cheung, Nai-Kong V.
Production of recombinant antibodies using bacteriophages
Recombinant antibody fragments such as Fab, scFv, diabodies, triabodies, single domain antibodies and minibodies have recently emerged as potential alternatives to monoclonal antibodies, which can be engineered using phage display technology. These antibodies match the strengths of conventionally produced monoclonal antibodies and offer advantages for the development of immunodiagnostic kits and assays. These fragments not only retain the specificity of the whole monoclonal antibodies but also easy to express and produce in prokaryotic expression system. Further, these antibody fragments are genetically stable, less expensive, easy to modify in response to viral mutations and safer than monoclonal antibodies for use in diagnostic and therapeutic applications. This review describes the potential of antibody fragments generated using phage display and their use as diagnostic reagents.
Shukra, A. M.; Sridevi, N. V.; Dev Chandran
Entanglement model of antibody viscosity.
Antibody solutions are typically much more viscous than solutions of globular proteins at equivalent volume fraction. Here we propose that this is due to molecular entanglements that are caused by the elongated shape and intrinsic flexibility of antibody molecules. We present a simple theory in which the antibodies are modeled as linear polymers that can grow via reversible bonds between the antigen binding domains. This mechanism explains the observation that relatively subtle changes to the interparticle interaction can lead to large changes in the viscosity. The theory explains the presence of distinct power law regimes in the concentration dependence of the viscosity as well as the correlation between the viscosity and the charge on the variable domain in our antistreptavidin IgG1 model system. PMID:24758234
Schmit, Jeremy D; He, Feng; Mishra, Shradha; Ketchem, Randal R; Woods, Christopher E; Kerwin, Bruce A
Molecular-specific urokinase antibodies
NASA Technical Reports Server (NTRS)
Antibodies have been developed against the different molecular forms of urokinase using synthetic peptides as immunogens. The peptides were synthesized specifically to represent those regions of the urokinase molecules which are exposed in the three-dimensional configuration of the molecule and are uniquely homologous to urokinase. Antibodies are directed against the lysine 158-isoleucine 159 peptide bond which is cleaved during activation from the single-chain (ScuPA) form to the bioactive double chain (54 KDa and 33 KDa) forms of urokinase and against the lysine 135 lysine 136 bond that is cleaved in the process of removing the alpha-chain from the 54 KDa form to produce the 33 KDa form of urokinase. These antibodies enable the direct measurement of the different molecular forms of urokinase from small samples of conditioned medium harvested from cell cultures.
Atassi, M. Zouhair (Inventor); Morrison, Dennis R. (Inventor)
Antibody testing in peripheral neuropathies.
Many autoantibodies have been described in association with peripheral neuropathy, but the use of antibody testing in clinical practice remains a matter of some debate. Serum autoantibodies to gangliosides or glycoproteins are implicated in a variety of sensory and motor neuropathy syndromes. Paraneoplastic antibodies help identify patients who have a neuropathy related to an underlying malignancy. Detection of an autoantibody in the right clinical setting provides some evidence that the peripheral nerve disturbance is immune mediated and that immunomodulatory therapy may be of benefit. PMID:17324719
Vernino, Steven; Wolfe, Gil I
Monoclonal Antibodies for the Treatment of Cancer
Over the past decade, the clinical utility of monoclonal antibodies has been realized and antibodies are now a mainstay for the treatment of cancer. Antibodies have the unique capacity to target and kill tumor cells while simultaneously activating immune effectors to kill tumor cells through the complement cascade or antibody-dependent cellular cytotoxicity (ADCC). This multifaceted mechanism of action combined with target specificity underlies the capacity of antibodies to elicit anti-tumor responses while minimizing the frequency and magnitude of adverse events. This review will focus on mechanisms of action, clinical applications and putative mechanisms of resistance to monoclonal antibody therapy in the context of cancer.
Shuptrine, Casey; Surana, Rishi; Weiner, Louis M.
Antibody profiling sensitivity through increased reporter antibody layering
A method for analyzing a biological sample by antibody profiling for identifying forensic samples or for detecting the presence of an analyte. In an embodiment of the invention, the analyte is a drug, such as marijuana, Cocaine (crystalline tropane alkaloid), methamphetamine, methyltestosterone, or mesterolone. The method comprises attaching antigens to a surface of a solid support in a preselected pattern to form an array wherein locations of the antigens are known; contacting the array with the biological sample such that a portion of antibodies in the sample reacts with and binds to the antigens in the array to form immune complexes; washing away antibodies that do form immune complexes; and detecting the immune complexes, to form an antibody profile. Forensic samples are identified by comparing a sample from an unknown source with a sample from a known source. Further, an assay, such as a test for illegal drug use, can be coupled to a test for identity such that the results of the assay can be positively correlated to the subject's identity.
Apel, William A; Thompson, Vicki S
9 CFR 113.452 - Erysipelothrix Rhusiopathiae Antibody.
Code of Federal Regulations, 2010 CFR
... false Erysipelothrix Rhusiopathiae Antibody. 113.452 Section 113.452 ...AND VECTORS STANDARD REQUIREMENTS Antibody Products § 113.452 Erysipelothrix Rhusiopathiae Antibody. Erysipelothrix Rhusiopathiae...
9 CFR 113.452 - Erysipelothrix Rhusiopathiae Antibody.
Code of Federal Regulations, 2010 CFR
... false Erysipelothrix Rhusiopathiae Antibody. 113.452 Section 113.452 ...AND VECTORS STANDARD REQUIREMENTS Antibody Products § 113.452 Erysipelothrix Rhusiopathiae Antibody. Erysipelothrix Rhusiopathiae...
[IgA antibodies in exogenous allergic alveolitis are more specific than IgG antibodies].
Nineteen sera from patients suffering from extrinsic allergic alveolitis (bird fancier's lung, farmer's lung) and 19 sera from asymptomatic persons demonstrating IgG-antibodies were examined for IgA-antibodies against pigeon serum, Micropolyspora faeni and Aspergillus fumigatus. The IgA-antibodies against pigeon serum and Micropolyspora faeni were less of ten false-positive than the corresponding IgG-antibodies. Hence the IgA-antibodies were more specific than the IgG-antibodies. However, the sensitivity of the IgA-antibodies was lower than that of the IgG-antibodies. On the other hand IgA-antibodies against Aspergillus fumigatus were scarcely more specific than the corresponding IgG-antibodies against pigeon and M. faeni. It would thus seem that IgA-antibodies can supplement the IgG-serodiagnosis of extrinsic allergic alveolitis. PMID:2367456
Sennekamp, J; Rust, M; Kroidl, R; Spyra, M
Rabbit polyclonal antibody to Peripherin
Peripherin is a Class III intermediate filament subunit found in both the peripheral and central nervous systems, though it is concentrated, as its name suggests, in the neurons of peripheral ganglia and their processes. Antibodies to peripherin can be used in identifying, classifying, and studying neurons in the nervous system. Peripherin is also a good diagnostic marker for ballooned axons
Species Reactivity
Polymyalgia rheumatica and antimitochondrial antibodies.
Antimitochondrial antibodies (AMA) were detected in the sera of 11 of 36 patients with a clinical diagnosis of polymyalgia rheumatica (PMR) in whom comprehensive autoantibody screening had been performed. AMA did not correlate with biochemical changes of hepatic dysfunction, which are common in PMR, nor with parameters of musculoskeletal inflammation. Possible explanations are discussed.
Sattar, M A; Cawley, M I; Hamblin, T J; Robertson, J C
1. A study has been made of the non-specific fluorescent staining of splenic imprints treated with fluorescent sheep antibody globulins. 2. In tissue imprints made with the spleens of antigen-stimulated animals, no morphological distinction was evident between areas showing non-specific fluorescence and specific fluorescence. 3. Elimination of non-specific fluorescence was not achieved by any one, or any combination of the following: (a) conjugating only gamma globulins with fluorescein isothiocyanate; (b) removal of dialyzable fluorescent products on sephadex, followed by concentration through the use of pressure dialysis; (c) use of crystalline preparations of fluorescein isothiocyanate. 4. Individual preparations of fluorescent antibodies were separated by gradient elution chromatography on diethylaminoethyl (DEAE) cellulose into fractions possessing different numbers of fluorescein radicals per molecule of globulin. 5. The coupling ratio of 50 mg fluorescein isothiocyanate (FITC) per gm of protein, as commonly advocated, can not be recommended for the precise localization of antibody globulin in tissues owing to the capacity of the coupled products to give non-specific fluorescent staining. When crystalline preparations of FITC are used instead of the amorphous product at 50 mg/gm protein, far too high non-specific fluorescence results. 6. A fraction with bright specific fluorescence and no or negligible nonspecific fluorescence was obtained from each fluorescent antibody that was prepared by using 6 to 8 mg of crystalline fluorescein isothiocyanate per gm of globulin and was then subjected to DEAE-cellulose chromatography and gradient elution to eliminate the most highly coupled molecules.
Goldstein, Gerald; Slizys, Irene S.; Chase, Merrill W.
Neuroticism and Antibody Responses in Military Recruits.
National Technical Information Service (NTIS)
The hypothesis that neurotic individuals produce lower levels of antibodies under stress than emotionally-stable individuals was tested by measuring neutralizing antibody responses to inoculations for adenoviruses 4 and 7 in military recruits (n = 24) dur...
R. R. Vickers L. K. Hervig R. Rappaport P. Linard
Humanization and simultaneous optimization of monoclonal antibody.
Antibody humanization is an essential technology for reducing the potential risk of immunogenicity associated with animal-derived antibodies and has been applied to a majority of the therapeutic antibodies on the market. For developing an antibody molecule as a pharmaceutical at the current biotechnology level, however, other properties also have to be considered in parallel with humanization in antibody generation and optimization. This section describes the critical properties of therapeutic antibodies that should be sufficiently qualified, including immunogenicity, binding affinity, physiochemical stability, expression in host cells and pharmacokinetics, and the basic methodologies of antibody engineering involved. By simultaneously optimizing the antibody molecule in the light of these properties, it should prove possible to shorten the research and development period necessary to identify a highly qualified clinical candidate and consequently accelerate the start of the clinical trial. PMID:24037839
Kuramochi, T; Igawa, T; Tsunoda, H; Hattori, K
Detection of Campylobacter species using monoclonal antibodies
NASA Astrophysics Data System (ADS)
A panel of species specific monoclonal antibodies were raised to Campylobacter coli, Campylobacter jejuni and Campylobacter lari. The isotypes, and cross-reactivity profiles of each monoclonal antibody against an extensive panel of micro- organisms, were determined.
Young, Colin R.; Lee, Alice; Stanker, Larry H.
Guinea pigs hyperimmunized with single protein antigens or hapten conjugates emulsified in complete adjuvants produced two types of precipitating antibodies with different electrophoretic mobilities. "Slow" migrating antibody generally appeared earlier and "fast" migrating antibody later in the course of immunization. Animals initially immunized by the intraperitoneal route with hapten conjugates without adjuvants produced primarily fast migrating antibody. Purified guinea pig antibodies were also separable into slow and fast migrating components by electrophoresis in supporting media. Using suitable antisera prepared in rabbits hyperimmunized with guinea pig serum, it was demonstrated that slow and fast antibodies have both common and distinct antigenic determinants. Analytical ultracentrifugation disclosed that both antibodies have sedimentation coefficients of approximately 7S. These antibodies have been designated guinea pig 7S?1 and 7S?2.
Benacerraf, Baruj; Ovary, Zoltan; Bloch, Kurt J.; Franklin, Edward C.
Uses of Monoclonal Antibody 8H9.
National Technical Information Service (NTIS)
This invention provides a composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a suitable carrier. This invention provides a pharmaceutical composition comprising an effective amount of monoclonal antibody 8H9 ...
N. K. Cheung
Platelet Fibrinogen-Specific Monoclonal Antibody.
National Technical Information Service (NTIS)
The invention is related generally to monoclonal antibodies. More particularly, the invention is related to a unique monoclonal antibody (MAb) directed against a specific antigen, the platelet fibrinogen, found on stimulated platelets, said MAb being desi...
H. Grelnick
Antibody approach of labeling blood cells.
National Technical Information Service (NTIS)
Although the science of blood cell labeling using monoclonal antibodies directed against specific cellular antigens is still in its early stages, considerable progress has recently been accomplished in this area. The monoclonal antibody approach offers th...
S. C. Srivastava
Progranulin antibodies in autoimmune diseases.
Systemic vasculitides constitute a heterogeneous group of diseases. Autoimmunity mediated by B lymphocytes and their humoral effector mechanisms play a major role in ANCA-associated vasculitis (AAV) as well as in non-ANCA associated primary systemic vasculitides and in the different types of autoimmune connective tissue disorders and rheumatoid arthritis. In order to detect autoantibodies in systemic vasculitides, we screened protein macroarrays of human cDNA expression libraries with sera from patients with ANCA-associated and ANCA-negative primary systemic vasculitides. This approach led to the identification of antibodies against progranulin, a 88 kDA secreted glycoprotein with strong anti-inflammatory activity in the course of disease of giant-cell arteritis/polymyalgia rheumatica (14/65), Takayasu's arteritis (4/13), classical panarteritis nodosa (4/10), Behcet's disease (2/6) and in the course of disease in granulomatosis with polyangiitis (31/75), Churg-Strauss syndrome (7/23) and in microscopic polyangiitis (7/19). In extended screenings the progranulin antibodies were also detected in other autoimmune diseases such as systemic lupus erythematosus (39/91) and rheumatoid arthritis (16/44). Progranulin antibodies were detected only in 1 of 97 healthy controls. Anti-progranulin positive patients with systemic vasculitides, systemic lupus erythematosus or rheumatoid arthritis had significant lower progranulin plasma levels, indicating a neutralizing effect. In light of the anti-inflammatory effects of progranulin, progranulin antibodies might exert pro-inflammatory effects thus contributing to the pathogenesis of the respective autoimmune diseases and might serve as a marker for disease activity. This hypothesis is supported by the fact that a positive progranulin antibody status was associated with active disease in granulomatosis with polyangiitis. PMID:23149338
Thurner, Lorenz; Preuss, Klaus-Dieter; Fadle, Natalie; Regitz, Evi; Klemm, Philipp; Zaks, Marina; Kemele, Maria; Hasenfus, Andrea; Csernok, Elena; Gross, Wolfgang L; Pasquali, Jean-Louis; Martin, Thierry; Bohle, Rainer Maria; Pfreundschuh, Michael
Nanobodies - the new concept in antibody engineering
Nanobodies are antibody-derived therapeutic proteins that contain the unique structural and functional properties of naturally occurring heavy-chain antibodies. The Nanobody technology was originally developed following the discovery that camelidae (camels and llamas) possess fully functional antibodies that lack light chains. These heavy-chain antibodies contain a single variable domain (VHH) and two constant domains (CH2 and CH3). Importantly, the cloned and
Khalissa Deffar; Hengliang Shi; Liang Li; Xingzhi Wang
Ocular symptoms in association with antiphospholipid antibodies
· Objective: To describe the clinical and serologic findings of 50 antiphospholipid antibody (APA)-positive patients within\\u000a a retrospective study.?· Methods: Measurement of visual acuity, slit-lamp biomicroscopy, tonometry, fundus examination and\\u000a perimetry. Laboratory tests were performed for detection of APA against thromboplastin and cardiolipin. Antinuclear antibodies\\u000a (ANA), antibodies to dsDNA, antithyroidal and antiparietal antibodies were also tested.?· Results: A combination of
Beate Leo-Kottler; Reinhild Klein; Peter A. Berg; Eberhart Zrenner
Development and characterization of antibodies against aflatoxins
A panel of ten monoclonal antibodies against aflatoxins B1, B2, and G2 was produced and comprehensively characterized. The\\u000a affinity and cross reactivity of these antibodies were determined using the methods of direct, indirect, and competitive ELISA.\\u000a The structures of monoclonal antibody genes were comprehensively studied and the variable and constant regions of the antibody\\u000a genes were cloned and sequenced. Sequencing
A. A. Kalinichenko; V. A. Toporova; A. A. Panina; T. K. Aliev; E. A. Kryukova; O. B. Shemchukova; O. N. Solopova; L. P. Pozdnyakova; P. G. Sveshnikov; D. A. Dolgikh; M. P. Kirpichnikov
Mathematical and Experimental Analyses of Antibody Transport in Hollow-Fiber-Based Specific Antibody Filters
We are developing hollow fiber-based specific antibody filters (SAFs) that selectively remove antibodies of a given specificity directly from whole blood, without separation of the plasma and cellular blood components and with minimal removal of plasma proteins other than the targeted pathogenic antibodies. A principal goal of our research is to identify the primary mechanisms that control antibody transport within
Mariah S. Hout; William J. Federspiel
Therapeutic antibodies for autoimmunity and inflammation
The development of therapeutic antibodies has evolved over the past decade into a mainstay of therapeutic options for patients with autoimmune and inflammatory diseases. Substantial advances in understanding the biology of human diseases have been made and tremendous benefit to patients has been gained with the first generation of therapeutic antibodies. The lessons learnt from these antibodies have provided the
Andrew C. Chan; Paul J. Carter
Recombinant antibody constructs in cancer therapy
Recombinant antibodies and their fragments now represent over 30% of all biological proteins undergoing clinical trials for diagnosis and therapy. The focus on antibodies as the ideal cancer-targeting reagents recently culminated in approval by the Food and Drugs Administration for the first engineered therapeutic antibodies. In the past year, important advances have been made in the design, selection and production
Peter J Hudson
Techniques to quantify TSH receptor antibodies
The presence of antibodies to TSH receptor (TSHR) is the hallmark of Graves disease (GD). These antibodies mimic the action of TSH, resulting in TSHR stimulation and hyperthyroidism, and have been associated with GD-associated extrathyroidal manifestations. TSH binding inhibition assays and bioassays for measurement of TSHR antibody levels have been used for clinical and research purposes. In the former, inhibition
AP Weetman; RA Ajjan
The role of antibodies in myasthenia gravis
Myasthenia gravis is an autoimmune disease associated with antibodies directed to the postsynaptic acetylcholine receptor. These antibodies reduce the number of receptors. Autoantibodies against AChR and other muscle antigens can be used for the diagnosis of myasthenia gravis and related disorders. The origin and the role of these antibodies in the disease are discussed.Experimental autoimmune myasthenia gravis, an experimental model
M De Baets; M. H. W Stassen
This book contains 8 chapters. Some of the titles are: Anti-DNA Antibodies in SLE: Historical Perspective; Specificity of Anti-DNA Antibodies in Systemic Lupus Erythematosus; Monoclonial Autoimmune Anti-DNA Antibodies; and Structure--Function Analyses of Anti-DNA Autoantibodies.
Voss, E.W.
Antibody pretargeting advances cancer radioimmunodetection and radioimmunotherapy.
This article reviews the methods of pretargeting, which involve separating the targeting antibody from the subsequent delivery of an imaging or therapeutic agent that binds to the tumor-localized antibody. This provides enhanced tumor:background ratios and the delivery of a higher therapeutic dose than when antibodies are directly conjugated with radionuclides, as currently practiced in cancer radioimmunotherapy. We describe initial promising clinical results using streptavidin-antibody constructs with biotin-radionuclide conjugates in the treatment of patients with malignant gliomas, and of bispecific antibodies with hapten-radionuclides in the therapy of tumors expressing carcinoembryonic antigen, such as medullary thyroid and small-cell lung cancers. PMID:16380412
Goldenberg, David M; Sharkey, Robert M; Paganelli, Giovanni; Barbet, Jacques; Chatal, Jean-François
Expression studies of catalytic antibodies
We have examined the positive influence of human constant regions on the folding and bacterial expression of active soluble mouse immunoglobulin variable domains derived form a number of catalytic antibodies. Expression yields of eight hybridoma-and myeloma-derived chimeric Fab fragments are compared in both shake flasks and high-density fermentation. In addition the usefulness of this system for the generation of in vivo expression libraries is examined by constructing and expressing combinations of heavy and light chain variable regions that were not selected as a pair during an immune response. A mutagenesis study of one of the recombinant catalytic Fab fragments reveals that single amino acid substitutions can have dramatic effects on the expression yield. This system should be generally applicable to the production of Fab fragments of catalytic and other hybridoma-derived antibodies for crystallographic and structure-function studies. 41 refs., 4 figs., 1 tab.
Ulrich, H.D.; Patten, P.A.; Yang, P.L. [Univ. of California, Berkeley, CA (United States)] [and others
The antineutrophil antibody in uveitis.
Ninety eight patients with uveitis of various types were tested for the presence of the antineutrophil antibody or ANCA by an indirect immunofluorescence method. This antibody is found in patients with diseases associated with small vessel vasculitis, including Wegener's granulomatosis and microscopic polyarteritis. Eleven true positive cases were found. A positive test was not associated with the anatomical site of the uveitis but was related to the time course of the disease. In particular single/repeated rather than nonrecurrent uveitis was associated with a positive test. Three groups of patients seem more likely to have positive tests: those with bilateral chronic posterior uveitis; a group with an anterior uveitis which is single/repeated who were younger than 46 at disease onset and had isolated eye disease; and an older group, aged 60 years or more at onset, with single/repeated uveitis and systemic features.
Young, D W
Antibody buffering of a ligand in vivo
NASA Astrophysics Data System (ADS)
Clearance is the practical limit on drug action. Here we propose a means of slowing clearance, thereby extending drug lifetime in vivo by "antibody buffering." In this process, a drug and an anti-drug antibody are coadministered. Most of the drug is bound to the antibody, preventing the drug from acting, but also preventing its elimination. A dynamic free drug pool is established by reversible dissociation from the antibody. The free drug is active and can be eliminated, but the free pool is constantly replenished by reequilibration from the antibody-drug complex, giving a long effective lifetime. Here we explore antibody buffering experimentally by using a model compound, 2-phenyloxazol-5-one--aminobutyrate (Ox), as a drug proxy. We show that antibody buffering can extend by an order of magnitude the plasma lifetime of Ox in rats, and that the steady-state Ox level depends on the molecular properties of the antibody used to buffer the Ox. In addition, the anti-Ox antibody can be recharged with drug in vivo to extend Ox lifetime without additional antibody administration, making this technique even more suitable for possible clinical application. drug delivery | pharmacokinetics | immunotherapy | antibody-antigen interactions
O'Hear, Carol E.; Foote, Jefferson
Toward aggregation-resistant antibodies by design.
Monoclonal antibodies are attractive therapeutics for treating a wide range of human disorders due to their exquisite binding specificity and high binding affinity. However, a limitation of antibodies is their highly variable and difficult-to-predict propensities to aggregate when concentrated during purification and delivery. Despite the large size and complex structure of antibodies, recent findings suggest that antibody solubility can be dramatically improved using rational design methods in addition to conventional selection methods. Here, we review key advances and unmet challenges in engineering the variable and constant regions of antibody fragments and full-length antibodies to resist aggregation without reducing their binding affinity. These experimental and computational discoveries should accelerate the development of robust algorithms for designing aggregation-resistant antibodies. PMID:23932102
Lee, Christine C; Perchiacca, Joseph M; Tessier, Peter M
Solubility evaluation of murine hybridoma antibodies
The successful development of antibody therapeutics depends on the molecules having properties that are suitable for manufacturing, as well as use by patients. Because high solubility is a desirable property for antibodies, screening for solubility has become an essential step during the early candidate selection process. In considering the screening process, we formed a hypothesis that hybridoma antibodies are filtered by nature to possess high solubility and tested this hypothesis using a large number of murine hybridoma-derived antibodies. Using the cross-interaction chromatography (CIC) method, we screened the solubility of 92 murine hybridoma-derived monoclonal antibodies and found that all of these molecules exhibited CIC profiles that are indicative of high solubility (>100mg/mL). Further investigations revealed that variable region N-linked glycosylation or isoelectric parameters are unlikely to contribute to the high solubility of these antibodies. These results support the general hypothesis that hybridoma monoclonal antibodies are highly soluble.
Spencer, Stacey; Bethea, Deidra; Raju, T. Shantha; Giles-Komar, Jill; Feng, Yiqing
The Role of Antibodies in Transplantation
For the past forty years T-cells have been considered the primary threat to the survival of allografts. However antibodies can induce severe vascular disease of organ transplants and this disease, particularly "antibody-mediated" rejection, has become a major clinical challenge. Not only do antibodies cause rejection, the rejection caused by antibodies resists treatment by conventional drug regimens. On the other hand, antibodies can induce a condition in which grafts seemingly resist antibody-mediated injury, which is accommodation. In this communication we discuss the role of antibodies in the diagnosis and pathogenesis of rejection and accommodation, and suggest what we consider the major gaps in knowledge and directions research into this subject might productively take.
Chang, Alexander T.; Platt, Jeffrey L.
Heterophilic antibody interference in immunometric assays.
Immunometric assays are inherently vulnerable to interference from heterophilic antibodies, endogenous antibodies that bind assay antibodies. The consequences of such interference can be devastating. In this review, we discuss strategies that reduce the damage caused by heterophilic antibodies. Clinicians should only order blood tests that are indicated for the patient and clinical setting at hand, and have the confidence to question laboratory results discordant with the clinical picture. Laboratorians should familiarize themselves with the vulnerability of the assays they offer, and be able to perform and interpret adequate confirmatory measures correctly. When designing immunoassays, the immunoassay industry should invest the necessary resources in specific protective measures against heterophilic antibody interference. Examples include using antibody fragments and the addition of effective blockers to assay reagents. The increasing use of modified monoclonal mouse antibodies both in therapy and diagnostics could present a particular challenge in the future. PMID:24094636
Bolstad, Nils; Warren, David J; Nustad, Kjell
Antibodies to HBIMF63 polypeptide
US Patent & Trademark Office Database
The present invention relates to novel human secreted proteins and isolated nucleic acids containing the coding regions of the genes encoding such proteins. Also provided are vectors, host cells, antibodies, and recombinant methods for producing human secreted proteins. The invention further relates to diagnostic and therapeutic methods useful for diagnosing and treating diseases, disorders, and/or conditions related to these novel human secreted proteins.
Bispecific Antibodies for Diagnostic Applications
\\u000a Bispecific monoclonal antibodies (BsMAb) are unique engineered macromolecules that have two different pre-determined binding\\u000a specificities. Their ability to simultaneously bind to a specific antigen and a given detection moiety enables them to function\\u000a as excellent bifunctional immunoprobes in diagnostic assays. BsMAb are being exploited for the development of simple, rapid,\\u000a and highly sensitive immunoassays for diagnosis of bacterial and viral
Archana Parashar; Susmita Sarkar; Advaita Ganguly; Sai Kiran Sharma; Mavanur R. Suresh
Antibodies to HWHGU54 polypeptides
US Patent & Trademark Office Database
The present invention relates to novel human secreted proteins and isolated nucleic acids containing the coding regions of the genes encoding such proteins. Also provided are vectors, host cells, antibodies, and recombinant methods for producing human secreted proteins. The invention further relates to diagnostic and therapeutic methods useful for diagnosing and treating diseases, disorders, and/or conditions related to these novel human secreted proteins.
Researchers at Pacific Northwest National Laboratory (PNNL) have constructed a nonimmune library consisting of 109 human antibody scFv fragments, which have been cloned and expressed on the surface of yeast. Nanomolar-affinity scFvs are routinely obtained by magnetic bead screening and flow cytometric sorting. The yeast library can be amplified 1010 fold without measurable loss of clonal diversity. This allows for indefinite expansion of the library. All scFv clones can be assessed directly on the yeast cell surface by immunofluorescent labeling and flow cytometry, obviating separate subcloning, expression, and purification steps. The ability to use multiplex library screening demonstrates the utility of this approach for high-throughput antibody isolation for proteomic applications. The yeast library may be used for research projects or teaching performed for U.S. Government purposes only. If you would like to request an aliquot of the single-chain antibody library for your research, please print and fill out the Materials Transfer Agreement (MTA) [PDF, 20K]. The website provides the contact information for mailing the MTA. [copied from http://www.sysbio.org/dataresources/singlechain.stm
Baird, Cheryl
Antibodies targeting cancer stem cells
Antibody targeting of cancer is showing clinical and commercial success after much intense research and development over the last 30 years. They still have the potential to delivery long-term cures but a shift in thinking towards a cancer stem cell (CSC) model for tumor development is certain to impact on how antibodies are selected and developed, the targets they bind to and the drugs used in combination with them. CSCs have been identified from many human tumors and share many of the characteristics of normal stem cells. The ability to renew, metabolically or physically protect themselves from xenobiotics and DNA damage and the range of locomotory-related receptors expressed could explain the observations of drug resistance and radiation insensitivity leading to metastasis and patient relapse. Targeting CSCs could be a strategy to improve the outcome of cancer therapy but this is not as simple as it seems. Targets such as CD133 and EpCAM/ESA could mark out CSCs from normal cells enabling specific intervention but indirect strategies such as interfering with the establishment of a supportive niche through anti-angiogenic or anti-stroma therapy could be more effective. This review will outline the recent discoveries for CSCs across the major tumor types highlighting the possible molecules for intervention. Examples of antibody-directed CSC therapies and the outlook for the future development of this emerging area will be given.
Kousparou, Christina A; Epenetos, Agamemnon A
Antiphospholipid antibody testing and standardization.
The laboratory criteria that define patients with antiphospholipid syndrome (APS) include lupus anticoagulant (LAC), anticardiolipin antibodies and anti-?2 glycoprotein I antibodies (a?2GPI). All assays show methodological shortcomings and the combination of the three tests, each with different sensitivity and specificity, and hence, differences in clinical utility make the laboratory diagnosis of APS challenging. Consensus guidelines and proposals for antiphospholipid antibodies (aPL) testing have been published in the last 20 years and have led to a substantial improvement. Despite efforts so far, standardization is not reached yet, but progress has been made. On-going efforts to reduce the interlaboratory/interassay variations remain important; even an absolute standardization cannot be feasibly achieved. Taking into account the methodological shortcomings of the means we have available, more detailed guidelines may help in adequate performance of aPL testing. This review will focus on the efforts and achievements in standardization and on the weaknesses and strengths of the current available laboratory methods. PMID:24750682
Devreese, K M J
Improved monoclonal antibodies to halodeoxyuridine
The development, method of production, characterization and methods of use of two hybridomas, CIdU-1 (ATCC Accession No. HB-8321) and CIdU-2 (ATCC Accession No. HB-8320), are described. These secrete IgG/sub 1/(K) immunoglobulins that react with halodeoxyuridine (HdU or halodU) such as bromo, chloro, fluoro and iodo deoxyuridine (BrdU, CldU, FdU and IdU), whether these are free in solution or incorporated into single stranded DNA in whole cells. The antibodies do not react with naturally occurring free nucleic acids or with deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) polymers. These antibodies are suitable for use in enzyme immunoassays for free CldU, FdU, IdU and BrdU and for detecting cells with these nucleotides incorporated into them. The monoclonal antibodies are useful in the detection of the sensitivity of tumor cells to specific chemotherapeutic agents, in the measurement of the rate of cellular DNA synthesis, in the measurement of the rate of proliferation of normal and malignant cells and in the detection of HPRT deficiency in cells. 1 tab.
Vanderlaan, M.; Dolbeare, F.A.; Gray, J.W.; Thomas, C.B.
Selective enrichment of monospecific polyclonal antibodies for antibody-based proteomics efforts.
A high stringency protocol, suitable for systematic purification of polyclonal antibodies, is described. The procedure is designed to allow the generation of target protein-specific antibodies suitable for functional annotation of proteins. Antibodies were generated by immunization with recombinantly produced affinity-tagged target proteins. To obtain stringent recovery of the antibodies, a two-step affinity chromatography principle was devised to first deplete the affinity tag-specific antibodies followed by a second step for affinity capture of the target protein-specific antibodies. An analytical dot-blot array system was developed to analyze the cross-reactivity of the affinity-purified antibodies. The results suggest that the protocol can be used in a highly parallel and automated manner to generate mono-specific polyclonal antibodies for large-scale, antibody-based proteomics efforts, i.e. affinity proteomics. PMID:15317410
Agaton, Charlotta; Falk, Ronny; Höidén Guthenberg, Ingmarie; Göstring, Lovisa; Uhlén, Mathias; Hober, Sophia
Generation of monospecific antibodies based on affinity capture of polyclonal antibodies
A method is described to generate and validate antibodies based on mapping the linear epitopes of a polyclonal antibody followed by sequential epitope-specific capture using synthetic peptides. Polyclonal antibodies directed towards four proteins RBM3, SATB2, ANLN, and CNDP1, potentially involved in human cancers, were selected and antibodies to several non-overlapping epitopes were generated and subsequently validated by Western blot, immunohistochemistry, and immunofluorescence. For all four proteins, a dramatic difference in functionality could be observed for these monospecific antibodies directed to the different epitopes. In each case, at least one antibody was obtained with full functionality across all applications, while other epitope-specific fractions showed no or little functionality. These results present a path forward to use the mapped binding sites of polyclonal antibodies to generate epitope-specific antibodies, providing an attractive approach for large-scale efforts to characterize the human proteome by antibodies.
Hjelm, Barbara; Forsstrom, Bjorn; Igel, Ulrika; Johannesson, Henrik; Stadler, Charlotte; Lundberg, Emma; Ponten, Fredrik; Sjoberg, Anna; Rockberg, Johan; Schwenk, Jochen M; Nilsson, Peter; Johansson, Christine; Uhlen, Mathias
Generation of monospecific antibodies based on affinity capture of polyclonal antibodies.
A method is described to generate and validate antibodies based on mapping the linear epitopes of a polyclonal antibody followed by sequential epitope-specific capture using synthetic peptides. Polyclonal antibodies directed towards four proteins RBM3, SATB2, ANLN, and CNDP1, potentially involved in human cancers, were selected and antibodies to several non-overlapping epitopes were generated and subsequently validated by Western blot, immunohistochemistry, and immunofluorescence. For all four proteins, a dramatic difference in functionality could be observed for these monospecific antibodies directed to the different epitopes. In each case, at least one antibody was obtained with full functionality across all applications, while other epitope-specific fractions showed no or little functionality. These results present a path forward to use the mapped binding sites of polyclonal antibodies to generate epitope-specific antibodies, providing an attractive approach for large-scale efforts to characterize the human proteome by antibodies. PMID:21898641
Hjelm, Barbara; Forsström, Björn; Igel, Ulrika; Johannesson, Henrik; Stadler, Charlotte; Lundberg, Emma; Ponten, Fredrik; Sjöberg, Anna; Rockberg, Johan; Schwenk, Jochen M; Nilsson, Peter; Johansson, Christine; Uhlén, Mathias
Distinction between MOG antibody-positive and AQP4 antibody-positive NMO spectrum disorders
Objective: To evaluate clinical features among patients with neuromyelitis optica spectrum disorders (NMOSD) who have myelin oligodendrocyte glycoprotein (MOG) antibodies, aquaporin-4 (AQP4) antibodies, or seronegativity for both antibodies. Methods: Sera from patients diagnosed with NMOSD in 1 of 3 centers (2 sites in Brazil and 1 site in Japan) were tested for MOG and AQP4 antibodies using cell-based assays with live transfected cells. Results: Among the 215 patients with NMOSD, 7.4% (16/215) were positive for MOG antibodies and 64.7% (139/215) were positive for AQP4 antibodies. No patients were positive for both antibodies. Patients with MOG antibodies represented 21.1% (16/76) of the patients negative for AQP4 antibodies. Compared with patients with AQP4 antibodies or patients who were seronegative, patients with MOG antibodies were more frequently male, had a more restricted phenotype (optic nerve more than spinal cord), more frequently had bilateral simultaneous optic neuritis, more often had a single attack, had spinal cord lesions distributed in the lower portion of the spinal cord, and usually demonstrated better functional recovery after an attack. Conclusions: Patients with NMOSD with MOG antibodies have distinct clinical features, fewer attacks, and better recovery than patients with AQP4 antibodies or patients seronegative for both antibodies.
Sato, Douglas Kazutoshi; Callegaro, Dagoberto; Lana-Peixoto, Marco Aurelio; Waters, Patrick J.; Jorge, Frederico M. de Haidar; Takahashi, Toshiyuki; Nakashima, Ichiro; Apostolos-Pereira, Samira Luisa; Talim, Natalia; Simm, Renata Faria; Lino, Angelina Maria Martins; Misu, Tatsuro; Leite, Maria Isabel; Aoki, Masashi
Radioimmunotherapy of malignancy using antibody targeted radionuclides.
Antibodies directed against tumour associated antigens provide a means for delivering preferentially cytotoxic radionuclides to the cells of primary and secondary tumours. The factors that influence the effectiveness of the radiation in the tumour compared with its effect on the radiosensitive normal tissues include the specificity of the antibody, the distribution of targeted energy within the tumour and the host's response to the injected foreign antibody. Recently some encouraging results from clinical trials of radioimmunotherapy have been reported in the literature. There is a continual search for more avid and specific antibodies, and the techniques of genetic engineering are being applied to the problem of reducing the antigenicity and mass of the carrier antibody. The improved efficiency of the labelled antibody needs to be supplemented by an identification of those tumours most likely to respond to this form of therapy.
Cobb, L. M.; Humm, J. L.
Phase Separation in Solutions of Monoclonal Antibodies
NASA Astrophysics Data System (ADS)
We report the observation of liquid-liquid phase separation (LLPS) in a solution of humanized monoclonal antibodies, IgG2, and the effects of human serum albumin, a major blood protein, on this phase separation. We find a significant reduction of phase separation temperature in the presence of albumin, and a preferential partitioning of the albumin into the antibody-rich phase. We provide a general thermodynamic analysis of the antibody-albumin mixture phase diagram and relate its features to the magnitude of the effective inter-protein interactions. Our analysis suggests that additives (HSA in this report), which have moderate attraction with antibody molecules, may be used to forestall undesirable protein condensation in antibody solutions. Our findings are relevant to understanding the stability of pharmaceutical solutions of antibodies and the mechanisms of cryoglobulinemia.
Benedek, George; Wang, Ying; Lomakin, Aleksey; Latypov, Ramil
Monoclonal Antibody Therapy for Cancer
\\u000a Since the approval of rituximab (Rituxan®) for the treatment of B-cell non-Hodgkin's lymphoma (B-NHL) in 1997, nine additional monoclonal antibodies (mAbs) have been\\u000a approved by the FDA for cancer therapy. Currently, more than 1,300 clinical studies registered at ClinicalTrials.gov investigate\\u000a mAb therapy of cancer, including more than 150 phase III clinical trials. In concert with their clinical acceptance, mAbs\\u000a in
Christoph Rader
Antibody engineering and therapeutics, The Annual Meeting of the Antibody Society: December 8-12, 2013, Huntington Beach, CA.
The 24th Antibody Engineering & Therapeutics meeting brought together a broad range of participants who were updated on the latest advances in antibody research and development. Organized by IBC Life Sciences, the gathering is the annual meeting of The Antibody Society, which serves as the scientific sponsor. Preconference workshops on 3D modeling and delineation of clonal lineages were featured, and the conference included sessions on a wide variety of topics relevant to researchers, including systems biology; antibody deep sequencing and repertoires; the effects of antibody gene variation and usage on antibody response; directed evolution; knowledge-based design; antibodies in a complex environment; polyreactive antibodies and polyspecificity; the interface between antibody therapy and cellular immunity in cancer; antibodies in cardiometabolic medicine; antibody pharmacokinetics, distribution and off-target toxicity; optimizing antibody formats for immunotherapy; polyclonals, oligoclonals and bispecifics; antibody discovery platforms; and antibody-drug conjugates. PMID:24589717
Almagro, Juan Carlos; Gilliland, Gary L; Breden, Felix; Scott, Jamie K; Sok, Devin; Pauthner, Matthias; Reichert, Janice M; Helguera, Gustavo; Andrabi, Raiees; Mabry, Robert; Bléry, Mathieu; Voss, James E; Laurén, Juha; Abuqayyas, Lubna; Barghorn, Stefan; Ben-Jacob, Eshel; Crowe, James E; Huston, James S; Johnston, Stephen Albert; Krauland, Eric; Lund-Johansen, Fridtjof; Marasco, Wayne A; Parren, Paul W H I; Xu, Kai Y
In vitro Reaction of Antibodies to Ragweed
Immunoglobulin E-rich fractions and IgE-depleted fractions were prepared from individual and pooled sera of patients allergic to ragweed. The IgE-depleted fractions contained antibodies to ragweed associated with the immunoglobulins G, A and M. The IgE-rich fraction contained only IgE antibodies to ragweed, although other immunoglobulins were present in this fraction. The amount of antibodies in these fractions was determined by
Yosuke Fujita; Konrad Wicher; John I. Wypych; Robert E. Reisman; Carl E. Arbesman
The Human Antibody Response Against WNV
\\u000a Experimental evidence has shown that antibody responses to West Nile virus (WNV) are critical for protection from WNV-mediated\\u000a disease. Antibody responses are also an important immune correlate of protection for the clinical evaluation of WNV vaccines.\\u000a However, little direct study has been carried out on the characteristics of the human antibody response to natural WNV infection.\\u000a Preliminary evidence suggests that
Mark Throsby; Jaap Goudsmit; John de Kruif
Antibody-Based Therapies for Solid Tumors
\\u000a Monoclonal antibodies have heralded a new era in cancer therapeutics, adding to standard cytotoxic chemotherapy without excess\\u000a side effects. Bevacizumab, an anti VEGF antibody, has shown substantial benefit in metastatic colorectal, breast and non-small-cell\\u000a lung cancer. The HER2 receptor antibody trastuzumab has improved survival in both the adjuvant and metastatic setting in HER2-overexpressing\\u000a breast cancer. The mechanisms of action, side
Satish Shanbhag; Barbara Burtness
Monoclonal Antibodies in Paediatric Acute Lymphoblastic Leukemia
\\u000a Development of monoclonal antibodies (moAbs) for treatment of haematological malignancies is a rapidly growing field. Whereas\\u000a unconjugated humanized antibodies are well tolerated and may be easily combined with chemotherapy, immunoconjugates delivering\\u000a toxic compounds right into the target cells exhibit more severe side effects. Highly and selectively expressed antigens are\\u000a ideal targets for antibody treatment and suitable for broad development in
Arend von Stackelberg
Immunoassays, Assay Methods, Antibodies and Method of Creating Antibodies for Detecting FGF-23.
National Technical Information Service (NTIS)
Immunoassays, assay methods, antibodies and methods of producing antibodies for the detection of fibroblast growth factor-23 (FGF-23). The immunoassay and assay method preferably comprise a non-competitive, sandwich-type assay utilizing a first bound anti...
H. Jueppner J. Lavigne R. Zahradnik
5th Annual Monoclonal Antibodies Conference
The conference, which was organized by Visiongain and held at the BSG Conference Center in London, provided an excellent opportunity for participants to exchange views on the development, production and marketing of therapeutic antibodies, and discuss the current business environment. The conference included numerous interactive panel and group discussions on topics such as isotyping for therapeutic antibodies (panel chair: Nick Pullen, Pfizer), prospects for fully human monoclonal antibodies (chair: Christian Rohlff, Oxford BioTherapeutics), perspectives on antibody manufacturing and development (chair: Bo Kara, Avecia), market impact and post-marketing issues (chair: Keith Rodgers, Bodiam Consulting) and angiogenesis inhibitors (chair: David Blakey, AstraZeneca).
Uses of monoclonal antibody 8H9
This invention provides a composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a suitable carrier. This invention provides a pharmaceutical composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a pharmaceutically acceptable carrier. This invention also provides an antibody other than the monoclonal antibody 8H9 comprising the complementary determining regions of monoclonal antibody 8H9 or a derivative thereof, capable of binding to the same antigen as the monoclonal antibody 8H9. This invention provides a substance capable of competitively inhibiting the binding of monoclonal antibody 8H9. This invention also provides an isolated scFv of monoclonal antibody 8H9 or a derivative thereof. This invention also provides the 8H9 antigen. This invention also provides a method of inhibiting the growth of tumor cells comprising contacting said tumor cells with an appropriate amount of monoclonal antibody 8H9 or a derivative thereof.
Cheung, Nai-Kong V
[Managing patients with therapeutic antibodies in odontostomatology].
Immunotherapies, particularly therapeutic antibodies, are increasingly used in the treatment of many autoimmune or oncological diseases. Patients treated with therapeutic antibodies may present with an increased risk of infection or of osteonecrosis of the jaws (ONJ). There is currently no consensus on the management of patients treated with therapeutic antibodies. These treatments are mainly used in hospitals, but they have been increasingly prescribed in ambulatory treatment for patients undergoing oral care. It is therefore important to establish therapeutic precautions for these patients. We had for aim to describe these antibody therapies, their indications, their potentially adverse effects in the oral cavity and to review the latest recommendations. PMID:24797731
Demoersman, J; Soueidan, A; Corre, P; Pers, J O
Anti-myeloperoxidase antibodies in systemic vasculitis.
Anti-neutrophil cytoplasmic antibodies (ANCA) are important diagnostic markers in vasculitic disorders. In a study of 164 patients with various clinical syndromes associated with vasculitis, 60 were found to have the antibody, as detected by indirect immunofluorescence; 23 had the so-called perinuclear antibody pattern, and of these, 15 had antibodies to neutrophil myeloperoxidase in an ELISA. These patients had multi-system involvement, and 14 of the 15 had histological evidence of small-vessel arteritis in the kidneys. In preliminary experiments, the isolated IgG from one patient was shown to inhibit luminol-dependent chemiluminescence of fluid phase myeloperoxidase. Images Fig. 3 Fig. 4
Lee, S S; Adu, D; Thompson, R A
Cancer therapy with bispecific antibodies: Clinical experience
The binding of at least two molecular targets simultaneously with a single bispecific antibody is an attractive concept. The use of bispecific antibodies as possible therapeutic agents for cancer treatment was proposed in the mid-1980s. The design and production of bispecific antibodies using antibody- and/or receptor-based platform technology has improved significantly with advances in the knowledge of molecular manipulations, protein engineering techniques, and the expression of antigens and receptors on healthy and malignant cells. The common strategy for making bispecific antibodies involves combining the variable domains of the desired mAbs into a single bispecific structure. Many different formats of bispecific antibodies have been generated within the research field of bispecific immunotherapeutics, including the chemical heteroconjugation of two complete molecules or fragments of mAbs, quadromas, F(ab')2, diabodies, tandem diabodies and single-chain antibodies. This review describes key modifications in the development of bispecific antibodies that can improve their efficacy and stability, and provides a clinical perspective on the application of bispecific antibodies for the treatment of solid and liquid tumors, including the promises and research limitations of this approach.
Thakur, Archana; Lum, Lawrence G
Monoclonal Antibody That Defines Human Myoepithelium
NASA Astrophysics Data System (ADS)
We have isolated a mouse monoclonal antibody that, upon immunohistochemical localization in frozen sections, displays specificity for human myoepithelial cells in the resting mammary gland, sweat glands, and salivary glands. Furthermore, this antibody was strongly and homogeneously reactive with frozen sections of 3 of 60 breast carcinoma specimens. Using immunolocalization techniques in conjunction with polyacrylamide gel electrophoresis, we have determined that the reactivity of this monoclonal antibody is directed toward a 51,000-dalton keratin polypeptide. The potential uses of this antibody in the prognosis of human mammary carcinoma and in understanding the role of the myoepithelium in development and differentiation are discussed.
Dairkee, Shahnaz Hashmi; Blayney, Carlene; Smith, Helene S.; Hackett, Adeline J.
Vibriobactin Antibodies: A Vaccine Strategy
A new target strategy in the development of bacterial vaccines, the induction of antibodies to microbial outer membrane ferrisiderophore complexes, is explored. A vibriobactin (VIB) analogue, with a thiol tether, 1-(2,3-dihydroxybenzoyl)-5,9-bis[[(4S,5R)-2-(2,3-dihydroxyphenyl)-4,5-dihydro-5-methyl-4-oxazolyl]carbonyl]-14-(3-mercaptopropanoyl)-1,5,9,14-tetraazatetradecane, was synthesized and linked to ovalbumin (OVA) and bovine serum albumin (BSA). The antigenicity of the VIB microbial iron chelator conjugates and their iron complexes was evaluated. When mice were immunized with the resulting OVA-VIB conjugate, a selective and unequivocal antigenic response to the VIB hapten was observed; IgG monoclonal antibodies specific to the vibriobactin fragment of the BSA and OVA conjugates were isolated. The results are consistent with the idea that the isolated adducts of siderophores covalently linked to their bacterial outer membrane receptors represent a credible target for vaccine development.
Bergeron, Raymond J.; Bharti, Neelam; Singh, Shailendra; McManis, James S.; Wiegand, Jan; Green, Linda G.
Selective enrichment of monospecific polyclonal antibodies for antibody-based proteomics efforts
A high stringency protocol, suitable for systematic purification of polyclonal antibodies, is described. The procedure is designed to allow the generation of target protein-specific antibodies suitable for functional annotation of proteins. Antibodies were generated by immunization with recombinantly produced affinity-tagged target proteins. To obtain stringent recovery of the antibodies, a two-step affinity chromatography principle was devised to first deplete the
Charlotta Agaton; Ronny Falk; Ingmarie Höidén Guthenberg; Lovisa Göstring; Mathias Uhlén; Sophia Hober
EFFECT OF COLCHICINE ON THE ANTIBODY RESPONSE I. Enhancement of Antibody Formation in Mice
In 1954, some experiments were carried out in this laboratory on the effect ofcolchicine (CC) 1 on antibody formation in rabbits. At that time, we had found that antibody formation was associated with rapid and extensive cell divisions in the plasma cell and its immediate precursors which had just been associated with antibody synthesis (1). We thought, therefore, that the
The production of antibody fragments and antibody fusion proteins by yeasts and filamentous fungi
In this review we will focus on the current status and views concerning the production of antibody fragments and antibody fusion proteins by yeasts and filamentous fungi. We will focus on single-chain antibody fragment production (scFv and VHH) by these lower eukaryotes and the possible applications of these proteins. Also the coupling of fragments to relevant enzymes or other components
Vivi Joosten; Christien Lokman; Cees AMJJ van den Hondel; Peter J Punt
An indirect hemagglutination antibody test to detect antibodies to Borrelia burgdorferi in patients with Lyme disease
An indirect hemagglutination antibody (IHA) test was evaluated for its ability to detect borrelial antibodies in serum samples from patients with Lyme disease. The key test reagent developed for this antibody detection system was tannic acid-treated and glutaraldehyde-fixed sheep red blood cells (SRBC) containing Borrelia burgdorferi (Bb) antigens attached to the outer surface of the SRBC. In order to establish
Charles S Pavia; Gary P Wormser; Susan Bittker; Denise Cooper
Nanopore-based kinetics analysis of individual antibody-channel and antibody-antigen interactions
BACKGROUND: The UNO\\/RIC Nanopore Detector provides a new way to study the binding and conformational changes of individual antibodies. Many critical questions regarding antibody function are still unresolved, questions that can be approached in a new way with the nanopore detector. RESULTS: We present evidence that different forms of channel blockade can be associated with the same antibody, we associate
Stephen Winters-hilt; Eric Morales; Iftekhar Amin; Alexander Stoyanov
Antibody humanization methods for development of therapeutic applications.
Recombinant antibody technologies are rapidly becoming available and showing considerable clinical success. However, the immunogenicity of murine-derived monoclonal antibodies is restrictive in cancer immunotherapy. Humanized antibodies can overcome these problems and are considered to be a promising alternative therapeutic agent. There are several approaches for antibody humanization. In this article we review various methods used in the antibody humanization process. PMID:24746146
Ahmadzadeh, Vahideh; Farajnia, Safar; Feizi, Mohammad Ali Hosseinpour; Nejad, Ramezan Ali Khavari
Optimized monoclonal antibodies against tissue factor pathway inhibitor (TFPI)
US Patent & Trademark Office Database
Isolated monoclonal antibodies that bind human tissue factor pathway inhibitor (TFPI) are provided. Isolated nucleic acid molecules encoding monoclonal antibodies that bind TFPI are also contemplated. Pharmaceutical compositions comprising the anti-TFPI monoclonal antibodies and methods of treating deficiencies or defects in coagulation by administration of the antibodies are also provided. Methods of producing the antibodies are also provided.
Engineered antibody fragments and the rise of single domains
With 18 monoclonal antibody (mAb) products currently on the market and more than 100 in clinical trials, it is clear that engineered antibodies have come of age as biopharmaceuticals. In fact, by 2008, engineered antibodies are predicted to account for >30% of all revenues in the biotechnology market. Smaller recombinant antibody fragments (for example, classic monovalent antibody fragments (Fab, scFv))
Philipp Holliger; Peter J Hudson
The antibody paradox: Trying on a pair of genes
Rodney Porter's separation of antibody molecules into Fab and Fc fragments engendered the notion that a single antibody polypeptide chain might be coded by two or more genes. This concept profoundly influenced the development of molecular immunology over the past 25 years. Our current knowledge of antibody gene organization has enabled investigators to recombine antibody genes to create "chimeric" antibodies
Julian B. Fleischman
Induction and detection of antibodies to squalene.
An enzyme-linked immunosorbent assay (ELISA) utilizing antigen coated on hydrophobic polyvinyldiene fluoride (PVDF) membranes is described for detecting antibodies that bind to squalene (SQE). Because of the prior lack of availability of validated antibodies to SQE, positive controls for the assay were made by immunization with formulations containing SQE to create monoclonal antibodies (mAbs) that reacted with SQE. Among eight immunogens tested, only two induced detectable murine antibodies to SQE: liposomes containing dimyristoyl phosphatidylcholine, dimyristoyl phosphatidylglycerol, 71% SQE, and lipid A [L(71% SQE+LA)], and, to a much lesser extent, an oil-in-water emulsion containing SQE, Tween 80, Span 85, and lipid A. In each case, lipid A served as an adjuvant, but neither SQE alone, SQE mixed with lipid A, liposomes containing 43% SQE and lipid A, nor several other emulsions containing both SQE and lipid A, induced antibodies that reacted with SQE. Monoclonal antibodies produced after immunizing mice with [L(71% SQE+LA)] served as positive controls for developing the ELISA. Monoclonal antibodies were produced that either recognized SQE alone but did not recognize squalane (SQA, the hydrogenated form of SQE), or that recognized both SQE and SQA. As found previously with other liposomal lipid antigens, liposomes containing lipid A also induced antibodies that reacted with the liposomal phospholipids. However, mAbs were also identified that reacted with SQE on PVDF membranes, but did not recognize either SQA or liposomal phospholipid. The polyclonal antiserum produced by immunizing mice with [L(71% SQE+LA)] therefore contained a mixed population of antibody specificities and, as expected, the ELISA of polyclonal antiserum with PVDF membranes detected antibodies both to SQE and SQA. We conclude that SQE is a weak antigen, but that antibodies that specifically bind to SQE can be readily induced by immunization with [L(71% SQE+LA)] and detected by ELISA with PVDF membranes coated with SQE. PMID:11042279
Matyas, G R; Wassef, N M; Rao, M; Alving, C R
The immune response suppressed by specific antibody
Homologous or heterologous anti-sheep erythrocyte serum given passively to normal rats markedly suppressed their spleen plaque-forming cell and serum antibody response to sheep erythrocytes. Passive immunization against bovine ?-globulin prevented `sensitization' by a first injection of the antigen. The suppressive effect of passive antibody was prevented or partially prevented by adjuvants, B. pertussis vaccine, S. typhi endotoxin or Freund's complete adjuvant. Passive antibody or adjuvants had relatively little effect on the primary response when given more than 24 hours after antigen or on the secondary response when given with antigen. The kinetics of the early spleen plaque-forming cell response were measured using sheep erythrocytes as antigen, homologous anti-sheep erythrocyte serum for passive immunization and B. pertussis vaccine as adjuvant. With a constant antigen dose, larger amounts of passive antibody caused increased suppression. Suppression apparently resulted from a decrease in the number of cells initially responding; the rate of proliferation of cells that did respond was not affected by passive antibody. If the amount of passive antibody was kept constant, an increase in antigen dose or addition of adjuvant to the antigen increased the rate of proliferation of the cells that did respond; an effect sufficient to completely mask suppression produced by smaller amounts of passive antibody. These findings can be accounted for by assuming that passive antibody and higher antigen doses or adjuvant affect different interactions required for the antibody response. Thus, the magnitude of the antibody response is dependent not only on the amounts of antigen and passively given antibody but also on the amount and activity of any intentionally or unintentionally introduced factor having adjuvant activity.
Rowley, D. A.; Fitch, F. W.; Axelrad, M. A.; Pierce, C. W.
IBC's 23rd Annual Antibody Engineering, 10th Annual Antibody Therapeutics International Conferences and the 2012 Annual Meeting of The Antibody Society
The 23rd Annual Antibody Engineering, 10th Annual Antibody Therapeutics international conferences, and the 2012 Annual Meeting of The Antibody Society, organized by IBC Life Sciences with contributions from The Antibody Society and two Scientific Advisory Boards, were held December 3–6, 2012 in San Diego, CA. The meeting drew over 800 participants who attended sessions on a wide variety of topics relevant to antibody research and development. As a prelude to the main events, a pre-conference workshop held on December 2, 2012 focused on intellectual property issues that impact antibody engineering. The Antibody Engineering Conference was composed of six sessions held December 3–5, 2012: (1) From Receptor Biology to Therapy; (2) Antibodies in a Complex Environment; (3) Antibody Targeted CNS Therapy: Beyond the Blood Brain Barrier; (4) Deep Sequencing in B Cell Biology and Antibody Libraries; (5) Systems Medicine in the Development of Antibody Therapies/Systematic Validation of Novel Antibody Targets; and (6) Antibody Activity and Animal Models. The Antibody Therapeutics conference comprised four sessions held December 4–5, 2012: (1) Clinical and Preclinical Updates of Antibody-Drug Conjugates; (2) Multifunctional Antibodies and Antibody Combinations: Clinical Focus; (3) Development Status of Immunomodulatory Therapeutic Antibodies; and (4) Modulating the Half-Life of Antibody Therapeutics. The Antibody Society's special session on applications for recording and sharing data based on GIATE was held on December 5, 2012, and the conferences concluded with two combined sessions on December 5–6, 2012: (1) Development Status of Early Stage Therapeutic Antibodies; and (2) Immunomodulatory Antibodies for Cancer Therapy.
Klohn, Peter-Christian; Wuellner, Ulrich; Zizlsperger, Nora; Zhou, Yu; Tavares, Daniel; Berger, Sven; Zettlitz, Kirstin A.; Proetzel, Gabriele; Yong, May; Begent, Richard H.J.; Reichert, Janice M
Humanized recombinant and monoclonal antibodies specific for the ectodomain of the influenza virus M2 ion channel protein are disclosed. The antibodies of the invention have anti-viral activity and may be useful as anti-viral therapeutics and/or prophylactic/vaccine agents for inhibiting influenza virus replication and for treating individuals infected with influenza.
Bradbury, Andrew M.
Humanized recombinant and monoclonal antibodies specific for the ectodomain of the influenza virus M2 ion channel protein are disclosed. The antibodies of the invention have anti-viral activity and may be useful as anti-viral therapeutics and/or prophylactic/vaccine agents for inhibiting influenza virus replication and for treating individuals infected with influenza.
Bradbury, Andrew M. (Santa Fe, NM)
The medical potential of catalytic antibodies
Catalytic antibodies are relatively slow catalysts with turnover numbers some 106 less than is common for enzymes. However, they have the advantage of high affinity for a pre-selected substrate and the ability to carry out a pre- determined chemical transformation with an efficiency that is adequate for medical application. In a feasibility study, we have chosen to investigate antibody catalysis
Michael Blackbum; Anita Datta; Lynda J. Partridge
Monoclonal antibodies for serotyping Leishmania strains.
Mouse monoclonal antibodies raised against Leishmania tropica major were found to precipitate with the excreted factor produced by this and other leishmanial species. We suggest that a classification system for Leishmania, based on selective precipitation reactions between monoclonal antibodies and the excreted factor, would remove many ambiguities that currently exist. Images
Greenblatt, C L; Slutzky, G M; de Ibarra, A A; Snary, D
Autoimmune Limbic Encephalitis With GAD Antibodies
The neurologic presentation of limbic encephalitis is variable and when it occurs due to a rare cause the diagnosis may be problematic. We present a case of autoimmune limbic encephalitis due to glutamic acid decarboxylase antibody and consider the magnetic resonance imaging and antineural antibody screening aspects in the diagnosis of this entity.
Finelli, Pasquale F.
Use of second antibody in radioimmunotherapy
In this study, a second antibody was directed against the first antitumor antibody to accelerate clearance of the /sup 131/I-labeled first antibody and improve tumor to normal tissue ratios of radioactivity. The value of this method in improving the therapeutic index of radioimmunotherapy with /sup 131/I-antibody to CEA has been investigated in nude mice bearing xenografts of human colon carcinoma and in 5 patients with colorectal cancer. The xenografts did not become saturated with anti-CEA as the administered dose was increased to therapeutic levels. At these high dose levels, the second antibody increased tumor to blood ratios to a maximum of 155:1, 48 times the level in controls that did not receive the second antibody. In 5 patients given 50 mCi of anti-CEA, there was no significant toxicity with the second antibody; clearance of radioactivity was accelerated; and tumor imaging was enhanced. The second antibody appears to have the potential to improve the therapeutic index of radioimmunotherapy.
Begent, R.H.; Bagshawe, K.D.; Pedley, R.B.; Searle, F.; Ledermann, J.A.; Green, A.J.; Keep, P.A.; Chester, K.A.; Glaser, M.G.; Dale, R.G.
Database on monoclonal antibodies to cytokeratins.
Cytokeratins (CK) are being extensively used as diagnostic markers for various malignancies and other diseases, including human oral precancer and cancer, due to their tissue specific expression. CK are epithelia specific intermediate filament (IF) proteins, which are expressed in a differentiation dependent and tissue specific manner. There are about 30 polypeptides of CK expressed by different human epithelia. Each type of epithelium expresses about 4-6 polypeptides. CK polypeptides share many common epitopes, due to which the antibodies developed against CK tend to cross react. Therefore, a large number of monoclonal and polyclonal antibodies have been developed to distinguish among these proteins. Many of these antibodies are not only monospecific but are also epitope specific. These antibodies are being used in pathology laboratories for routine diagnosis using immunohistochemistry. A number of fixatives are used for fixation of tissue sections prior to the use of these antibodies. Sometimes, this leads in epitope masking. Hence, it becomes necessary to use a battery of monoclonal antibodies (MAb) for accurate diagnosis. Apart from the use of these antibodies in diagnostics, they are also being used in basic research for the study of CK function and their interactions with associated proteins and membrane proteins. In the present communication an effort has been made to make a comprehensive list of MAb to CK giving information like cross-reactivity, epitope specificity, various fixatives used, etc. along with the source of the antibodies, which will provide useful information to the users. PMID:14747055
Upasani, Ojaswini S; Vaidya, Milind M; Bhisey, Avinash N
Receptor Monoclonal Antibodies that Inhibit Tumor Angiogenesis.
National Technical Information Service (NTIS)
The purpose of this project is to generate monoclonal antibodies to receptor molecules for the angiogenic growth factor VEGF. These antibodies will be tested for their potential as inhibitors of VEGF-stimulated angiogenesis, which is thought to play an im...
J. D. Sato
7th Annual European Antibody Congress 2011
The 7th European Antibody Congress (EAC), organized by Terrapin Ltd., was again held in Geneva, Switzerland, following on the tradition established with the 4th EAC. The 2011 version of the EAC was attended by nearly 250 delegates who learned of the latest advances and trends in the global development of antibody-based therapeutics. The first day focused on advances in understanding structure-function relationships, choosing the best format, glycoengineering biobetter antibodies, improving the efficacy and drugability of mAbs and epitope mapping. On the second day, the discovery of novel targets for mAb therapy, clinical pipeline updates, use of antibody combinations to address resistance, generation and identification of mAbs against new targets and biosimilar mAb development were discussed. Antibody-drug conjugates, domain antibodies and new scaffolds and bispecific antibodies were the topics of the third day. In total, nearly 50 speakers provided updates of programs related to antibody research and development on-going in the academic, government and commercial sectors.
Antinuclear antibodies in dogs with leishmaniasis.
An indirect immunofluorescence method using rat liver as material was developed to determine the incidence of antinuclear antibodies in serum from 44 adult dogs naturally infected with leishmaniasis and, for comparative purposes, in a control group of 30 healthy dogs. Animals in both groups were of different breeds with a similar age distribution. Antinuclear antibodies were not detected in the healthy dogs and only 15.9% of the diseased dogs, 12.0% males and 21.1% females, were positive. The results indicate that in contrast to previously reported figures, the incidence of antinuclear antibodies is low in canine leishmaniasis at least when diagnosis is first made. In order to investigate whether antinuclear antibodies may play a role in the development of the renal damage observed in canine leishmaniasis, the concentrations of serum creatinine were related to the presence of antinuclear antibodies. Only 28.5% of the antinuclear antibodies positive infected dogs showed hypercreatininemia as did 32.4% of the antinuclear antibodies negative infected dogs. Thus, antinuclear antibodies are not significantly cause-effect related to the development of the renal lesions seen in canine leishmaniasis. PMID:8767735
Lucena, R; Ginel, P J; Lopez, R; Novales, M; Martin, E; Molleda, J M
Antibody neutralization and escape by HIV1
Neutralizing antibodies (Nab) are a principal component of an effective human immune response to many pathogens, yet their role in HIV-1 infection is unclear. To gain a better understanding of this role, we examined plasma from patients with acute HIV infection. Here we report the detection of autologous Nab as early as 52 days after detection of HIV-specific antibodies. The
Xiping Wei; Julie M. Decker; Shuyi Wang; Huxiong Hui; John C. Kappes; Xiaoyun Wu; Jesus F. Salazar-Gonzalez; Maria G. Salazar; J. Michael Kilby; Michael S. Saag; Natalia L. Komarova; Martin A. Nowak; Beatrice H. Hahn; Peter D. Kwong; George M. Shaw
Antibody-mediated organ-allograft rejection
Recent studies show that alloantibodies mediate a substantial proportion of graft-rejection episodes, contributing to both early and late graft loss. Rejection that is caused by antibody is mediated by different mechanisms from rejection that is caused by T cells, thereby requiring other approaches to treatment and prevention. Antibody induces rejection acutely through the fixation of complement, resulting in tissue injury
R. Neal Smith; Robert B. Colvin
Production of human monoclonal antibodies to myeloperoxidase.
Two mouse-human heterohybridomas secreting human antibodies to myeloperoxidase (MPO) were derived from the peripheral blood of a patient who developed microscopic polyarteritis as the result of long-term treatment with hydralazine. Forty-five immunoglobulin-secreting lines were obtained from the fusion of patient lymphocytes with the CB-F7 heteromyeloma cell line. Of these, two antibodies, one IgG and one IgM, bound to myeloperoxidase in solid phase ELISA and gave a perinuclear staining pattern on ethanol-fixed human neutrophil cytospin preparations. The staining patterns were similar to those seen with serum from the patient. Antigen-inhibition studies revealed that the affinity of the IgG monoclonal antibody was 28 times higher (k = 1.4 x 10(-7)) than the IgM antibody (k = 5 x 10(-5)). Cross-inhibition studies further suggested that the two monoclonal antibodies recognized the same epitope on MPO. Of the other secreting cell lines, none produced antibody which reacted with the panel of autoantigens used for testing. Neither mononuclear antibody reacted with this panel indicating that they were not simply polyreactive natural autoantibodies. These are the first human monoclonal antibodies to native myeloperoxidase to be reported. Images Figure 2 Figure 3
Ehrenstein, M R; Leaker, B; Isenberg, D; Cambridge, G
Novel Monoclonal Antibody against Human Platelets.
National Technical Information Service (NTIS)
The production of a monoclonal antibody against human platelets is described in the invention. Unique features of the described monoclonal antibody are that it belongs to the IgGl subclass; it binds only to activated platelets, recognizing a 148,000 kilod...
H. Gralnick
454 antibody sequencing - error characterization and correction
Background 454 sequencing is currently the method of choice for sequencing of antibody repertoires and libraries containing large numbers (106 to 1012) of different molecules with similar frameworks and variable regions which poses significant challenges for identifying sequencing errors. Identification and correction of sequencing errors in such mixtures is especially important for the exploration of complex maturation pathways and identification of putative germline predecessors of highly somatically mutated antibodies. To quantify and correct errors incorporated in 454 antibody sequencing, we sequenced six antibodies at different known concentrations twice over and compared them with the corresponding known sequences as determined by standard Sanger sequencing. Results We found that 454 antibody sequencing could lead to approximately 20% incorrect reads due to insertions that were mostly found at shorter homopolymer regions of 2-3 nucleotide length, and less so by insertions, deletions and other variants at random sites. Correction of errors might reduce this population of erroneous reads down to 5-10%. However, there are a certain number of errors accounting for 4-8% of the total reads that could not be corrected unless several repeated sequencing is performed, although this may not be possible for large diverse libraries and repertoires including complete sets of antibodies (antibodyomes). Conclusions The experimental test procedure carried out for assessing 454 antibody sequencing errors reveals high (up to 20%) incorrect reads; the errors can be reduced down to 5-10% but not less which suggests the use of caution to avoid false discovery of antibody variants and diversity.
Genetically engineered antibody molecules and their application.
Immunoglobulin genes can be efficiently expressed following transfection into myeloma cells. Using protoplast fusion, transfection frequencies greater than 10(-3) can be achieved. Compatible plasmids containing two different selectible markers are used to simultaneously deliver heavy and light chain genes to the same cell. To produce molecules with differing specificities the rearranged and expressed variable regions can be cloned from the appropriate hybridoma. In some cases, variable regions from cDNAs can be inserted into the expression vectors. It is possible to manipulate the immunoglobulin genes and produce novel antibody molecules. Antibodies have been produced in which the variable regions from mouse antibodies have been joined to human constant regions. In addition, antibodies with altered constant regions have been produced. These genetically engineered antibodies provide a unique set of reagents to study structure-function relationships within the molecule. They also can potentially be used in the diagnosis and therapy of human disease. PMID:3327412
Morrison, S L; Wims, L; Wallick, S; Tan, L; Oi, V T
Dual targeting strategies with bispecific antibodies
Monoclonal antibodies are widely used for the treatment of cancer, inflammatory and infectious diseases and other disorders. Most of the marketed antibodies are monospecific and therefore capable of interacting and interfering with a single target. However, complex diseases are often multifactorial in nature, and involve redundant or synergistic action of disease mediators or upregulation of different receptors, including crosstalk between their signaling networks. Consequently, blockade of multiple, different pathological factors and pathways may result in improved therapeutic efficacy. This result can be achieved by combining different drugs, or use of the dual targeting strategies applying bispecific antibodies that have emerged as an alternative to combination therapy. This review discusses the various dual targeting strategies for which bispecific antibodies have been developed and provides an overview of the established bispecific antibody formats.
Antibody to intermediate filaments of the cytoskeleton.
IgM antibodies against cultures of intermediate filaments (IMF) of the cytoskeleton were demonstrated by immunofluorescence in the sera of 94 (80%) of 118 patients with seropositive rheumatoid arthritis. These antibodies reacted with IMF in cultures of both human fetal fibroblasts and laryngeal carcinoma (HEp2) cells. Of 10 patients from whom paired synovial fluids were also available 8 had anti-IMF antibodies in both serum and fluid. In seronegative RA the incidence of anti-IMF was 40%, in ankylosing spondylitis 25%, in osteoarthrosis 16%, and in normal subjects 14%. Only a minority of RA sera positive for anti-IMF antibodies were also positive for smooth muscle antibody. Absorption experiments suggest that in RA anti-IMF is directed at the intermediate filament protein, vimentin. Images
Osung, O A; Chandra, M; Holborow, E J
[Radiolabeled antibodies for cancer treatment].
The first treatment ever by radio-immunotherapy (RIT) was performed by William H. Beierwaltes in 1951 and was a success. Fifty years later, the main question is to find ways of extending the success of radiolabelled anti-CD20 antibodies in indolent non-Hodgkin's lymphoma to other forms of cancer. Solid tumours are much more radioresistant than lymphomas, but they respond to RIT if the lesions are small. Clinical situations of residual or minimal disease are thus the most likely to benefit from RIT in the adjuvant or consolidation settings. For disseminated disease, like leukemias or myelomas, the problem is different: beta- particles emitted by the radioactive atoms classically used for cancer treatment (iodine-131 or yttrium-90) disperse their energy in large volumes (ranges 1 mm to 1 cm) and are not very effective against isolated cells. Advances in RIT progress in two directions. One is the development of pretargeting strategies in which the antibody is not labelled but used to provide binding sites to small molecular weight radioactivity vectors (biotin, haptens). These techniques have been shown to increase tumour to non-target uptake ratios and anti-tumour efficacy has been demonstrated in the clinic. The other approach is the use of radionuclides adapted to the various clinical situations. Lutetium-177 or copper-67, because of the lower energy of their emission, their relatively long half-life and good gamma emission, may significantly improve RIT efficacy and acceptability. Beyond that, radionuclides emitting particles such as alpha particles or Auger electrons, much more efficient to kill isolated tumour cells, are being tested for RIT in the clinic. Finally, RIT should be integrated with other cancer treatment approaches in multimodality protocols. Thus RIT, now a mature technology, should enter a phase of well designed and focused clinical developments that may be expected to afford significant therapeutic advances. PMID:20035676
Barbet, Jacques; Chatal, Jean-François; Kraeber-Bodéré, Françoise
Comparison of physical chemical properties of llama VHH antibody fragments and mouse monoclonal antibodies.
Antigen specific llama VHH antibody fragments were compared to antigen specific mouse monoclonal antibodies with respect to specificity, affinity and stability. The llama VHH antibody fragments and the mouse monoclonal antibodies investigated were shown to be highly specific for the protein antigen hCG or the hapten antigen RR-6. The affinity of the interaction between monovalent llama VHH antibody fragments and their antigen is close to the nanomolar range, similar to the bivalent mouse monoclonal antibodies studied. Llama VHH antibody fragments are similar to mouse monoclonal antibodies with respect to antigen binding in the presence of ammonium thiocyanate and ethanol. The results show that relative to antigen specific mouse monoclonal antibodies, antigen specific llama VHH fragments are extremely temperature stable. Two out of six llama VHHs are able to bind antigen specifically at temperatures as high as 90 degrees C, whereas four out of four mouse monoclonal antibodies are not functional at this temperature. Together with the finding that llama VHH fragments can be produced at high yield in Saccharomyces cerevisiae, these findings indicate that in the near future antigen specific llama VHH fragments can be used in for antibodies unexpected products and processes. PMID:10209277
van der Linden, R H; Frenken, L G; de Geus, B; Harmsen, M M; Ruuls, R C; Stok, W; de Ron, L; Wilson, S; Davis, P; Verrips, C T
Monoclonal Antibodies to Herpes Simplex Virus Type I Polypeptides.
National Technical Information Service (NTIS)
It is now possible to make pure antibodies to proteins by application of the 'monoclonal antibody' or 'hybridoma' technique. The present invention is one such new technique which embodies the monoclonal antibody mechanism. About 50 proteins have been disc...
B. Hampar M. Zweig S. D. Showalter
The Clinical Proteomic Technologies for Cancer | Antibody Scientific Committee
The Antibody Scientific Committee provides scientific insight and guidance to the National Cancer Institute's Antibody Characterization Program. Specifically the members of this committee evaluate request from the external scientific community for development and characterization of antibodies by the program.
Radiohalogenated half-antibodies and maleimide intermediate therefor
N-(m-radiohalophenyl) maleimide can be conjugated with a reduced antibody having a mercapto group to provide a radiolabeled half-antibody having immunological specific binding characteristics of whole antibody. No Drawings
Kassis, A.I.; Khawli, L.A.
Recombinant human polyclonal antibodies: A new class of therapeutic antibodies against viral infections.
The mammalian immune system eliminates pathogens by generating a specific antibody response. Polyclonality is a key feature of this immune response: the immune system produces antibodies which bind to different structures on a given pathogen thereby increasing the likelihood of its elimination. The vast majority of current recombinant antibody drugs rely on monospecific monoclonal antibodies. Inherently, such antibodies do not represent the benefits of polyclonality utilized by a natural immune system and this has impeded the identification of efficacious antibody drugs against infectious agents, including viruses. The development of novel technologies has allowed the identification and manufacturing of antigen-specific recombinant polyclonal human antibodies, so-called symphobodies. This review describes the rationale for designing drugs based on symphobodies against pathogenic viruses, including HIV, vaccinia and smallpox virus, and respiratory syncytial virus. PMID:16787244
Bregenholt, Sřren; Jensen, Allan; Lantto, Johan; Hyldig, Sara; Haurum, John S
Antibody-based resistance to plant pathogens.
Plant diseases are a major threat to the world food supply, as up to 15% of production is lost to pathogens. In the past, disease control and the generation of resistant plant lines protected against viral, bacterial or fungal pathogens, was achieved using conventional breeding based on crossings, mutant screenings and backcrossing. Many approaches in this field have failed or the resistance obtained has been rapidly broken by the pathogens. Recent advances in molecular biotechnology have made it possible to obtain and to modify genes that are useful for generating disease resistant crops. Several strategies, including expression of pathogen-derived sequences or anti-pathogenic agents, have been developed to engineer improved pathogen resistance in transgenic plants. Antibody-based resistance is a novel strategy for generating transgenic plants resistant to pathogens. Decades ago it was shown that polyclonal and monoclonal antibodies can neutralize viruses, bacteria and selected fungi. This approach has been improved recently by the development of recombinant antibodies (rAbs). Crop resistance can be engineered by the expression of pathogen-specific antibodies, antibody fragments or antibody fusion proteins. The advantages of this approach are that rAbs can be engineered against almost any target molecule, and it has been demonstrated that expression of functional pathogen-specific rAbs in plants confers effective pathogen protection. The efficacy of antibody-based resistance was first shown for plant viruses and its application to other plant pathogens is becoming more established. However, successful use of antibodies to generate plant pathogen resistance relies on appropriate target selection, careful antibody design, efficient antibody expression, stability and targeting to appropriate cellular compartments. PMID:11252378
Schillberg, S; Zimmermann, S; Zhang, M Y; Fischer, R
Identification of measles virus-specific hemolysis-inihibiting antibodies separate from hemagglutination-inhibiting antibodies.
The occurrence of antibodies giving hemolysis inhibition (HLI) but not hemagglutination inhibition (HI) was examined in human convalescent and rabbit hyperimmune sera. HI antibodies, which through their interaction with hemagglutinin components display HLI activity, were removed by absorption with Tween 80-ether (TE)-treated measles virus material. This absorption did not change the titer of non-HI HLI antibodies. After removal of HI antibodies from 16 late measles convalescent sera and three batches of gamma globulin. HLI antibody titers showed a two- to eightfold reduction. The titers of neutralizing antibodies were reduced from 1/4 to 1/20 of the original titers. There was a good correlation between the titers of neutralizing and HLI antibodies both in sera from which HI antibodies had been removed by absorption and in sera spontaneously showing markedly higher HLI than HI antibody titers. HLI antibodies with these characteristics could be identified in HI tests when whole virus instead of TE-treated material was used an antigen and anti-antiserum was added to the tests. In contrast to the situation in human sera, antibodies remaining after removal of HI antibodies from rabbit hyperimmune sera against purified virus particles were detectable in neutralization and HLI tests only in the presence of anti-antiserum. However, virus particles from which the major fraction of all envelope projections had been removed by treatment with 0.004% trypsin induced the production of non-HI HLI antibodies active also in the absence of anti-antiserum. TE and formalin treatment destroyed the hemolytic activity of virus preparations and also their capacity to induce a production of non-HI HLI antibodies. Images
Norrby, E; Gollmar, Y
Antibody production by molecular farming in plants.
"Molecular farming" is the production of pharmaceutical proteins in transgenic plants and has great potential for the production of therapeutic anti-cancer antibodies and recombinant therapeutic proteins. Plants make fully functional recombinant human or animal antibodies. Cultivating transgenic plants on an agricultural scale will produce almost unlimited supplies of recombinant proteins for uses in medicine. Combinatorial library technology is a key tool for the generation and optimisation of therapeutic antibodies ahead of their expression in plants. Optimised antibody expression can be rapidly verified using transient expression assays in plants before creation of transgenic suspension cells or plant lines. Subcellular targeting signals that increase expression levels and optimise protein stability can be identified and exploited using transient expression to create high expresser plant lines. When high expresser lines have been selected, the final step is the development of efficient purification methods to retrieve functional antibody. Antibody production on an industrial scale is then possible using plant suspension cell culture in fermenters, or by the propagation of stably transformed plant lines in the field. Recombinant proteins can be produced either in whole plants or in seeds and tubers, which can be used for the long-term storage of both the protein and its production system. The review will discuss these developments and how we are moving toward the molecular farming of therapeutic antibodies becoming an economic and clinical reality. PMID:10841283
Fischer, R; Hoffmann, K; Schillberg, S; Emans, N
Antibody testing in peripheral nerve disorders.
The identification of autoantibodies associated with dysimmune neuropathies was a major contribution to the characterization of peripheral nerve disorders, the understanding of their pathophysiology, and the clinical diagnosis of neuropathies. Antibodies directed to GM1, GQ1b, and disyalilated gangliosides, and anti-MAG antibodies are very useful in the diagnosis of acute or chronic motor or sensory-motor neuropathies with or without monoclonal IgM. Anti-onconeural anti-Hu and anti-CV2/CRMP antibodies allow when they are detected the diagnosis of paraneoplastic neuropathies. This chapter focuses on the description of these antibodies as diagnostic markers and on their immunopathogenesis. We give a background overview on the origin of these antibodies, their detection, and review those studies, which clearly show that these antibodies are capable of binding to the target tissues in peripheral nerve and thereby can exert a variety of pathophysiological effects. The corresponding electrophysiological and histological changes observed both in human and animal models are exemplified in order to get a better understanding of the immune mechanisms of these antibody-mediated neuropathies. PMID:23931781
Steck, Andreas; Yuki, Nobuhiro; Graus, Francesc
Next generation of antibody therapy for cancer
Monoclonal antibodies (mAbs) have become a major class of therapeutic agents providing effective alternatives to treating various human diseases. To date, 15 mAbs have been approved by regulatory agencies in the world for clinical use in oncology indications. The selectivity and specificity, the unique pharmacokinetics, and the ability to engage and activate the host immune system differentiate these biologics from traditional small molecule anticancer drugs. mAb-based regimens have brought clinical benefits, including improvements in overall survival, to patients with a variety of cancers. Many challenges still remain, however, to fully realize the potential of these new medicines. With our further understanding of cancer biology, mechanism of antibody action, and advancement of antibody engineering technologies, many novel antibody formats or antibody-derived molecules are emerging as promising new generation therapeutics. Carefully designed and engineered, they retain the advantage of specificity and selectivity of original antibodies, but in the meantime acquire additional special features such as improved pharmacokinetics, increased selectivity, and enhanced anticancer efficacy. Promising clinical results are being generated with these newly improved antibody-based therapeutics.
Zhu, Zhenping; Yan, Li
Anti-sulphatide antibodies in peripheral neuropathy.
A study was carried out on 135 patients with chronic idiopathic neuropathy (63), neuropathy associated with monoclonal gammopathy (51, including eight with anti-MAG antibody activity) and the Guillain-Barré syndrome (GBS) (21). Serum IgM, IgG and IgA anti-sulphatide antibody titres were compared with titres in 304 patients with other neurological or immunological diseases and in 50 normal subjects. Titres were presented a) as the highest serum dilution at which reactivity could be detected, and b) in the linear region of the optical density curve. A substantial number of patients with neurological or immunological diseases had higher titres than normal subjects. Compared with normal and disease controls, five patients with neuropathy associated with IgMk monoclonal gammopathy had raised titres of IgM anti-sulphatide antibodies and one patient with GBS had raised IgM, IgG and IgA anti-sulphatide antibodies in the acute phase of the disease. Two patients had a predominantly axonal sensory neuropathy with presenting symptoms of painful paresthesiae and minimal neurological deficit. Three patients had a predominantly demyelinating sensorimotor neuropathy associated with anti-MAG antibody activity. The patient with GBS had extensive sensory loss and antibody titres returned to normal within three weeks. Raised titres of anti-sulphatide antibodies occurred in several types of neuropathy, but all had some degree of sensory impairment and associated immunological abnormality.
van den Berg, L H; Lankamp, C L; de Jager, A E; Notermans, N C; Sodaar, P; Marrink, J; de Jong, H J; Bar, P R; Wokke, J H
Antibodies in the Pathogenesis of Hypertension
It has long been known that circulating levels of IgG and IgM antibodies are elevated in patients with essential and pregnancy-related hypertension. Recent studies indicate these antibodies target, and in many cases activate, G-protein coupled receptors and ion channels. Prominent among these protein targets are AT1 receptors, ?1-adrenoceptors, ?1-adrenoceptors, and L-type voltage operated Ca2+ channels, all of which are known to play key roles in the regulation of blood pressure through modulation of vascular tone, cardiac output, and/or Na+/water reabsorption in the kidneys. This suggests that elevated antibody production may be a causal mechanism in at least some cases of hypertension. In this brief review, we will further describe the protein targets of the antibodies that are elevated in individuals with essential and pregnancy-related hypertension and the likely pathophysiological consequences of antibody binding to these targets. We will speculate on the potential mechanisms that underlie elevated antibody levels in hypertensive individuals and, finally, we will outline the therapeutic opportunities that could arise with a better understanding of how and why antibodies are produced in hypertension.
Chan, Christopher T.; Lieu, Maggie; Toh, Ban-Hock; Kyaw, Tin S.; Bobik, Alexander; Sobey, Christopher G.; Drummond, Grant R.
Structure Based Antibody-Like Peptidomimetics
Biologics such as monoclonal antibodies (mAb) and soluble receptors represent new classes of therapeutic agents for treatment of several diseases. High affinity and high specificity biologics can be utilized for variety of clinical purposes. Monoclonal antibodies have been used as diagnostic agents when coupled with radionuclide, immune modulatory agents or in the treatment of cancers. Among other limitations of using large molecules for therapy the actual cost of biologics has become an issue. There is an effort among chemists and biologists to reduce the size of biologics which includes monoclonal antibodies and receptors without a reduction of biological efficacy. Single chain antibody, camel antibodies, Fv fragments are examples of this type of deconstructive process. Small high-affinity peptides have been identified using phage screening. Our laboratory used a structure-based approach to develop small-size peptidomimetics from the three-dimensional structure of proteins with immunoglobulin folds as exemplified by CD4 and antibodies. Peptides derived either from the receptor or their cognate ligand mimics the functions of the parental macromolecule. These constrained peptides not only provide a platform for developing small molecule drugs, but also provide insight into the atomic features of protein-protein interactions. A general overview of the reduction of monoclonal antibodies to small exocyclic peptide and its prospects as a useful diagnostic and as a drug in the treatment of cancer are discussed.
Murali, Ramachandran; Greene, Mark I.
Progress towards recombinant anti-infective antibodies
The global market for monoclonal antibody therapeutics reached a total of $11.2 billion in 2004, with an impressive 42% growth rate over the previous five years and is expected to reach ~$34 billion by 2010. Coupled with this growth are stream-lined product development, production scale-up and regulatory approval processes for the highly conserved antibody structure. While only one of the 21 current FDA-approved antibodies, and one of the 38 products in advanced clinical trials target infectious diseases, there is increasing academic, government and commercial interest in this area. Synagis, an antibody neutralizing respiratory syncitial virus (RSV), garnered impressive sales of $1.1 billion in 2006 in spite of its high cost and undocumented effects on viral titres in human patients. The success of anti-RSV passive immunization has motivated the continued development of anti-infectives to treat a number of other infectious diseases, including those mediated by viruses, toxins and bacterial/fungal cells. Concurrently, advances in antibody technology suggest that cocktails of several monoclonal antibodies with unique epitope specificity or single monoclonal antibodies with broad serotype specificity may be the most successful format. Recent patents and patent applications in these areas will be discussed as predictors of future anti-infective therapeutics.
Pai, Jennifer C.; Sutherland, Jamie N.; Maynard, Jennifer A.
Antibody binding loop insertions as diversity elements
In the use of non-antibody proteins as affinity reagents, diversity has generally been derived from oligonucleotide-encoded random amino acids. Although specific binders of high-affinity have been selected from such libraries, random oligonucleotides often encode stop codons and amino acid combinations that affect protein folding. Recently it has been shown that specific antibody binding loops grafted into heterologous proteins can confer the specific antibody binding activity to the created chimeric protein. In this paper, we examine the use of such antibody binding loops as diversity elements. We first show that we are able to graft a lysozyme-binding antibody loop into green fluorescent protein (GFP), creating a fluorescent protein with lysozyme-binding activity. Subsequently we have developed a PCR method to harvest random binding loops from antibodies and insert them at predefined sites in any protein, using GFP as an example. The majority of such GFP chimeras remain fluorescent, indicating that binding loops do not disrupt folding. This method can be adapted to the creation of other nucleic acid libraries where diversity is flanked by regions of relative sequence conservation, and its availability sets the stage for the use of antibody loop libraries as diversity elements for selection experiments.
Kiss, Csaba; Fisher, Hugh; Pesavento, Emanuele; Dai, Minghua; Valero, Rosa; Ovecka, Milan; Nolan, Rhiannon; Phipps, M. Lisa; Velappan, Nileena; Chasteen, Leslie; Martinez, Jennifer S.; Waldo, Geoffrey S.; Pavlik, Peter; Bradbury, Andrew R.M.
Labeling of monoclonal antibodies with radionuclides
Antibodies, specifically monoclonal antibodies, are potentially very useful and powerful carriers of therapeutic agents to target tissues and diagnostic agents. The loading or charging of antibodies with agents, especially radiotracers, is reviewed here. The choice of radioisotope for immunodetection and/or immunotherapy is based on its availability, half-life, nature of the radiation emitted, and the metabolic pathways of the radionuclide in the body. Most important of all are the derivatization techniques available for labeling the antibody with the given radionuclide. Isotopes of iodine and divalent metal ions are the most commonly used radionuclides. Antibodies labeled with iodine at tyrosine residues are metabolized rapidly in vivo. This leads to the incorporation of metabolized radioactive iodine into various tissues, mainly the thyroid gland and stomach, and to the accumulation of high levels of circulating iodine in the blood, which masks tumor uptake considerably. To overcome these limitations, the use of iodohippurate as an iodine-anchoring molecule to the protein should be considered. When divalent or multivalent metal ions are used as the preferred radionuclide, bifunctional chelating reagents such as EDTA or DTPA are first coupled to the protein or antibody. These chelating molecules are attached to the protein by formation of an isopeptide linkage between the carboxylate of the chelating reagent and the amino group of the protein. Several procedures are available to generate the isopeptide linkage. When the anchoring of the chelating agent through isopeptide linkage results in the inactivation of the antibody, periodate oxidation of the carbohydrate moiety of the antibody, followed by reductive coupling of chelator, could be considered as an alternative. There is still a need for better, simpler, and more direct methods for labeling antibodies with radionuclides. 78 references.
Bhargava, K.K.; Acharya, S.A. (Albert Einstein College of Medicine-Montefiore Medical Center, Bronx, NY (USA))
Coeliac Disease-Associated Antibodies in Psoriasis
Background The possible relationship between psoriasis and coeliac disease (CD) has been attributed to the common pathogenic mechanisms of the two diseases and the presence of antigliadin antibodies in patients has been reported to increase the incidence of CD. Objective The aim of this report was to study CD-associated antibodies serum antigliadin antibody immunoglobulin (Ig)A, IgG, anti-endomysial antibody IgA and anti-transglutaminase antibody IgA and to demonstrate whether there is an increase in the frequency of those markers of CD in patients with psoriasis. Methods Serum antigliadin antibody IgG and IgA, antiendomysial antibody IgA and anti-transglutaminase antibody IgA were studied in 37 (19 males) patients with psoriasis and 50 (23 males) healthy controls. Upper gastrointestinal endoscopy and duodenal biopsies were performed in patients with at least one positive marker. Results Antigliadin IgA was statistically higher in the psoriasis group than in the controls (p<0.05). Serological markers were found positive in 6 patients with psoriasis and 1 person from the control group. Upper gastrointestinal endoscopy was performed in all these persons, with biopsies collected from the duodenum. The diagnosis of CD was reported in only one patient with psoriasis following the pathological examination of the biopsies. Whereas one person of the control group was found to be positive for antigliadin antibody IgA, pathological examination of the duodenal biopsies obtain from this patient were found to be normal. Conclusion Antigliadin IgA prominently increases in patients diagnosed with psoriasis. Patients with psoriasis should be investigated for latent CD and should be followed up.
Akbulut, Sabiye; Gur, Gunes; Topal, Firdevs; Topal, Fatih Esad; Alli, Nuran; Saritas, Ulku
Antibodies Against Three Forms of Urokinase
NASA Technical Reports Server (NTRS)
Antibodies that bind to preselected regions of the urokinase molecule have been developed. These antibodies can be used to measure small quantities of each of three molecular forms of urokinase that could be contained in microsamples or conditioned media harvested from cultures of mammalian cells. Previously available antibodies and assay techniques do not yield both clear distinctions among, and measurements of, all three forms. Urokinase is a zymogen that is synthesized in a single-chain form, called ScuPA, which is composed of 411 amino acid residues (see figure). ScuPA has very little enzyme activity, but it can be activated in two ways: (1) by cleavage of the peptide bond lysine 158/isoleucine 159 and the loss of lysine 158 to obtain the high molecular-weight (HMW) form of the enzyme or (2) by cleavage of the bond lysine 135/lysine 136 to obtain the low-molecular-weight (LMW) form of the enzyme. The antibodies in question were produced in mice and rabbits by use of peptides as immunogens. The peptides were selected to obtain antibodies that bind to regions of ScuPA that include the lysine 158/isoleucine 159 and the lysine 135/lysine 136 bonds. The antibodies include monoclonal and polyclonal ones that yield indications as to whether either of these bonds is intact. The polyclonal antibodies include ones that preferentially bind to the HMW or LMW forms of the urokinase molecule. The monoclonal antibodies include ones that discriminate between the ScuPA and the HMW form. A combination of these molecular-specific antibodies will enable simultaneous assays of the ScuPA, HMW, and LMW forms in the same specimen of culture medium.
Morrison, Dennis R.; Atassi, M. Zouhair
Reshaping Human Antibodies: Grafting an Antilysozyme Activity
NASA Astrophysics Data System (ADS)
The production of therapeutic human monoclonal antibodies by hybridoma technology has proved difficult, and this has prompted the ``humanizing'' of mouse monoclonal antibodies by recombinant DNA techniques. It was shown previously that the binding site for a small hapten could be grafted from the heavy-chain variable domain of a mouse antibody to that of a human myeloma protein by transplanting the hypervariable loops. It is now shown that a large binding site for a protein antigen (lysozyme) can also be transplanted from mouse to human heavy chain. The success of such constructions may be facilitated by an induced-fit mechanism.
Verhoeyen, Martine; Milstein, Cesar; Winter, Greg
Status of antithyroid antibodies in Bangladesh.
To study autoimmunity among thyroid diseases, 397 thyroid patients (age 30 (13) years; M/F 75/322) from two referral centres in Bangladesh and 94 healthy controls (age 30 (13) years; M/F 24/70) were studied for antimicrosomal and antithyroglobulin antibodies. Thyroid patients were clinically grouped as suspected autoimmune thyroid disease (AITD), non-autoimmune, or indeterminate groups (where no decision could be reached). Antimicrosomal antibody was strongly positive in 19.4% and weakly positive in 7.3% of patients but only 4.3% and 2.1% respectively in the controls (chi(2) = 17.852; p = 0.000) whereas strong and weak positivity were 27.2% and 6. 8% in patients compared with 8.5% and 4.3% respectively in the controls (chi(2) = 16.916; p = 0.000) for antithyroglobulin antibody. Antibodies were positive in 63.0% with Hashimoto's thyroiditis, 36.4% with Graves' disease, and 44.7% with atrophic thyroiditis among the autoimmune group. In the non-autoimmune group antibodies were positive in 100% with multinodular hypothyroidism, 46.7% with subacute thyroiditis, 40.0% with suspected iodine deficiency goitre, 31.3% with toxic multinodular goitre, 30.8% with non-toxic solitary nodules, and 19.4% with simple diffuse goitre. None was positive for antimicrosomal antibody without being positive for antithyroglobulin antibody. The two antibodies strongly correlated in both patients (r = 0.977, p = 0.000) and controls (r = 0.986, p = 0.000). About 9% (36/397) of patients were mismatched with the final diagnosis on antibody measurement; most of them had Hashimoto's thyroiditis (33/36). Prevalence of AITD among thyroid patients was 48.36%. Specificity of antimicrosomal and antithyroglobulin antibodies were 93% and 87%. It was concluded that AITD is not uncommon in Bangladesh; antimicrosomal antibody is a useful marker for AITD and unless antibodies are checked, an appreciable number of patients with AITDs will remain undetected. PMID:10824048
Hasanat, M A; Rumi, M A; Alam, M N; Hasan, K N; Salimullah, M; Salam, M A; Fariduddin, M; Mahtab, H; Khan, A K
Status of antithyroid antibodies in Bangladesh
To study autoimmunity among thyroid diseases, 397 thyroid patients (age 30 (13) years; M/F 75/322) from two referral centres in Bangladesh and 94 healthy controls (age 30 (13) years; M/F 24/70) were studied for antimicrosomal and antithyroglobulin antibodies. Thyroid patients were clinically grouped as suspected autoimmune thyroid disease (AITD), non-autoimmune, or indeterminate groups (where no decision could be reached). Antimicrosomal antibody was strongly positive in 19.4% and weakly positive in 7.3% of patients but only 4.3% and 2.1% respectively in the controls (?2 = 17.852; p = 0.000) whereas strong and weak positivity were 27.2% and 6.8% in patients compared with 8.5% and 4.3% respectively in the controls (?2 = 16.916; p = 0.000) for antithyroglobulin antibody. Antibodies were positive in 63.0% with Hashimoto's thyroiditis, 36.4% with Graves' disease, and 44.7% with atrophic thyroiditis among the autoimmune group. In the non-autoimmune group antibodies were positive in 100% with multinodular hypothyroidism, 46.7% with subacute thyroiditis, 40.0% with suspected iodine deficiency goitre, 31.3% with toxic multinodular goitre, 30.8% with non-toxic solitary nodules, and 19.4% with simple diffuse goitre. None was positive for antimicrosomal antibody without being positive for antithyroglobulin antibody. The two antibodies strongly correlated in both patients (r = 0.977, p = 0.000) and controls (r = 0.986, p = 0.000). About 9% (36/397) of patients were mismatched with the final diagnosis on antibody measurement; most of them had Hashimoto's thyroiditis (33/36). Prevalence of AITD among thyroid patients was 48.36%. Specificity of antimicrosomal and antithyroglobulin antibodies were 93% and 87%. It was concluded that AITD is not uncommon in Bangladesh; antimicrosomal antibody is a useful marker for AITD and unless antibodies are checked, an appreciable number of patients with AITDs will remain undetected.???Keywords: thyroid autoimmunity; diagnostic dilemma
Hasanat, M; Rumi, M; Alam, M; Hasan, K; Salimullah, M; Salam, M; Fariduddin, M; Mahtab, H.; Khan, A
The antibody approach of labeling blood cells
Although the science of blood cell labeling using monoclonal antibodies directed against specific cellular antigens is still in its early stages, considerable progress has recently been accomplished in this area. The monoclonal antibody approach offers the promise of greater selectivity and enhanced convenience since specific cell types can be labeled in vivo, thus eliminating the need for complex and damaging cell separation procedures. This article focuses on these developments with primary emphasis on antibody labeling of platelets and leukocytes. The advantages and the shortcomings of the recently reported techniques are criticality assessed and evaluated.
Srivastava, S.C.
The antibody approach of labeling blood cells
Although the science of blood cell labeling using monoclonal antibodies directed against specific cellular antigens is still in its early stages, considerable progress has recently been accomplished in this area. The monoclonal antibody approach offers the promise of greater selectivity and enhanced convenience since specific cell types can be labeled in vivo, thus eliminating the need for complex and damaging cell separation procedures. This article focuses on these developments with primary emphasis on antibody labeling of platelets and leukocytes. The advantages and the shortcomings of the recently reported techniques are criticality assessed and evaluated.
Srivastava, S.C.
Fc engineering of antibodies and antibody derivatives by primary sequence alteration and their functional characterization.
Therapeutic antibodies used in the treatment of cancer patients are able to mediate diverse effector mechanisms. Dependent on tumor entity, localization, and tumor burden different effector mechanisms may contribute to the in vivo antitumor activity to a variable degree. Especially Fc-mediated effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) have been suggested as being important for the in vivo activity of therapeutic antibodies like rituximab or trastuzumab. In recent years, several strategies have been pursued to further optimize the cytotoxic potential of monoclonal antibodies by modifying their Fc part (Fc engineering) with the ultimate goal to enhance antibody therapy.Since Fc engineering approaches are applicable to any Fc-containing molecule, strategies to enhance CDC or ADCC activity of full antibodies or scFv-Fc fusion proteins by altering the primary Fc sequence are described. PMID:24515488
Derer, Stefanie; Kellner, Christian; Rösner, Thies; Klausz, Katja; Glorius, Pia; Valerius, Thomas; Peipp, Matthias
Generalized platform for antibody detection using the antibody catalyzed water oxidation pathway.
Infectious diseases, such as influenza, present a prominent global problem including the constant threat of pandemics that initiate in avian or other species and then pass to humans. We report a new sensor that can be specifically functionalized to detect antibodies associated with a wide range of infectious diseases in multiple species. This biosensor is based on electrochemical detection of hydrogen peroxide generated through the intrinsic catalytic activity of all antibodies: the antibody catalyzed water oxidation pathway (ACWOP). Our platform includes a polymer brush-modified surface where specific antibodies bind to conjugated haptens with high affinity and specificity. Hydrogen peroxide provides an electrochemical signal that is mediated by Resorufin/Amplex Red. We characterize the biosensor platform, using model anti-DNP antibodies, with the ultimate goal of designing a versatile device that is inexpensive, portable, reliable, and fast. We demonstrate detection of antibodies at concentrations that fall well within clinically relevant levels. PMID:24410628
Welch, M Elizabeth; Ritzert, Nicole L; Chen, Hongjun; Smith, Norah L; Tague, Michele E; Xu, Youyong; Baird, Barbara A; Abruńa, Héctor D; Ober, Christopher K
Antiphospholipid antibodies in critically ill patients
Antiphospholipid antibodies are responsible for a wide spectrum of clinical manifestations. Venous, arterial and microvascular thrombosis and severe catastrophic cases account for a large morbidly/mortality. Through the connection between the immune, inflammatory and hemostatic systems, it is possible that these antibodies may contribute to the development of organ dysfunction and are associated with poor short and long-term prognoses in critically ill patients. We performed a search of the PubMed/MedLine database for articles written during the period from January 2000 to February 2013 to evaluate the frequency of antiphospholipid antibodies in critically ill patients and their impact on the outcomes of these patients. Only eight original studies involving critically ill patients were found. However, the development of antiphospholipid antibodies in critically ill patients seems to be frequent, but more studies are necessary to clarify their pathogenic role and implications for clinical practice.
Vassalo, Juliana; Spector, Nelson; de Meis, Ernesto; Soares, Marcio; Salluh, Jorge Ibrain Figueira
Antibodies against degradation products of polysaccharide antigens
Specific antibodies against periodate oxidized capsular polysaccharides isolated from different Klebsiella types, have been demonstrated in rabbit immune sera against five out of eleven Klebsiella types investigated. It is assumed that an oxidation similar to the periodate oxidation must occur in vivo. Antibodies specifically directed against the oxidized polysaccharide may appear early in the immunization period, and decrease or disappear again later. Reduction and alkylation of the antibodies against oxidized polysaccharide in one serum indicated that they probably belonged to the ?M-immunoglobulins, whereas the antibodies against the unoxidized polysaccharide appear to be mainly ?G. ImagesFIG. 1FIG. 3FIG. 4FIG. 5FIG. 6FIG. 9FIG. 10FIG. 11
Eriksen, Jorunn
Localization of tumors by radiolabelled antibodies
A method of utilizing radiolabelled antibodies to carcinoembryonic antigens for determining the site of tumors which produce or are associated with carcinoembryonic antigen is disclosed. 3 claims, no drawings.
H. J. Hansen; F. J. Primus
Microtiter Plate Agglutination Test for 'Salmonella' Antibodies.
National Technical Information Service (NTIS)
Similar results were obtained when testing human sera for Salmonella antibodies by the tube agglutination test and by the microtiter plate agglutination test. The plate test was easier to perform and saved time, space, antigen, and serum. (Author)
I. S. Barsoum A. Y. Awad
Monoclonal Antibodies to Cholesterol and Methods.
National Technical Information Service (NTIS)
This patent application relates to monoclonal antibodies which demonstrate specific reactivity to cholesterol and methods for the detection of high levels of cholesterol by contacting biological specimens containing cholesterol with the monoclonal antibod...
C. R. Alving G. M. Swartz
Polynucleotides encoding anti-sulfotyrosine antibodies
The invention provides anti-sulfotyrosine specific antibodies capable of detecting and isolating polypeptides that are tyrosine-sulfated. The sulfotyrosine antibodies and antibody fragments of the invention may be used to discriminate between the non-sulfated and sulfated forms of such proteins, using any number of immunological assays, such ELISAs, immunoblots, Western Blots, immunoprecipitations, and the like. Using a phage-display system, single chain antibodies (scFvs) were generated and screened against tyrosine-sulfated synthetic peptide antigens, resulting in the isolation of scFvs that specifically recognize sulfotyrosine-containing peptides and/or demonstrate sulfotyrosine-specific binding in tyrosine sulfated proteins. The VH and VL genes from one such sulfotyrosine-specific scFv were employed to generate a full length, sulfotyrosine-specific immunoglobulin.
Bertozzi, Carolyn R. (Berkeley, CA); Kehoe, John (Saint Davids, PA); Bradbury, Andrew M. (Santa Fe, NM)
Antigen-antibody interactions in capillary electrophoresis.
Immunoreactions in combination with separations by capillary electrophoresis (CE) are increasingly being used to quantitate specific analytes in biological fluids. Both competitive and non-competitive approaches have been used for the purpose and, in selected cases, now compare favorably with conventional quantitative immunoassays with respect to concentration limits of detection. CE is also a useful method to evaluate antigen-antibody binding on-line and offers unique possibilities for binding constant estimates, also for weakly binding antibodies and antibody fragments. In this review we cover recent developments in the use of antigen-antibody interactions in conjunction with CE and conclude that continued development of miniaturization, on-line preconcentration and more sensitive detection schemes will contribute to the further dissemination of CE-based immunoassays building on already established affinity CE approaches. PMID:11939562
Heegaard, Niels H H; Kennedy, Robert T
Selection of antibody fragments by yeast display.
The critical need for renewable, high-quality affinity reagents in biological research, as well as for diagnostic and therapeutic applications, has required the development of new platforms of discovery. Yeast display is one of the main methods of in vitro display technology with phage display. Yeast display has been chosen by numerous groups to refine both affinity and specificity of antibodies because it enables fine discrimination between mutant clones of similar affinity. In addition, the construction of display libraries of antibody fragments in yeast permit to sample the immune antibody repertoire more fully than using phage. This chapter gives an updated overview of the available systems of yeast display platforms and libraries, followed up by technical descriptions of selection methods of antibody fragments by yeast display. PMID:22907357
Scholler, Nathalie
HIV antibody testing in hospitalized patients.
We retrospectively reviewed the charts of 190 hospitalized patients who had human immunodeficiency virus (HIV) antibody testing at our medical center in 1986 and 1987. From 1986 to 1987, HIV antibody testing increased fourfold based on total hospital discharges. Nine patients (5%) tested positive by enzyme immunoassay and the Western blot method. No risk factor for HIV infection was identified in 30% of cases. Documentation of patients' consent for testing was found in 14% of cases. We conclude that HIV antibody testing of hospitalized patients has increased. However, the indication for testing is often not clear by chart review, and consent for testing is poorly documented in the absence of a formal hospital policy requiring consent before testing. Further physician education and establishment of hospital policies addressing these important aspects of HIV antibody testing are indicated. PMID:2300830
Manian, F A; Rinke, D
Feeder Cells for Monoclonal Antibody Production.
National Technical Information Service (NTIS)
Monoclonal antibodies are important tools with a wide variety of diagnostic and therapeutic applications. They are also extremely useful in many biochemical research procedures. The invention relates to a cell line which efficiently supports the growth of...
H. Mischak W. Kolch F. Hofer U. R. Rapp
Non-fucosylated therapeutic antibodies: the next generation of therapeutic antibodies
Therapeutic antibody IgG1 has two N-linked oligosaccharide chains bound to the Fc region. The oligosaccharides are of the complex biantennary type, composed of a trimannosyl core structure with the presence or absence of core fucose, bisecting N-acetylglucosamine (GlcNAc), galactose, and terminal sialic acid, which gives rise to structural heterogeneity. Both human serum IgG and therapeutic antibodies are well known to be heavily fucosylated. Recently, antibody-dependent cellular cytotoxicity (ADCC), a lytic attack on antibody-targeted cells, has been found to be one of the critical effector functions responsible for the clinical efficacy of therapeutic antibodies such as anti-CD20 IgG1 rituximab (Rituxan®) and anti-Her2/neu IgG1 trastuzumab (Herceptin®). ADCC is triggered upon the binding of lymphocyte receptors (Fc?Rs) to the antibody Fc region. The activity is dependent on the amount of fucose attached to the innermost GlcNAc of N-linked Fc oligosaccharide via an ?-1,6-linkage, and is dramatically enhanced by a reduction in fucose. Non-fucosylated therapeutic antibodies show more potent efficacy than their fucosylated counterparts both in vitro and in vivo, and are not likely to be immunogenic because their carbohydrate structures are a normal component of natural human serum IgG. Thus, the application of non-fucosylated antibodies is expected to be a powerful and elegant approach to the design of the next generation therapeutic antibodies with improved efficacy. In this review, we discuss the importance of the oligosaccharides attached to the Fc region of therapeutic antibodies, especially regarding the inhibitory effect of fucosylated therapeutic antibodies on the efficacy of non-fucosylated counterparts in one medical agent. The impact of completely non-fucosylated therapeutic antibodies on therapeutic fields will be also discussed.
Mori, Katsuhiro; Iida, Shigeru; Yamane-Ohnuki, Naoko; Kanda, Yutaka; Kuni-Kamochi, Reiko; Nakano, Ryosuke; Imai-Nishiya, Harue; Okazaki, Akira; Shinkawa, Toyohide; Natsume, Akihito; Niwa, Rinpei; Shitara, Kenya
Antibody radiolabeling techniques to optimize cellular retention.
Radiolabeling of antibodies and antibody fragments facilitates the development of new targeted therapeutics or tracking and validation of biosimilars. The typical metal ion chelators that can be used for radiolabeling reactions have residualizing properties in tissues/tumors. A team at Genentech has developed an elegant new technique for combining iodine radiolabeling with an azamacrocyclic chelator to confer residualizing properties on the radioiodine metabolites. Robust protocols, such as this example, are required for the future development of protein based drugs. PMID:24283797
Archibald, Stephen J
Antibodies to Chlamydia trachomatis in acute salpingitis.
Recent isolation studies have shown Chlamydia trachomatis to be an important aetiological agent in acute salpingitis in women. The present serological study indicates that C. trachomatis is the probable aetiological agent in two-thirds of 143 women with pelvic inflammatory disease (PID). In general, high levels of chlamydial antibody were found in sera and fluids aspirated from the pouch of Douglas and such antibody titres were shown to correlate with the severity of clinically graded tubal inflammation. PMID:427512
Treharne, J D; Ripa, K T; Mĺrdh, P A; Svensson, L; Weström, L; Darougar, S
Monoclonal antibodies to human malignant mesothelioma
Murine monoclonal antibodies were used to identify tumor-cell membrane antigens on a new human mesothelioma cell line. Hybridomas were constructed by fusing SP2\\/0 mouse myeloma cells with spleen cells from Balb\\/C mice immunized by the human mesothelioma cell line MT-1. Hybridoma antibody was detected in 55\\/672 microculture wells that reacted to these MT-1 tumor cells by an indirect125I-protein A binding
Timothy M. Anderson; E. Carmack Holmes; Calvin J. Kosaka; Lorna Cheng; Romaine E. Saxton
Naturally occurring antibodies devoid of light chains
RANDOM association of VL and VH repertoires contributes considerably to antibody diversity1. The diversity and the affinity are then increased by hypermutation in B cells located in germinal centres2. Except in the case of 'heavy chain' disease3, naturally occurring heavy-chain antibodies have not been described, although antigen binding has been demonstrated for separated heavy chains4 or cloned VH domains5. Here
C. Hamers-Casterman; T. Atarhouch; S. Muyldermans; G. Robinson; C. Hammers; E. Bajyana Songa; N. Bendahman; R. Hammers
Selective Detection of Autoimmune Antibodies to Single
Presence in serum of anti-double-stranded deoxyribonucleic acid antibodies (anti-dsDNA) is one of the diagnostic criteria for systemic lupus erythematosus. Anti-single stranded DNA antibodies (anti-ssDNA) also occur, but their clinical significance is unclear. Use of enzyme immunoassay (EIA) kits that are specific for anti-dsDNA is desirable but problematic, since preparation of dsDNA of high integrity is difficult (ie, regions of ssDNA
Gregory S. Makowski; Melinda L. Ramsby
A novel strategy for generating monoclonal antibodies from single, isolated lymphocytes producing antibodies of defined specificities.
We report a novel approach to the generation of monoclonal antibodies based on the molecular cloning and expression of immunoglobulin variable region cDNAs generated from single rabbit or murine lymphocytes that were selected for the production of specific antibodies. Single cells secreting antibodies for a specific peptide either from gp116 of the human cytomegalovirus or from gp120 of HIV-1 or for sheep red blood cells were selected using antigen-specific hemolytic plaque assays. Sheep red blood cells were coated with specific peptides in a procedure applicable to any antigen that can be biotinylated. Heavy- and light-chain variable region cDNAs were rescued from single cells by reverse transcription-PCR and expressed in the context of human immunoglobulin constant regions. These chimeric murine and rabbit monoclonal antibodies replicated the target specificities of the original antibody-forming cells. The selected lymphocyte antibody method exploits the in vivo mechanisms that generate high-affinity antibodies. This method can use lymphocytes from peripheral blood, can exploit a variety of procedures that identify individual lymphocytes producing a particular antibody, and is applicable to the generation of monoclonal antibodies from many species, including humans. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4
Babcook, J S; Leslie, K B; Olsen, O A; Salmon, R A; Schrader, J W
Characterization of a monoclonal anti-idiotype antibody to human anti-factor VIII antibodies.
Approximately 15% of individuals with hemophilia A develop antibodies (inhibitors) to therapeutically infused factor VIII that interfere with F.VIII coagulant activity. By using isoelectric focusing and immunospecific detection of anti-factor VIII antibodies, inhibitor plasma showed varied patterns of reactivity characteristic of a polyclonal response. Inhibitor plasma from patient Bt was observed to have an isolated banding pattern, or spectrotype, at pI 8.4 (SP8.4) distinct from his remaining anti-factor VIII antibodies. SP8.4 antibodies from this patient were partially purified and used to prepare monoclonal anti-idiotype antibodies. Monoclonal antibody Mab20-2H was found to detect a spectrotype in isoelectric-focused Bt plasma identical to SP8.4 and to bind anti-factor VIII antibodies. Furthermore, Mab20-2H binding could inhibit the binding of these anti-factor VIII antibodies to antigen, indicating that Mab20-2H recognizes an idiotope associated with antigen binding. Mab20-2H was also found to recognize antibodies from another inhibitor patient. This and other anti-idiotype reagents will be useful for defining genetic factors involved in the human immune response to factor VIII and in designing approaches to prevent or ameliorate this response. Images
Lubahn, B C; Reisner, H M
Molecular farming of recombinant antibodies in plants.
Antibodies represent a large proportion of therapeutic drugs currently in development. In most cases, they are produced in mammalian cell lines or transgenic animals because these have been shown to fold and assemble the proteins correctly and generate authentic glycosylation patterns. However, such expression systems are expensive, difficult to scale up and there are safety concerns due to potential contamination with pathogenic organisms or oncogenic DNA sequences. Plants represent an inexpensive, efficient and safe alternative for the production of recombinant antibodies. Research over the last 10 years has shown that plants can produce a variety of functional antibodies and there is now intense interest in scaling up production to commercial levels. In this review, we discuss the advantages of plants over traditional expression systems, describe how antibody expression in plants is achieved and optimized and then consider the practical issues concerning large-scale molecular farming in plants. The first plant-produced therapeutic antibodies are already in clinical trials, and, given the economic benefits of this production system, we are likely to see many more recombinant antibodies produced in this manner in the future. PMID:12737305
Schillberg, S; Fischer, R; Emans, N
Antibody-mediated resistance against plant pathogens.
Plant diseases have a significant impact on the yield and quality of crops. Many strategies have been developed to combat plant diseases, including the transfer of resistance genes to crops by conventional breeding. However, resistance genes can only be introgressed from sexually-compatible species, so breeders need alternative measures to introduce resistance traits from more distant sources. In this context, genetic engineering provides an opportunity to exploit diverse and novel forms of resistance, e.g. the use of recombinant antibodies targeting plant pathogens. Native antibodies, as a part of the vertebrate adaptive immune system, can bind to foreign antigens and eliminate them from the body. The ectopic expression of antibodies in plants can also interfere with pathogen activity to confer disease resistance. With sufficient knowledge of the pathogen life cycle, it is possible to counter any disease by designing expression constructs so that pathogen-specific antibodies accumulate at high levels in appropriate sub-cellular compartments. Although first developed to tackle plant viruses and still used predominantly for this purpose, antibodies have been targeted against a diverse range of pathogens as well as proteins involved in plant-pathogen interactions. Here we comprehensively review the development and implementation of antibody-mediated disease resistance in plants. PMID:21872654
Safarnejad, Mohammad Reza; Jouzani, Gholamreza Salehi; Tabatabaei, Meisam; Tabatabaie, Meisam; Twyman, Richard M; Schillberg, Stefan
Antibody-based therapeutics in oncology.
The recent clinical and commercial success of anticancer antibodies, such as rituximab (Rituxan) and trastuzumab (Herceptin) has created great interest in antibody-based therapeutics for hematopoietic malignancies and solid tumors. Given the likely lower toxicity for antibodies versus small molecules, the potential increase in efficacy by conjugation to radioisotopes and other cellular toxins and the ability to characterize the target with clinical laboratory diagnostics to improve the drug's clinical performance, it is anticipated that current and future antibody therapeutics will find substantial roles alone and in combination therapy strategies for the treatment of patients with cancer. It is also likely that conjugation strategies will add new radiolabeled and toxin-linked products to the market to complement the recent approvals of ibritumomab tiuxetan (Zevalin) and gemtuzumab ozogamycin (Mylotarg). However, although there are a large number of agents in both early and later stages of clinical development, only a handful will make it through regulatory approval and become successful products. This review considers the structure of anticancer therapeutic antibodies, the techniques used to reduce their antigenicity, factors that influence efficacy and toxicity, conjugation with isotopes and toxins and antibody target validation. PMID:12597355
Ross, Jeffreys; Gray, Karen; Schenkein, David; Greene, Barry; Gray, Gary S; Shulok, Jeanine; Worland, Peter J; Celniker, Abbie; Rolfe, Mark
Antibody Responses during Hepatitis B Viral Infection.
Hepatitis B is a DNA virus that infects liver cells and can cause both acute and chronic disease. It is believed that both viral and host factors are responsible for determining whether the infection is cleared or becomes chronic. Here we investigate the mechanism of protection by developing a mathematical model of the antibody response following hepatitis B virus (HBV) infection. We fitted the model to data from seven infected adults identified during acute infection and determined the ability of the virus to escape neutralization through overproduction of non-infectious subviral particles, which have HBs proteins on their surface, but do not contain nucleocapsid protein and viral nucleic acids. We showed that viral clearance can be achieved for high anti-HBV antibody levels, as in vaccinated individuals, when: (1) the rate of synthesis of hepatitis B subviral particles is slow; (2) the rate of synthesis of hepatitis B subviral particles is high but either anti-HBV antibody production is fast, the antibody affinity is high, or the levels of pre-existent HBV-specific antibody at the time of infection are high, as could be attained by vaccination. We further showed that viral clearance can be achieved for low equilibrium anti-HBV antibody levels, as in unvaccinated individuals, when a strong cellular immune response controls early infection. PMID:25078553
Ciupe, Stanca M; Ribeiro, Ruy M; Perelson, Alan S
Decay of maternal antibodies in broiler chickens.
The objective of this study was to determine the decay rate of maternal antibodies against major broiler chicken pathogens. A total of 30 one-day-old broiler chicks were obtained from a commercial hatchery and reared in isolation. These chicks were retrieved from a parent flock that received a routine vaccination program. Chicks were bled at hatch and sequentially thereafter every 5 d through 30 d of age. Maternal antibody titers were measured by ELISA for avian encephalomyelitis (AEV), avian influenza virus (AIV), chicken anemia virus (CAV), infectious bursal disease virus (IBDV), infectious bronchitis virus (IBV), infectious laryngotracheitis virus (ILTV), Mycoplasma gallisepticum (MG), Mycoplasma synoviae (MS), and reovirus (Reo). Maternal antibody titers for Newcastle disease virus (NDV) were measured using a hemagglutination inhibition test. Half-life estimates of maternal antibody titers were 5.3, 4.2, 7, 5.1, 3.9, 3.8, 4.9, 4.1, 6.3, and 4.7 d for AEV, AIV, CAV, IBDV, IBV, ILTV, MG, MS, NDV, and Reo, respectively. The statistical analysis revealed significant differences among half-lives of maternal antibody titers against certain pathogens. Furthermore, all maternal antibody titers were depleted by 10 d of age except for IBDV. PMID:23960115
Gharaibeh, Saad; Mahmoud, Kamel
Anti Transglutaminase Antibodies Cause Ataxia in Mice
Background Celiac disease (CD) is an autoimmune gastrointestinal disorder characterized by the presence of anti-transglutaminase 2 (TG2) and anti-gliadin antibodies. Amongst the neurological dysfunctions associated with CD, ataxia represents the most common one. Methods We analyzed by immunohistochemistry, the anti-neural reactivity of the serum from 20 CD patients. To determine the role of anti-TG2 antibodies in ataxia, two anti-TG2 single chain variable fragments (scFv), isolated from a phage-display IgA antibody library, were characterized by immunohistochemistry and ELISA, and injected in mice to study their effects on motor coordination. We found that 75% of the CD patient population without evidence of neurological involvement, has circulating anti-neural IgA and/or IgG antibodies. Two anti-TG2 scFvs, cloned from one CD patient, stained blood vessels but only one reacted with neurons. This anti-TG2 antibody showed cross reactivity with the transglutaminase isozymes TG3 and TG6. Intraventricular injection of the anti-TG2 or the anti-TG2/3/6 cross-reactive scFv provoked transient, equally intensive ataxia in mice. Conclusion The serum from CD patients contains anti-TG2, TG3 and TG6 antibodies that may potentially cause ataxia.
Boscolo, Sabrina; Lorenzon, Andrea; Sblattero, Daniele; Florian, Fiorella; Stebel, Marco; Marzari, Roberto; Not, Tarcisio; Aeschlimann, Daniel; Ventura, Alessandro; Hadjivassiliou, Marios; Tongiorgi, Enrico
Broad neutralization coverage of HIV by multiple highly potent antibodies
Broadly neutralizing antibodies against highly variable viral pathogens are much sought after to treat or protect against global circulating viruses. Here we probed the neutralizing antibody repertoires of four human immunodeficiency virus (HIV)-infected donors with remarkably broad and potent neutralizing responses and rescued 17 new monoclonal antibodies that neutralize broadly across clades. Many of the new monoclonal antibodies are almost
Laura M. Walker; Michael Huber; Katie J. Doores; Emilia Falkowska; Robert Pejchal; Jean-Philippe Julien; Sheng-Kai Wang; Alejandra Ramos; Po-Ying Chan-Hui; Matthew Moyle; Jennifer L. Mitcham; Phillip W. Hammond; Ole A. Olsen; Pham Phung; Steven Fling; Chi-Huey Wong; Sanjay Phogat; Terri Wrin; Melissa D. Simek; Wayne C. Koff; Ian A. Wilson; Dennis R. Burton; Pascal Poignard
Neutralizing antibodies against HIV – back in the major leagues?
The past few months have seen encouraging successes for neutralizing antibodies against HIV; human monoclonal antibodies targeting conserved HIV envelope epitopes potently neutralized primary virus isolates, including strains of different clades. In primates, passive immunization with combinations containing human monoclonal antibodies completely prevented infection, even after mucosal virus challenges. Epitopes recognized by the protective monoclonal antibodies are important determinants for
Flavia Ferrantelli; Ruth M Ruprecht
Therapeutic monoclonal antibody for sporotrichosis
Sporotrichosis is a chronic subcutaneous mycosis that affects both humans and animals worldwide. This subcutaneous mycosis had been attributed to a single etiological agent, Sporothrix schenckii. S. schenckii exhibits considerable genetic variability, and recently, it was suggested that this taxon consists of a complex of species. Sporotrichosis is caused by traumatic inoculation of the fungus, which is a ubiquitous environmental saprophyte that can be isolated from soil and plant debris. The infection is limited to cutaneous forms, but recently, more severe clinical forms of this mycosis have been described, especially among immunocompromised individuals. The immunological mechanisms involved in the prevention and control of sporotrichosis are not well understood. Some studies suggest that cell-mediated immunity plays an important role in protecting the host against S. schenckii. In contrast, the role of the humoral immune response in protection against this fungus has not been studied in detail. In a previous study, we showed that antigens secreted by S. schenckii induced a specific humoral response in infected animals, primarily against a 70-kDa molecule, indicating a possible role of specific antibodies against this molecule in infection control. In another study by our group, we produced a mAb against a 70-kDa glycoprotein of S. schenckii to better understand the effect of the passive immunization of mice infected with S. schenckii. The results showed a significant reduction in the number of CFUs in various mice organs when the mAb was injected before or during S. schenckii infection. Similar results were observed when T-cell-deficient mice were used. The drugs of choice in the treatment of sporotrichosis require long periods, and relapses are frequently observed, primarily in immunocompromised patients. The strong protection induced by the mAb against a 70-kDa glycoprotein makes it a strong candidate as a therapeutic vaccine against sporotrichosis.
Almeida, Sandro R.
Rabbit monoclonal antibody: potential application in cancer therapy
By targeting antigens specifically, monoclonal antibodies represent a new class of therapeutic agents for the clinical management of various diseases including cancers. Monoclonal antibody technology has been greatly developed by reducing murine content in antibodies to minimize side effects in clinical applications. However, several intrinsic disadvantages of antibodies with murine origin limit the clinical efficacy of monoclonal antibodies based targeted therapy. The development of rabbit monoclonal antibody technology provides an alternative source of monoclonal antibodies with higher specificity and less cost for the development of routine targeted therapy against cancers.
Feng, Lifeng; Wang, Xian; Jin, Hongchuan
[Advances in the study of natural small molecular antibody].
Small molecule antibodies are naturally existed and well functioned but not structurally related to the conventional antibodies. They are only composed of heavy protein chains or light chains, much smaller than common antibody. The first small molecule antibody, called Nanobody was engineered from heavy-chain antibodies found in camelids. Cartilaginous fishes also have heavy-chain antibodies (IgNAR, "immunoglobulin new antigen receptor"), from which single-domain antibodies called Vnar fragments can be obtained. In addition, free light chain (FLC) antibodies in human bodies are being developed as therapeutic and diagnostic agents. Comparing to intact antibodies, common advantages of small molecule antibodies are with better solubility, tissue penetration, stability towards heat and enzymes, and comparatively low production costs. This article reviews the structural characteristics and mechanism of action of the Nanobody, IgNAR and FLC. PMID:23289139
Zhu, Lei; Zhang, Da-peng
[Idiotype-antiidiotypic interaction of antibodies in the bloodstream].
A problem of "prozone" formation in agglutination of corpuscular antibodies by bivalent antibodies was considered. Using a new coordinate system, which was proposed by us earlier, we analyzed theoretical and experimental curves that describe relations between bivalent antibody concentration and some blocking factors. It was shown that occupation of antibody paratopes by a blocking factor or by antiidiotypic antibodies can induce "prozone" formation. It was also demonstrated that experimental titration curves for a mixture of antibodies and corresponding antigens coincide with theoretical curves that were calculated according to our theory. Our data also demonstrate that major part of serum antibodies are blocked by antiidiotypic antibodies when maximum of antibody formation is over and antibody titers come down. PMID:24868915
Bobrovnik, S A
Binding of antibody to the surface of B lymphocytes was shown to involve the Fc piece of the immunoglobulin molecule. This property was not shared equally by all immunoglobulin classes as revealed by direct binding and inhibition studies. Total IgG globulin was found to label cells more heavily than IgM, and IgG1-containing fractions more heavily than IgG2 fractions lacking IgG1. The ability of various purified myeloma proteins to inhibit attachment of antibody to B cells was examined. Pretreatment of B cells with excess IgG2a, IgA, or light chain proteins failed to inhibit, whereas IgG1 proteins and to a lesser extent Ig2b and IgM proteins at the same concentrations did so. At lower protein concentrations, IgG1 myeloma protein alone retained the capacity to inhibit binding. The conclusion was reached that the receptor on B cells for antibody has a marked predilection for the IgG1 class. Although IgM and IgG2b antibody may bind, they do so with lower avidity and probably in insignificant amounts if IgG1 antibody is present in excess. No evidence was found to implicate complement in the binding process. For example, heat-inactivated sera at high dilution retained the ability to label B cells, while the use of purified low molecular weight anticomplementary factor, a potent inhibitor of C'3, did not interfere with the formation of the bond between antibody and cell surface. The failure of anti-mouse immunoglobulin F(ab)'2 fragments to prevent access of antibody to B cells implied that the antibody-binding receptor and antigen-binding (immunoglobulin) receptor are discrete entities on the B cell membrane. Despite this, a marked similarity between their surface distribution was observed on electron microscopy. The antibody-binding receptor was shown to be a marker for mature B cells. It did not appear to be present on hematopoietic precursor stem cells and was lost during differentiation to antibody-forming cells.
Basten, A.; Warner, N. L.; Mandel, T.
Cross-reactive broadly neutralizing antibodies: timing is everything
The recent surge of research into new broadly neutralizing antibodies in HIV-1 infection has recharged the field of HIV-1 vaccinology. In this review we discuss the currently known broadly neutralizing antibodies and focus on factors that may shape these antibodies in natural infection. We further discuss the role of these antibodies in the clinical course of the infection and consider immunological obstacles in inducing broadly neutralizing antibodies with a vaccine.
Euler, Zelda; Schuitemaker, Hanneke
Neutralization of HIV by Milk Expressed Antibody
Background In some areas of the world mother-to-child transmission of HIV remains a significant problem in part due to widespread breastfeeding which is essential due to scarce supply of a safe replacement, protection conferred by breast milk against many enteric illnesses, and cultural norms. We propose that sustained, adequate levels of protective antibodies in breast milk will prevent transmission of HIV. Methods The HIV neutralizing human monoclonal antibody b12 (IgG1) has been expressed as an IgA2 in CHO cells and shown to retain full immunoreactivity and neutralizing activity as the parental IgG1. The expression plasmids containing the b12 heavy and light chains were also used to construct milk specific expression vectors using the GTC goat ?-casein expression vector to direct expression of linked genes to the mammary gland with subsequent secretion into the milk. Female transgenic mice were generated and following parturition, their milk was tested for antibody immunoreactivity with gp120 and neutralization of HIV. Results When compared to CHO derived b12 IgA2 (or IgG1), immunoreactivity was retained. When tested for neutralization, milk derived b12 IgA2 was at least comparable to CHO derived antibody and in some cases superior to CHO derived antibody. Furthermore, milk that expressed b12 IgA2 was significantly more effective at mediating antibody dependent cell killing. Conclusions These results suggest it is possible to achieve functional HIV-specific mAb in the milk of transgenic mice and further investigations are warranted to explore ways for inducing this type of antibody response in the breast milk of HIV infected women.
Yu, Xiaocong; Pollock, Daniel; Duval, Mark; Lewis, Christopher; Joseph, Kristin; Meade, Harry; Cavacini, Lisa
Discovery of diverse and functional antibodies from large human repertoire antibody libraries.
Phage display antibody libraries have a proven track record for the discovery of therapeutic human antibodies, increasing the demand for large and diverse phage antibody libraries for the discovery of new therapeutics. We have constructed naďve antibody phage display libraries in both Fab and scFv formats, with each library having more than 250 billion clones that encompass the human antibody repertoire. These libraries show high fidelity in open reading frame and expression percentages, and their V-gene family distribution, VH-CDR3 length and amino acid usage mirror the natural diversity of human antibodies. Both the Fab and scFv libraries show robust sequence diversity in target-specific binders and differential V-gene usage for each target tested, supporting the use of libraries that utilize multiple display formats and V-gene utilization to maximize antibody-binding diversity. For each of the targets, clones with picomolar affinities were identified from at least one of the libraries and for the two targets assessed for activity, functional antibodies were identified from both libraries. PMID:23454004
Schwimmer, Lauren J; Huang, Betty; Giang, Hoa; Cotter, Robyn L; Chemla-Vogel, David S; Dy, Francis V; Tam, Eric M; Zhang, Fangjiu; Toy, Pamela; Bohmann, David J; Watson, Susan R; Beaber, John W; Reddy, Nithin; Kuan, Hua-Feng; Bedinger, Daniel H; Rondon, Isaac J
Association of Antipituitary Antibody and Type 2 Iodothyronine Deiodinase Antibody in Patients with Autoimmune Thyroid Disease
Antipituitary antibody (APA) has been reported to be detected in patients with autoimmune thyroid disease. Type 2 iodothyronine deiodinase (D2) is expressed in both pituitary gland and thyroid gland. We studied the association of APA and D2 peptide antibody in patients with autoimmune thyroid disease. Rat pituitary gland homogenate and D2 peptide were used as antigens in the present study.
Rieko NAKAHARA; Katsuhiko TSUNEKAWA; Shigeki YABE; Misa NARA; Koji SEKI; Takayuki KASAHARA; Takayuki OGIWARA; Michio NISHINO; Isao KOBAYASHI; Masami MURAKAMI
The antibody mining toolbox: an open source tool for the rapid analysis of antibody repertoires.
In vitro selection has been an essential tool in the development of recombinant antibodies against various antigen targets. Deep sequencing has recently been gaining ground as an alternative and valuable method to analyze such antibody selections. The analysis provides a novel and extremely detailed view of selected antibody populations, and allows the identification of specific antibodies using only sequencing data, potentially eliminating the need for expensive and laborious low-throughput screening methods such as enzyme-linked immunosorbant assay. The high cost and the need for bioinformatics experts and powerful computer clusters, however, have limited the general use of deep sequencing in antibody selections. Here, we describe the AbMining ToolBox, an open source software package for the straightforward analysis of antibody libraries sequenced by the three main next generation sequencing platforms (454, Ion Torrent, MiSeq). The ToolBox is able to identify heavy chain CDR3s as effectively as more computationally intense software, and can be easily adapted to analyze other portions of antibody variable genes, as well as the selection outputs of libraries based on different scaffolds. The software runs on all common operating systems (Microsoft Windows, Mac OS X, Linux), on standard personal computers, and sequence analysis of 1-2 million reads can be accomplished in 10-15 min, a fraction of the time of competing software. Use of the ToolBox will allow the average researcher to incorporate deep sequence analysis into routine selections from antibody display libraries. PMID:24423623
D'Angelo, Sara; Glanville, Jacob; Ferrara, Fortunato; Naranjo, Leslie; Gleasner, Cheryl D; Shen, Xiaohong; Bradbury, Andrew R M; Kiss, Csaba
The indirect fluorescent antibody technique as a method for detecting antibodies in aborted fetuses.
In this investigation the indirect fluorescent antibody technique was used to titrate antibodies in bovine sera to parainfluenza 3, infectious bovine rhinotracheitis virus and bovine viral diarrhea virus. These results were compared to those determined on the same samples by hemagglutination inhibition for parainfluenza 3 virus and serum neutralization for bovine virus diarrhea and infectious bovine rhinotracheitis virus. The results of the serological methods agreed closely. The indirect fluorescent antibody technique is a rapid and sensitive method for detecting antibodies and the procedure lends itself to use in diagnostic laboratories. In addition to the above viruses the presence or absence of antibodies to bovine coronavirus and bovine adenovirus 3 were determined by the indirect fluorescent antibody technique in thoracic fluids from 100 aborted fetuses and 50 nonaborted fetuses. Results on these samples were not compared to hemagglutination inhibition or serum neutralization as the condition of fluid samples from aborted fetuses renders interpretation of such tests unreliable. Antibodies to one or more viruses were detected in 30 of the 100 aborted fetuses and in seven of the 50 nonaborted fetuses. Antibodies to more than one agent were detected in eleven of the 100 aborted and in one of the 50 nonaborted fetuses. Reasons for this occurrence and application of the test in determination of causes of abortion are discussed. PMID:226243
Miller, R B; Wilkie, B N
The indirect fluorescent antibody technique as a method for detecting antibodies in aborted fetuses.
In this investigation the indirect fluorescent antibody technique was used to titrate antibodies in bovine sera to parainfluenza 3, infectious bovine rhinotracheitis virus and bovine viral diarrhea virus. These results were compared to those determined on the same samples by hemagglutination inhibition for parainfluenza 3 virus and serum neutralization for bovine virus diarrhea and infectious bovine rhinotracheitis virus. The results of the serological methods agreed closely. The indirect fluorescent antibody technique is a rapid and sensitive method for detecting antibodies and the procedure lends itself to use in diagnostic laboratories. In addition to the above viruses the presence or absence of antibodies to bovine coronavirus and bovine adenovirus 3 were determined by the indirect fluorescent antibody technique in thoracic fluids from 100 aborted fetuses and 50 nonaborted fetuses. Results on these samples were not compared to hemagglutination inhibition or serum neutralization as the condition of fluid samples from aborted fetuses renders interpretation of such tests unreliable. Antibodies to one or more viruses were detected in 30 of the 100 aborted fetuses and in seven of the 50 nonaborted fetuses. Antibodies to more than one agent were detected in eleven of the 100 aborted and in one of the 50 nonaborted fetuses. Reasons for this occurrence and application of the test in determination of causes of abortion are discussed.
Miller, R B; Wilkie, B N
Monoclonal antibodies against farmer's lung antigens having specific binding to IgG antibodies.
Hypersensitivity pneumonitis resulting from environmental exposure to Saccharopolyspora rectivirgula (Micropolyspora faeni) among farmers has been well recognized. The diagnosis of the disease depends on demonstration of circulating antibodies against S. rectivirgula. However, dependable pure antigens are not available for serodiagnosis. In the present study we have employed hybridoma technology to obtain monoclonal antibodies against S. rectivirgula antigens. These monoclonal antibodies were employed to purify antigens through affinity chromatography. When tested in ELISA, high levels of antibodies were demonstrated against these antigens in farmer's lung patient sera compared to exposed but asymptomatic individuals from the same household. In Western blots patient sera reacted with components of crude antigens with molecular masses of 28, 35, 60, 65 and 68 kD and 4 components above 100 kD, while the monoclonal antibodies reacted only with the 60-kD protein. These purified antigens can be used as reliable reagents in the specific diagnosis of farmer's lung disease. PMID:8400887
Kumar, A; Elms, N; Kurup, V P
A mutagenesis study of a catalytic antibody.
We have generated seven site-specific mutations in the genes encoding the variable region of the heavy chain domain (VH) of the phosphocholine-binding antibody S107. S107 is a member of a family of well-characterized highly homologous antibodies that bind phosphorylcholine mono- and diesters. Two of these antibodies, MOPC-167 and T15, have previously been shown to catalyze the hydrolysis of 4-nitrophenyl N-trimethylammonioethyl carbonate. Two conserved heavy-chain residues, Tyr-33 and Arg-52, were postulated to be involved in binding and hydrolysis of 4-nitrophenylcholine carbonate esters. To more precisely define the catalytic roles of these residues, three Arg-52 mutants (R52K, R52Q, R52C) and four Tyr-33 mutants (Y33H, Y33F, Y33E, Y33D) of antibody S107 were generated. The genes encoding the VH binding domain of S107 were inserted into plasmid pUC-fl, and in vitro mutagenesis was performed. The wild-type and mutant S107 antibodies were expressed in P-3X63-Ag8.653 (P-3) myeloma cells by using a modified SV2 shuttle vector. The catalytic properties of wild-type antibody S107 are similar to those of the phosphocholine-specific antibody T15, which has the same VH protein sequence. In general, mutations at Tyr-33 had little effect on catalytic activity, whereas mutations at Arg-52 that result in loss of the positively charged side chain significantly lower the catalytic activity of S107. One mutant, Y33H, catalyzed the hydrolysis of 4-nitrophenyl N-trimethylammonioethyl carbonate with a kcat of 5.7 min-1 and a Km of 1.6 mM at pH 7.5. These results not only demonstrate the importance of electrostatic interactions in catalysis by antibody S107 but also show that catalytic side chains can be introduced into antibodies to enhance their catalytic efficiency.
Jackson, D Y; Prudent, J R; Baldwin, E P; Schultz, P G
Single-domain antibodies and their utility.
Engineered monoclonal antibody fragments have gained market attention due to their versatility and tailor-made potential and are now considered to be an important part of future immunobiotherapeutics. Single-domain antibodies (sdAbs), also known as nanobodies, are derived from VHHs [variable domains (V) of heavy-chain-only antibodies (HCAb)] of camelid heavy-chain antibodies. These nature-made sdAbs are well suited for various applications due to their favorable characteristics such as small size, ease of genetic manipulation, high affinity and solubility, overall stability, resistance to harsh conditions (e.g., low pH, high temperature), and low immunogenicity. Most importantly, sdAbs have the feature of penetrating into cavities and recognizing hidden epitopes normally inaccessible to conventional antibodies, mainly due to their protruding CDR3/H3 loops. In this unit, we will present and discuss comprehensive and step-by-step protocols routinely practiced in our laboratory for isolating sdAbs from immunized llamas (or other members of the Camelidae family) against target antigens using phage-display technology. Expression, purification, and characterization of the isolated sdAbs will then be described, followed by presentation of several examples of applications of sdAbs previously characterized in our laboratory and elsewhere. PMID:24510545
Baral, Toya Nath; MacKenzie, Roger; Arbabi Ghahroudi, Mehdi
Defining antibody targets in Streptococcus oralis infection.
Immunoblotting of sera from 12 neutropenic patients with Streptococcus oralis septicemia and 18 patients with endocarditis due to viridans group streptococci revealed immunodominant S. oralis antigens at 85 and 180 kDa. The former cross-reacted with a mouse monoclonal antibody to hsp90. The latter was identified by sequencing positive clones obtained by screening a genomic expression library of S. oralis with pooled sera from patients who had been infected with S. oralis. Antibody eluted from one of these clones reacted with the 180-kDa antigen of S. oralis. Southern blotting confirmed the origin of the clone from S. oralis. The derived amino acid sequence showed 76.2% homology with the PAc protein precursor of Streptococcus mutans and 73.8% homology with the SpaA protein precursor of Streptococcus sobrinus. Epitope mapping of the derived amino acid sequence with sera from patients with viridans group streptococcal endocarditis delineated nine epitopes. Peptides 1 (TMYPNRQPGSGWDSS) and 2 (WYSLNGKIRAVDVPK), representing two of these epitopes, and peptide 3 (YEVEKPLEPAPVAPS), representing the repeat proline region, were synthesized. These three peptides were used to screen a phage antibody display library derived from a patient who had recovered from S. oralis infection. Two of the human recombinant antibodies produced (SORAL 3 and SORAL 4 against peptide 3) and a human recombinant antibody (B3.7) against the conserved epitope (LKVIRK) of hsp90 gave statistically significant protection, compared with control groups, in a mouse model of lethal S. oralis infection.
Burnie, J P; Brooks, W; Donohoe, M; Hodgetts, S; al-Ghamdi, A; Matthews, R C
Antibody phage display technology and its applications.
In recent years, the use of display vectors and in vitro selection technologies has transformed the way in which we generate ligands, such as antibodies and peptides, for a given target. Using this technology, we are now able to design repertoires of ligands from scratch and use the power of phage selection to select those ligands having the desired (biological) properties. With phage display, tailor-made antibodies may be synthesized and selected to acquire the desired affinity of binding and specificity for in vitro and in vivo diagnosis, or for immunotherapy of human disease. This review addresses recent progress in the construction of, and selection from phage antibody libraries, together with novel approaches for screening phage antibodies. As the quality of large naďve and synthetic antibody repertoires improves and libraries becomes more generally available, new and exciting applications are pioneered such as the identification of novel antigens using differential selection and the generation of receptor a(nta)gonists. A combination of the design and generation of millions to billions of different ligands, together with phage display for the isolation of binding ligands and with functional assays for identifying (and possibly selecting) bio-active ligands, will open even more challenging applications of this inspiring technology, and provide a powerful tool for drug and target discovery well into the next decade. PMID:9661810
Hoogenboom, H R; de Bruďne, A P; Hufton, S E; Hoet, R M; Arends, J W; Roovers, R C
Antibody Phage Display Libraries: Contributions to Oncology
Since the advent of phage display technology, dating back to 1985, antibody libraries displayed on filamentous phage surfaces have been used to identify specific binders for many different purposes, including the recognition of tumors. Phage display represents a high-throughput technique for screening billions of random fusion antibodies against virtually any target on the surface or inside cancer cells, or even soluble markers found in patient serum. Many phage display derived binders targeting important tumor markers have been identified. Selection directed to tumoral cells' surfaces lead to the identification of unknown tumoral markers. Also the improvement of methods that require smaller amounts of cells has opened the possibility to use this approach on patient samples. Robust techniques combining an antibody library displayed on the phage surface and protein microarray allowed the identification of auto antibodies recognized by patient sera. Many Ab molecules directly or indirectly targeting angiogenesis have been identified, and one of them, ramucirumab, has been tested in 27 phase I–III clinical trials in a broad array of cancers. Examples of such antibodies will be discussed here with emphasis on those used as probes for molecular imaging and other clinical trials.
Dantas-Barbosa, Carmela; de Macedo Brigido, Marcelo; Maranhao, Andrea Queiroz
Anti-ribosomal P antibody in schizophrenia.
Background: A series of epidemiological, clinical and laboratory findings suggest an autoimmune process in schizophrenia and include, among others, high titers of various autoantibodies in the sera of patients. Antiribosomal P antibody is known to exist in systemic lupus erythematosus (SLE) patients with a psychiatric presentation, including psychosis, rationalizing the examination of its existence in patients with schizophrenia. Methods: Sera of 59 patients, 48 diagnosed with schizophrenia and 11 diagnosed with a schizoaffective disorder, were examined for the presence of antiribosomal P antibody titers using ELISA. The control group consisted of 94 healthy subjects with similar age and gender distribution. Results: Anti-ribosomal P antibody titers were below cut-off level in 58 patients and borderline in one patient, similar to the low titers of the control group. Conclusions: Previous investigations have demonstrated high specificity for anti-ribosomal P antibody in SLE patients with psychosis. In view of the results of this study, however, anti-ribosomal P antibody is not a biological marker for schizophrenia. PMID:22572091
Gilat, Yaron; Shoenfeld, Yehuda; Kotler, Moshe; Iancu, Iulian
Antibody Fragments as Probe in Biosensor Development
Today's proteomic analyses are generating increasing numbers of biomarkers, making it essential to possess highly specific probes able to recognize those targets. Antibodies are considered to be the first choice as molecular recognition units due to their target specificity and affinity, which make them excellent probes in biosensor development. However several problems such as difficult directional immobilization, unstable behavior, loss of specificity and steric hindrance, may arise from using these large molecules. Luckily, protein engineering techniques offer designed antibody formats suitable for biomarker analysis. Minimization strategies of antibodies into Fab fragments, scFv or even single-domain antibody fragments like VH, VL or VHHs are reviewed. Not only the size of the probe but also other issues like choice of immobilization tag, type of solid support and probe stability are of critical importance in assay development for biosensing. In this respect, multiple approaches to specifically orient and couple antibody fragments in a generic one-step procedure directly on a biosensor substrate are discussed.
Saerens, Dirk; Huang, Lieven; Bonroy, Kristien; Muyldermans, Serge
Novel antibody therapy in acute lymphoblastic leukemia.
The treatment of adult acute lymphoblastic leukemia (ALL) poses a tremendous challenge for hematologists. The use of pediatric-based chemotherapy regimens in young adults up to the age of 45 years has resulted in improved outcomes when compared retrospectively with historical controls treated with adult therapy. A better understanding of the molecular landscape of ALL and advances in the field of monoclonal antibody therapy have resulted in the development of several new agents that may provide for a reduction in the toxicity inherent in pediatric-like regimens. The anti-CD20 antibody, rituximab, anti CD22 antibody, epratuzumab, anti-CD22 antibody-drug conjugate, Inotuzumab ozogamicin, the bi-specific T-cell engager (BiTE) antibody, Blinatumomab, and chimeric receptor antigen (CAR) therapy are among the emerging agents that have demonstrated the potential to improve response rate and decrease toxicity when used alone or in combination with chemotherapy. Several role-defining phase II and phase III clinical trials with these agents are currently underway in the relapsed/refractory and newly diagnosed ALL settings. PMID:24623281
Kochuparambil, Samith T; Litzow, Mark R
Monoclonal antibodies for the treatment of asthma.
Asthma is a chronic inflammatory disease of the airways which can have a detrimental effect on quality of life and in extreme cases cause death. Although the majority of patients can control their asthma symptoms with a combination of steroids and beta agonists there is still a group of patients whose asthma remains symptomatic despite the best available treatment. These severe asthmatic patients represent the unmet medical need in asthma and are the focus of those developing novel monoclonal antibody based drugs. The complex networks of cytokines and cells involved in the pathology of asthma provide plenty of scope for intervention with monoclonal antibody based drugs which are able to block cytokine or chemokine receptor interactions, deplete cells expressing a specific receptor or block cell/cell interactions. At present anti-IgE (Xolair©) is the only monoclonal antibody based drug approved for the treatment of asthma. However, a number of other antibody based drugs have been clinically tested in asthma including anti-IL-5, anti-IL-4, anti-IL-13, anti-TNF?, anti-CCR3, anti-CCR4 and anti-OX40L. This review will examine the development of these monoclonal antibody based therapies. Since many of these therapies have targeted key pathways in asthma pathology these studies provide information on patient stratification and asthma pathology. PMID:21944943
Catley, Matthew C; Coote, Julie; Bari, Mohamed; Tomlinson, Kate L
Homogeneity of Antibody Responses in Tuberculosis Patients
The goals of the present study were twofold: (i) to compare the repertoires of antigens in culture filtrates of in vitro-grown Mycobacterium tuberculosis that are recognized by antibodies from noncavitary and cavitary tuberculosis (TB) patients and (ii) to determine the extent of variation that exists between the antigen profiles recognized by individual TB patients. Lipoarabinomannan-free culture filtrate proteins of M. tuberculosis were fractionated by one-dimensional (1-D) and 2-D polyacrylamide gel electrophoresis, and the Western blots were probed with sera from non-human immunodeficiency virus (non-HIV)-infected cavitary and noncavitary TB patients and from HIV-infected, noncavitary TB patients. In contrast to earlier studies based on recombinant antigens of M. tuberculosis which suggested that antibody responses in TB patients were heterogeneous (K. Lyashchenko et al., 1998, Infect. Immun. 66:3936–3940, 1998), our studies with native culture filtrate proteins show that the antibody responses in TB patients show significant homogeneity in being directed against a well-defined subset of antigens. Thus, there is a well-defined subset of culture filtrate antigens that elicits antibodies during noncavitary and cavitary disease. In addition, another set of antigens is recognized primarily by cavitary TB patients. The mapping with individual patient sera presented here suggests that serodiagnostic tests based on the subset of antigens recognized during both noncavitary and cavitary TB will enhance the sensitivity of antibody detection in TB patients, especially in difficult-to-diagnose, smear-negative, noncavitary TB patients.
Samanich, K.; Belisle, J. T.; Laal, S.
Production of camel-like antibodies in plants.
Transgenic plants for the production of high-value recombinant complex and/or glycosylated proteins are a promising alternative for conventional systems, such as mammalian cells and bacteria. Many groups use plants as production platform for antibodies and antibody fragments. Here, we describe how bivalent camel-like antibodies can be produced in leaves and seeds. Camel-like antibodies are fusions of the antigen-binding domain of heavy chain camel antibodies (VHH) with an Fc fragment of choice. Transient expression in Nicotiana benthamiana leaves allows the production of VHH-Fc antibodies within a few days after the expression plasmid has been obtained. Generation of stable Arabidopsis thaliana transformants allows production of scalable amounts of VHH-Fc antibodies in seeds within a year. Further, we describe how the in planta-produced VHH-Fc antibodies can be quantified by Western blot analysis with Fc-specific antibodies. PMID:22886260
De Buck, Sylvie; Virdi, Vikram; De Meyer, Thomas; De Wilde, Kirsten; Piron, Robin; Nolf, Jonah; Van Lerberge, Els; De Paepe, Annelies; Depicker, Ann
Further characterization of cooperative interactions of monoclonal antibodies.
We reported previously that mixtures of some monoclonal antibodies directed against the glycoprotein hormone human chorionic gonadotropin (hCG) had a higher affinity for the antigen than either monoclonal antibody separately. The synergistic interaction could no longer be detected when one of the antibodies was replaced with its F(ab) fragment. This cooperative interaction has now been further characterized. One-half of 10 possible pairs prepared from five IgG1 monoclonal antibodies against hCG result in a synergistic interaction. The addition of an IgG2b monoclonal antibody to one of the IgG1 monoclonal antibodies also induces a cooperative interaction, which shows that the effect is not subclass restricted. Cooperative interactions between antibodies are also not restricted to solution conditions; adsorption of one antibody to a solid support appears to increase the cooperative effect. Indeed, one pair of antibodies that failed to bind hCG synergistically in solution did so when one antibody was bound to a solid surface. The liquid phase antibody also has an effect on the specificity of the solid phase antibody. The sensitivity of the solid phase assay system has enabled us to develop a rapid method of determining if two monoclonal antibodies can bind to an antigen simultaneously. A quantitative theoretical model has been devised that successfully predicts the cooperative behavior observed between antibodies and should be useful in devising conditions that result in sensitive solid phase radioimmunoassays. PMID:6619545
Ehrlich, P H; Moyle, W R; Moustafa, Z A
Production of therapeutic antibodies in plants.
Antibodies are versatile tools for the diagnosis and treatment of many diseases. Their use has increased dramatically with the advent of recombinant antibody (rAb) technology, allowing the production of immunological reagents with improved and novel properties. The main challenge now lies in achieving cost-effective production on a large scale. Over the past 15 years, the potential of plants for the production of pharmaceutical proteins has become well-established. Plants represent an inexpensive, efficient and safe alternative to traditional systems used for the commercial-scale synthesis of rAbs. This review describes the current status of antibody production in plants, focusing on their advantages compared with other expression systems and the remaining obstacles to widespread acceptance. PMID:14519078
Nölke, Greta; Fischer, Rainer; Schillberg, Stefan
Antibody-based bacterial toxin detection
NASA Astrophysics Data System (ADS)
Fiber optic evanescent fluorosensors are under investigation in our laboratory for the study of drug-receptor interactions for detection of threat agents and antibody-antigen interactions for detection of biological toxins. In a one step assay, antibodies against Cholera toxin or Staphylococcus Enterotoxin B were noncovalently immobilized on quartz fibers and probed with fluorescein-isothiocyanate (FITC)-labeled toxins. In the two-step assay, Cholera toxin or Botulinum toxoid A was immobilized onto the fiber, followed by incubation in an antiserum or partially purified antitoxin IgG. These were then probed with FITC-anti-IgG antibodies. Unlabeled toxins competed with labeled toxins or antitoxin IgG in a dose-dependent manner and the detection of the toxins was in the nanomolar range.
Menking, Darrell E.; Heitz, Jonathon M.; Anis, Nabil A.; Thompson, Roy G.
Monoclonal antibodies against the aster yellows agent.
Hybridoma clones secreting specific monoclonal antibodies against the aster yellows agent, a mycoplasma-like organism, were produced by using partially purified salivary gland preparations from infected leafhopper vectors as the immunogen. After 3947 hybridomas from 20 independent fusions were screened for specific antibody against the aster yellows agent, two table clones were obtained. With these monoclonal antibodies the aster yellows agent in diseased lettuce, periwinkles, and inoculative insects was specifically identified by enzyme-linked immunosorbent assay. The aster yellows agent was serologically differentiated from the mycoplasma-like organisms associated with ash yellows, loofah witches'-broom, paulownia witches'-broom, sweet potato witches'-broom, peanut rosette, maize bushy stunt, and elm phloem necrosis. PMID:17757867
Lin, C P; An Chen, T
Method for preparation of single chain antibodies
This invention provides a method for identifying cells expressing a target single chain antibody (scFv) directed against a target antigen from a collection of cells that includes cells that do not express the target scFv, comprising the step of combining the collection of cells with an anti-idiotype directed to an antibody specific for the target antigen and detecting interaction, if any, of the anti-idiotype with the cells, wherein the occurrence of an interaction identifies the cell as one which expresses the target scFv. This invention also provides a method for making a single chain antibody (scFv) directed against an antigen, wherein the selection of clones is made based upon interaction of those clones with an appropriate anti-idiotype, and heretofore inaccessible scFv so made. This invention provides the above methods or any combination thereof. Finally, this invention provides various uses of these methods.
Cheung, Nai-Kong V. (New York, NY); Guo, Hong-fen (New York, NY)
Method for altering antibody light chain interactions
A method for recombinant antibody subunit dimerization including modifying at least one codon of a nucleic acid sequence to replace an amino acid occurring naturally in the antibody with a charged amino acid at a position in the interface segment of the light polypeptide variable region, the charged amino acid having a first polarity; and modifying at least one codon of the nucleic acid sequence to replace an amino acid occurring naturally in the antibody with a charged amino acid at a position in an interface segment of the heavy polypeptide variable region corresponding to a position in the light polypeptide variable region, the charged amino acid having a second polarity opposite the first polarity. Nucleic acid sequences which code for novel light chain proteins, the latter of which are used in conjunction with the inventive method, are also provided.
Stevens, Fred J. (Naperville, IL); Stevens, Priscilla Wilkins (Evanston, IL); Raffen, Rosemarie (Elmhurst, IL); Schiffer, Marianne (Downers Grove, IL)
Multiplicity of antibodies in myositis sera.
Serologic studies on 114 patients with polymyositis and dermatomyositis revealed that 89% had either a precipitating antibody to antigens in calf thymus extract or a positive immunofluorescent reaction on HEp-2 cells, a human tissue culture line. Previously, the greatest proportion of polymyositis sera demonstrating positive serologic results (i.e., the proportion of patients' sera forming precipitates with calf thymus extract) was reported to be 60%. Use of the HEp-2 cell as immunofluorescent substrate enabled the detection of antibody in 89 (78%) of the sera, providing the additional probe which demonstrated specific antibody. Nuclear, cytoplasmic, and nucleolar staining are the most common patterns of immunofluorescence. The immunofluorescent patterns and individual precipitin reactions are related to each other and to the clinical syndromes in which they appear. PMID:6386003
Reichlin, M; Arnett, F C
SUMMMARY Patterns of serum and C.S.F. IgG, IgA and IgM, and haemagglutination inhibiting (HI) antibodies and complement fixing (CF) antibodies against rubella and cytomegaloviruses respectively and total CSF protein were investigated in 30 depressives, 20 each in neurological and surgical patients. Significant changes in total CSF protein and immunoglobulins in depressives and neurological patients in comparison to surgical subjects indicate an etiological similarity between the two groups. Failure to detect antibodies in C.S.F. of these subjects and statistically insignificant seropositivity refute the claim of viral hypothesis for depression but similar alterations in these body proteins in depressives and neurological patients raise other aetiological possibilities.
Tiwari, S.C.; Lal, N.; Trivedi, J.K.; Chaturvedi, U.C.; Varma, S.L.; Bahuguna, L.M.
Antibody-Based Therapies in Multiple Myeloma
The unmet need for improved multiple myeloma (MM) therapy has stimulated clinical development of monoclonal antibodies (mAbs) targeting either MM cells or cells of the bone marrow (BM) microenvironment. In contrast to small-molecule inhibitors, therapeutic mAbs present the potential to specifically target tumor cells and directly induce an immune response to lyse tumor cells. Unique immune-effector mechanisms are only triggered by therapeutic mAbs but not by small molecule targeting agents. Although therapeutic murine mAbs or chimeric mAbs can cause immunogenicity, the advancement of genetic recombination for humanizing rodent mAbs has allowed large-scale production and designation of mAbs with better affinities, efficient selection, decreasing immunogenicity, and improved effector functions. These advancements of antibody engineering technologies have largely overcome the critical obstacle of antibody immunogenicity and enabled the development and subsequent Food and Drug Administration (FDA) approval of therapeutic Abs for cancer and other diseases.
Tai, Yu-Tzu; Anderson, Kenneth C.
Proteomic identification of monoclonal antibodies from serum.
Characterizing the in vivo dynamics of the polyclonal antibody repertoire in serum, such as that which might arise in response to stimulation with an antigen, is difficult due to the presence of many highly similar immunoglobulin proteins, each specified by distinct B lymphocytes. These challenges have precluded the use of conventional mass spectrometry for antibody identification based on peptide mass spectral matches to a genomic reference database. Recently, progress has been made using bottom-up analysis of serum antibodies by nanoflow liquid chromatography/high-resolution tandem mass spectrometry combined with a sample-specific antibody sequence database generated by high-throughput sequencing of individual B cell immunoglobulin variable domains (V genes). Here, we describe how intrinsic features of antibody primary structure, most notably the interspersed segments of variable and conserved amino acid sequences, generate recurring patterns in the corresponding peptide mass spectra of V gene peptides, greatly complicating the assignment of correct sequences to mass spectral data. We show that the standard method of decoy-based error modeling fails to account for the error introduced by these highly similar sequences, leading to a significant underestimation of the false discovery rate. Because of these effects, antibody-derived peptide mass spectra require increased stringency in their interpretation. The use of filters based on the mean precursor ion mass accuracy of peptide-spectrum matches is shown to be particularly effective in distinguishing between "true" and "false" identifications. These findings highlight important caveats associated with the use of standard database search and error-modeling methods with nonstandard data sets and custom sequence databases. PMID:24684310
Boutz, Daniel R; Horton, Andrew P; Wine, Yariv; Lavinder, Jason J; Georgiou, George; Marcotte, Edward M
Anticardiolipin antibodies in patients with Behcet's disease.
The aims of this study are to determine anticardiolipin antibodies in patients with Sy Behcet and to determine correlation between the levels of anticardiolipin antibodies in serum in patients with clinic systemic and ocular manifestations. The study was conducted on 11 patients with Behcet disease (group I), and on 11 healthy subjects (group II). Anticardiolipin antibodies -aCL were determined by the standard ELISA method, where 1GPL= 1 microgram/ml IgG aCL and 1 MPL= 1 microgram/ ml IgM, and were considered negative < 10 GPL or MPL, low positive (10-40 GPL and MPL), or high positive (>40 GPL and MPL). In the group of 11 patients with the diagnosis Sy Behcet, 6 of them were (54.5%) with values of anticardiolipin antibodies over 10 positive. In the control group of the healthy examinees aCl were positive in 2 cases (18.2%). There are no statistically significant differences in the presence of systemic clinic characteristics between aCl positive and negative patients. All the patients with SY Behcet in whom anticardiolipn antibodies were found have extremely severe visual damage which is not present in the group of those patients where the values of aCl were low. The difference is statistically significant. The level of anticardiolipin antibodies is increased in the patients with Behcet. There are no statistically significant differences in the presence of systemic clinical characteristics between aCL positive and negative patients. Visual acuity in patients with SY Behcet is statistically significantly much lower in patients who had increased values of aCL. PMID:21342144
Zivkovic, Maja; Zlatanovic, Marko; Zlatanovic, Gordana; Djordjevic-Jocic, Jasmina; Cekic, Sonja
Behaviour of Non-Donor Specific Antibodies during Rapid Re-Synthesis of Donor Specific HLA Antibodies after Antibody Incompatible Renal Transplantation
Background HLA directed antibodies play an important role in acute and chronic allograft rejection. During viral infection of a patient with HLA antibodies, the HLA antibody levels may rise even though there is no new immunization with antigen. However it is not known whether the converse occurs, and whether changes on non-donor specific antibodies are associated with any outcomes following HLA antibody incompatible renal transplantation. Methods 55 patients, 31 women and 24 men, who underwent HLAi renal transplant in our center from September 2005 to September 2010 were included in the studies. We analysed the data using two different approaches, based on; i) DSA levels and ii) rejection episode post transplant. HLA antibody levels were measured during the early post transplant period and corresponding CMV, VZV and Anti-HBs IgG antibody levels and blood group IgG, IgM and IgA antibodies were quantified. Results Despite a significant DSA antibody rise no significant non-donor specific HLA antibody, viral or blood group antibody rise was found. In rejection episode analyses, multiple logistic regression modelling showed that change in the DSA was significantly associated with rejection (p?=?0.002), even when adjusted for other antibody levels. No other antibody levels were predictive of rejection. Increase in DSA from pre treatment to a post transplant peak of 1000 was equivalent to an increased chance of rejection with an odds ratio of 1.47 (1.08, 2.00). Conclusion In spite of increases or decreases in the DSA levels, there were no changes in the viral or the blood group antibodies in these patients. Thus the DSA rise is specific in contrast to the viral, blood group or third party antibodies post transplantation. Increases in the DSA post transplant in comparison to pre-treatment are strongly associated with occurrence of rejection.
Krishnan, Nithya S.; Zehnder, Daniel; Daga, Sunil; Lowe, Dave; Lam, F. T.; Kashi, Habib; Tan, Lam Chin; Imray, Christopher; Hamer, Rizwan; Briggs, David; Raymond, Neil; Higgins, Robert M.
Antibody-drug conjugates: present and future.
Antibody-drug conjugates (ADCs) are becoming an increasingly important sub-class of antibody-related therapeutics. Two ADCs, brentuximab vedotin (Adcetris) and ado-trastuzumab emtansine (Kadcyla), were recently approved for marketing both by the US Food and Drug Administration (FDA) and the European Medicine Agency (EMA). Brentuximab vedotin is marketed as therapy for hematological malignancies (Hodgkin lymphoma, systemic anaplastic large cell lymphoma), while ado-trastuzumab emtansine is marketed for treatment of a solid tumor (breast cancer). The approvals of these two ADCs followed the mitigated success of gemtuzumab ozogamicin (Mylotarg), which was withdrawn from the US market in 2010, ten years after approval by the FDA. PMID:24423577
Beck, Alain; Reichert, Janice M
Stimulating and inhibiting antibodies for bacterial penicillinase
The effect of specific antisera on the activity of penicillinases from Bacillus licheniformis and Bacillus cereus depends on the strain of penicillinase used, the type of substrate undergoing hydrolysis and the proportion of antiserum to antigen present. Enzyme function may be either decreased or increased. The antisera appear to contain at least two types of antibody, present in different proportions, capable of acting as stimulators or inhibitors of enzyme function, according to the nature of the substrate. The observed effects seem best explained by supposing that combination with antibody involves conformational changes in the enzyme which profoundly affect the properties of the active site. ImagesFIG. 2
Pollock, M. R.
Passive Immunization with Allergen-Specific Antibodies
\\u000a The induction of allergen-specific IgG antibodies has been identified as a major mechanism responsible for the reduction of\\u000a allergic inflammation in allergic patients treated by allergen-specific immunotherapy. Several studies suggest that allergen-specific\\u000a IgG antibodies induced by vaccination with allergens block mast cell and basophil degranulation, IgE-facilitated allergen\\u000a presentation to T cells and IgE production. The availability of recombinant allergens and
Sabine Flicker; Elisabeth Gadermaier; Christoph Madritsch; Rudolf Valenta
[Use of monoclonal antibodies in HLA immunology].
The author gives an account of the classification and use of HLA monoclonal antibodies. In experimental work the author demonstrates the contribution of investigations of the expressivity of histocompatible antigens class I and II and their changes in sound and tumourous tissues. In clinical practice they are important in particular in transplantology--cleaning of bone marrow, follow up of the GvH reaction, monitoring of patients. HLA monoclonal antibodies serve to reveal the polymorphism in the HLA sphere and the follow up of general biological laws. PMID:2327089
Prazák, J
Monoclonal Antibody Strategies for Targeting HER2
The HER family is composed of four receptors, HER1 to HER4, is dysregulated, and\\/or shows abnormal signaling activity in a\\u000a broad range of human tumors. The essential role of HER2 in the HER signaling network led to the development of anti-HER2 monoclonal\\u000a antibodies (mAb) for cancer therapy. In particular, the humanized antibody trastuzumab (Herceptin™) has antitumor activity\\u000a against HER2-overexpressing breast
Joan Albanell; Jeffrey S. Ross; Linda Pronk; Pere Gascon
Isolated Chorea Associated with LGI1 Antibody
Background Leucine-rich glioma inactivated 1 (LGI1) antibody produces a syndrome of limbic encephalitis, hyponatremia, and facio-brachial dystonic seizures that is non-paraneoplastic and responsive to corticosteroids. Parkinsonism, tremor, and generalized chorea are rare manifestations of LGI1, but, when present, commonly accompany other signs of limbic encephalitis. Case Report We present a case of LGI1-related isolated chorea in a 53-year-old Japanese male. His chorea responded to high-dose steroids, suggesting a potential role for this synaptic antibody in triggering chorea. Discussion This case highlights a new treatable etiology of chorea.
Ramdhani, Ritesh A.; Frucht, Steven J.
Effects of interferon on antibody formation
NASA Technical Reports Server (NTRS)
Studies of the effects of interferon on primary and secondary antibody responses and of the relationship of interferon to other cytokines, or cell products, are presented. Dosage- and timing-dependent immunoenhancing and immunosuppressive activities of interferon are documented for mouse spleen cell cultures and for mice infected with murine hepatitis virus (MHV-3). A possibility that altered interferon production might lead to immunopathological disorders, such as lupus erythematosus, AIDS, arthritis, etc., is discussed. Latest technological developments are presented that indicate that interferon does apparently play a major role in the regulation of antibody responses.
Sonnenfeld, G.
Sarcoidosis presenting as antinuclear antibody positive glomerulonephritis.
A 43 year old woman who initially presented with the nephrotic syndrome, glomerulonephritis, and antinuclear antibodies (ANAs) was given the diagnosis of systemic lupus erythematosus (SLE). One year later the patient developed progressive subcutaneous nodules on her forearms, with histopathology of non-caseating granulomas. Further evaluation of the patient showed mediastinal lymphadenopathy and interstitial lung disease with numerous granulomas, establishing the diagnosis of sarcoidosis. The presence of autoimmune antibodies and glomerulonephritis has been reported in sarcoidosis, but this case is believed to be the first in which both glomerulonephritis and ANAs are present in a sarcoid patient.
Soto-Aguilar, M C; Boulware, D W
[Inhibition of adenovirus reproduction in cell culture by specific antibodies].
The capacity of specific antibodies to inhibit the reproduction of homo- and heterologous adenoviruses in Hela cell added to culture medium after virus adsorption was studied. The inhibiting effect of polyclonal antivirus and monospecific antihexone antibodies to homo- and heterologous adenoviruses was shown. The effect was more expressed when using antibodies to homologous antibodies. The intensity of inhibition depended on antibodies concentration in the medium and infecting dose of the virus. Essential reduction of the quantity of infected cells and a decrease of the titer of adenovirus synthesized in the presence of homo- and heterologous antibodies was shown but adenovirus reproduction was not inhibited completely. PMID:19663330
Povnytsia, O Iu; Nosach, L M; Zhovnovata, V L; Zahorodnia, S D; Vantsak, N P; Tokarchuk, L V; Polishchuk, O M; Diachenko, N S
Application of recombinant antibody fragments for troponin I measurements.
The cardiac isoform of troponin I is a reliable biomarker of damaged cardiomyocytes that accompanies such severe cardiovascular diseases as myocardial infarction. Monoclonal antibody 19C7 recognizes troponin I in the bloodstream with high affinity and specificity. Recombinant antibodies can be used to improve detection systems based on monoclonal antibodies produced with hybridoma technology. In the present study, we compare the properties of monoclonal antibody 19C7 and its recombinant fragments. It is shown that the recombinant antibody fragments demonstrate similar affinity values as monoclonal antibodies and can be applied for troponin I detection. PMID:23244731
Altshuler, E P; Vylegzhanina, A V; Katrukha, I A; Bereznikova, A V; Serebryanaya, D V
Anti-transferrin receptor antibody and antibody-drug conjugates cross the blood-brain barrier
Delivery of nonlipophilic drugs to the brain is hindered by the tightly apposed capillary endothelial cells that make up the blood-brain barrier. The authors have examined the ability of a monoclonal antibody (OX-26), which recognizes the rat transferrin receptor, to function as a carrier for the delivery of drugs across the blood-brain barrier. This antibody, which was previously shown to bind preferentially to capillary endothelial cells in the brain after intravenous administration, labels the entire cerebrovascular bed in a dose-dependent manner. The initially uniform labeling of brain capillaries becomes extremely punctate {approximately} 4 hr after injection, suggesting a time-dependent sequestering of the antibody. Capillary-depletion experiments, in which the brain is separated into capillary and parenchymal fractions, show a time-dependent migration of radiolabeled antibody from the capillaries into the brain parenchyma, which is consistent with the transcytosis of compounds across the blood-brain barrier. Antibody-methotrexate conjugates were tested in vivo to assess the carrier ability of this antibody. Immunohistochemical staining for either component of an OX-26-methotrexate conjugate revealed patterns of cerebrovascular labeling identical to those observed with the unaltered antibody. Accumulation of radiolabeled methotrexate in the brain parenchyma is greatly enhanced when the drug is conjugated to OX-26.
Friden, P.M.; Walus, L.R.; Musso, G.F.; Taylor, M.A.; Malfroy, B.; Starzyk, R.M. (Alkermes, Inc., Cambridge, MA (USA))
Identifying functional anti-Staphylococcus aureus antibodies by sequencing antibody repertoires of patient plasmablasts.
Infection by Staphylococcus aureus is on the rise, and there is a need for a better understanding of host immune responses that combat S. aureus. Here we use DNA barcoding to enable deep sequencing of the paired heavy- and light-chain immunoglobulin genes expressed by individual plasmablasts derived from S. aureus-infected humans. Bioinformatic analysis of the antibody repertoires revealed clonal families of heavy-chain sequences and enabled rational selection of antibodies for recombinant expression. Of the ten recombinant antibodies produced, seven bound to S. aureus, of which four promoted opsonophagocytosis of S. aureus. Five of the antibodies bound to known S. aureus cell-surface antigens, including fibronectin-binding protein A. Fibronectin-binding protein A-specific antibodies were isolated from two independent S. aureus-infected patients and mediated neutrophil killing of S. aureus in in vitro assays. Thus, our DNA barcoding approach enabled efficient identification of antibodies involved in protective host antibody responses against S. aureus. PMID:24589749
Lu, Daniel R; Tan, Yann-Chong; Kongpachith, Sarah; Cai, Xiaoyong; Stein, Emily A; Lindstrom, Tamsin M; Sokolove, Jeremy; Robinson, William H
Association of antipituitary antibody and type 2 iodothyronine deiodinase antibody in patients with autoimmune thyroid disease.
Antipituitary antibody (APA) has been reported to be detected in patients with autoimmune thyroid disease. Type 2 iodothyronine deiodinase (D2) is expressed in both pituitary gland and thyroid gland. We studied the association of APA and D2 peptide antibody in patients with autoimmune thyroid disease. Rat pituitary gland homogenate and D2 peptide were used as antigens in the present study. APA and D2 peptide antibodies were measured by enzyme-linked immunosorbent assay (ELISA) in sera obtained from 42 patients with Hashimoto's disease, 26 patients with Graves' disease and 70 healthy control subjects. Moreover, D2 activity precipitation assay was performed in some patients with Hashimoto's disease. APA and D2 peptide antibody were elevated in patients with Hashimoto's disease and patients with Graves' disease, compared with control subjects. APA was positive in 32.4% (22/68), D2 peptide antibody was positive in 26.5% (18/68) of patients with autoimmune thyroid disease. APA was positive in 31.0% (13/42) of patients with Hashimoto's disease and 34.6% (9/26) of patients with Graves' disease. D2 peptide antibody was positive in 26.2% (11/42) of patients with Hashimoto's disease and 26.9% (7/26) of patients with Graves' disease. D2 peptide antibody was correlated with APA in patients with autoimmune thyroid disease. Moreover, precipitation of D2 activity was increased in some patients with Hashimoto's disease including a patient who also had idiopathic diabetes insipidus, and was correlated with D2 peptide antibody. These results suggest that D2 antibody may be associated with APA in patients with autoimmune thyroid disease. PMID:16410660
Nakahara, Rieko; Tsunekawa, Katsuhiko; Yabe, Shigeki; Nara, Misa; Seki, Koji; Kasahara, Takayuki; Ogiwara, Takayuki; Nishino, Michio; Kobayashi, Isao; Murakami, Masami
A Dynamic Landscape for Antibody Binding Modulates Antibody-Mediated Neutralization of West Nile Virus
Neutralizing antibodies are a significant component of the host's protective response against flavivirus infection. Neutralization of flaviviruses occurs when individual virions are engaged by antibodies with a stoichiometry that exceeds a required threshold. From this "multiple-hit" perspective, the neutralizing activity of antibodies is governed by the affinity with which it binds its epitope and the number of times this determinant is displayed on the surface of the virion. In this study, we investigated time-dependent changes in the fate of West Nile virus (WNV) decorated with antibody in solution. Experiments with the well-characterized neutralizing monoclonal antibody (MAb) E16 revealed a significant increase in neutralization activity over time that could not be explained by the kinetics of antibody binding, virion aggregation, or the action of complement. Additional kinetic experiments using the fusion-loop specific MAb E53, which has limited neutralizing activity because it recognizes a relatively inaccessible epitope on mature virions, identified a role of virus "breathing" in regulating neutralization activity. Remarkably, MAb E53 neutralized mature WNV in a time- and temperature-dependent manner. This phenomenon was confirmed in studies with a large panel of MAbs specific for epitopes in each domain of the WNV envelope protein, with sera from recipients of a live attenuated WNV vaccine, and in experiments with dengue virus. Given enough time, significant inhibition of infection was observed even for antibodies with very limited, or no neutralizing activity in standard neutralization assays. Together, our data suggests that the structural dynamics of flaviviruses impacts antibody-mediated neutralization via exposure of otherwise inaccessible epitopes, allowing for antibodies to dock on the virion with a stoichiometry sufficient for neutralization.
Durbin, Anna P.; Whitehead, Stephen S.; Pierson, Theodore C.
B cells contribute to MS pathogenesis through antibody-dependent and antibody-independent mechanisms.
For many years, central dogma defined multiple sclerosis (MS) as a T cell-driven autoimmune disorder; however, over the past decade there has been a burgeoning recognition that B cells contribute to the pathogenesis of certain MS disease subtypes. B cells may contribute to MS pathogenesis through production of autoantibodies (or antibodies directed at foreign bodies, which unfortunately cross-react with self-antigens), through promotion of T cell activation via antigen presentation, or through production of cytokines. This review highlights evidence for antibody-dependent and antibody-independent B cell involvement in MS pathogenesis. PMID:22690126
Wilson, Heather L
Ongoing Development of Monoclonal Antibodies and Antibody Drug-Conjugates in Lymphoma
Rituximab, a monoclonal antibody (mAb) directed against CD20, has changed practices in the treatment of patients with B-cell\\u000a lymphoma. The large success of rituximab has contributed to validate immunotherapy with monoclonal antibodies as a valuable\\u000a strategy in lymphoma. Recently, better-engineered anti-CD20-mAbs have been designed to improve efficacy and safety. Also,\\u000a new antibodies targeting other lymphoma subtypes including T-cell lymphoma and
Eileen M. Boyle; Franck Morschhauser
B cells contribute to MS pathogenesis through antibody-dependent and antibody-independent mechanisms
For many years, central dogma defined multiple sclerosis (MS) as a T cell-driven autoimmune disorder; however, over the past decade there has been a burgeoning recognition that B cells contribute to the pathogenesis of certain MS disease subtypes. B cells may contribute to MS pathogenesis through production of autoantibodies (or antibodies directed at foreign bodies, which unfortunately cross-react with self-antigens), through promotion of T cell activation via antigen presentation, or through production of cytokines. This review highlights evidence for antibody-dependent and antibody-independent B cell involvement in MS pathogenesis.
Wilson, Heather L
Antitubulin antibody in healthy adults and patients with infectious mononucleosis and its relationship to smooth muscle antibody (SMA).
Antibody to tubulin in man has been studied using a specific radioimmunoassay, affinity chromatography radioimmunoassay but markedly increased levels were noted in patients with infectious mononucleosis where the antibody was predominantly IgM in type. This finding was confirmed on fluorescence microscopy. Affinity chromatography purified antibody produced characteristic microtubular staining of fixed 3T3 cells, but in addition, produced weak staining of cryostat sections of rat tissue, similar in distribution to that of smooth muscle antibody. Our studies indicate that the IgM smooth muscle antibody found in infectious mononucleosis by IF techniques is at least in part due to an antitubulin antibody. Images FIG. 1
Mead, G M; Cowin, P; Whitehouse, J M
Let your Antibody Work -- Immunize Early.
National Technical Information Service (NTIS)
It is suggested that immunization against dental caries by given before the primary teeth fully erupt into the mouth, or before the tooth surfaces become available for colonization by Streptococcus mutans. It would appear to be easier for the antibody to ...
I. L. Shklair H. J. Keene
Antibody-based metabolic engineering in plants.
Genetic engineering is a powerful tool for the manipulation of cellular metabolism and the development of plant varieties with enhanced biological and nutrional functions. Several strategies are available for the in vivo modulation of enzymatic activities, allowing metabolic flux to be directed towards desired biochemical products. Such strategies include the simultaneous expression and/or suppression of multiple genes encoding rate-limiting enzymes, ectopic expression of transcription factors, and the RNA-based inhibition of catabolic enzymes. As an alternative approach, recombinant antibodies expressed in plants have been used to inactivate or sequestrate specific host proteins or compounds, resulting in significant changes to metabolic pathways. The impact of this approach depends on prudent selection of the target antigen, careful antibody design, appropriate subcellular targeting and stable accumulation of the recombinant antibodies in planta. Here, we describe the current status of antibody-based metabolic engineering in plants, discuss procedures for the optimisation of this technology and consider the remaining challenges to its widespread use. PMID:16698105
Nölke, Greta; Fischer, Rainer; Schillberg, Stefan
Molecular farming of recombinant antibodies in plants.
'Molecular farming' is the production of recombinant proteins in plants. It is intended to harness the power of agriculture to cultivate and harvest transgenic plants producing recombinant therapeutics. Molecular farming has the potential to provide virtually unlimited quantities of recombinant antibodies for use as diagnostic and therapeutic tools in both health care and the life sciences. Importantly, recombinant antibody expression can be used to modify the inherent properties of plants, for example by using expressed antipathogen antibodies to increase disease resistance. Plant transformation is technically straightforward for model plant species and some cereals, and the functional expression of recombinant proteins can be rapidly analyzed using transient expression systems in intact or virally infected plants. Protein production can then be increased using plant suspension cell production in fermenters, or by the propagation of stably transformed plant lines in the field. Transgenic plants can be exploited to produce organs rich in a recombinant protein for its long-term storage. This demonstrates the promise of using transgenic plants as bioreactors for the 'molecular farming' of recombinant therapeutics, blood substitutes and diagnostics, such as recombinant antibodies. PMID:10494831
Fischer, R; Liao, Y C; Hoffmann, K; Schillberg, S; Emans, N
Molecularly imprinted microspheres as antibody binding mimics
Molecular imprinting is an emerging technique for preparing artificial antibodies that have potential applications in affinity-based separations, biomimetic sensors and assays. The conventional imprinting methods either deliver a monolith that is mechanically ground to irregular particles — a time-consuming process with low yield, or involves complicated polymerization process by which spherical beads may be obtained, but with frequently compromised binding
Lei Ye; Klaus Mosbach
A Monoclonal Antibody to Limbic System Neurons
A monoclonal antibody produced against hippocampal cell membranes labeled the surface of neurons in the rat limbic system. With a few exceptions, all nonlimbic components were unstained. This specific distribution of immunopositive neurons provides strong evidence of molecular specificity among functionally related neurons in the mammalian brain and supports the concept of a limbic system.
Pat Levitt
Degenerate interfaces in antigen-antibody complexes.
In most of the work dealing with the analysis of protein-protein interfaces, a single X-ray structure is available or selected, and implicitly it is assumed that this structure corresponds to the optimal complex for this pair of proteins. However, we have found a degenerate interface in a high-affinity antibody-antigen complex: the two independent complexes of the camel variable domain antibody fragment cAb-Lys3 and its antigen hen egg white lysozyme present in the asymmetric unit of our crystals show a difference in relative orientation between antibody and antigen, leading to important differences at the protein-protein interface. A third cAb-Lys3-hen lysozyme complex in a different crystal form adopts yet another relative orientation. Our results show that protein-protein interface characteristics can vary significantly between different specimens of the same high-affinity antibody-protein antigen complex. Consideration should be given to this type of observation when trying to establish general protein-protein interface characteristics. PMID:11676532
Decanniere, K; Transue, T R; Desmyter, A; Maes, D; Muyldermans, S; Wyns, L
Antichlamydial antibodies in pelvic inflammatory disease.
The role of Chlamydia trachomatis in pelvic inflammatory disease (PID) diagnosed without laparoscopy was assessed by measuring antichlamydial antibodies in the patient's serum and by comparing the results with those in patients with uncomplicated non-specific genital infection (NSGI) and gonorrhoea and in non-infected controls. A modified microimmunofluorescence test was used. Patients with severe PID had significantly more positive antichlamydial IgG and IgM results than did control subjects, patients with gonorrhoea, and patients with NSGI. Less severe PID was associated with significantly raised levels of antichlamydial IgG antibodies compared with NSGI and controls and with raised levels of IgM antibodies compared with controls. Two patients with PID had lower genital tract gonorrhoea, one of whom had raised antichlamydial antibody levels. These findings may indicate a mixed infection and therapy should be reviewed in such patients. A serological diagnosis of chlamydial infection is relatively easy and cheap and enables a rapid diagnosis of chlamydial infection to be made. PMID:526845
Simmons, P D; Forsey, T; Thin, R N; Treharne, J D; Darougar, S; Langlet, F; Pandhi, R K
Neuropsychological deficits associated with antiphospholipid antibodies
Antiphospholipid antibodies (APAS) are circulating immunoglobulins that lead to a hypercoagulant state and recurrent thromboembolic events. The cerebral circulation is the most common site of occlusion with neurologic events including amaurosis fugax, migrainous cephalalgia, transient ischemic attacks, stroke, ischemic encephalopathy, and vascular dementia. There are numerous case studies citing neurocognitive and neuropsychiatric symptoms associated with this syndrome in a much
Mark William Jacobson
New antibody conjugates in cancer therapy.
Targeting of radiation, drugs, and protein toxins to cancers selectively with monoclonal antibodies (MAbs) has been a topic of considerable interest and an area of continued development. Radioimmunotherapy (RAIT) of lymphoma using directly labeled MAbs is of current interest after approval of two radiolabeled anti-CD20 MAbs, as illustrated with the near 100% overall response rate obtained in a recent clinical trial using an investigational radiolabeled anti-CD22 MAb, 90Y-epratuzumab. The advantage of pretargeted RAIT over directly labeled MAbs is continuing to be validated in preclinical models of lymphoma and solid tumors. Importantly, the advantages of combining RAIT with radiation sensitizers, with immunotherapy, or a drug conjugate targeting a different antigen are being studied clinically and preclinically. The area of drug-conjugated antibodies is progressing with encouraging data published for the trastuzumab-DM1 conjugate in a phase I clinical trial in HER2-positive breast cancer. The Dock-and-Lock platform technology has contributed to the design and the evaluation of complex antibody-cytokine and antibody-toxin conjugates. This review describes the advances made in these areas, with illustrations taken from advances made in the authors' institutions. PMID:20953556
Govindan, Serengulam V; Goldenberg, David M
Antibody-catalyzed anaerobic destruction of methamphetamine
Methamphetamine [(+)-2] abuse has emerged as a fast-rising global epidemic, with immunopharmacotherapeutic approaches being sought for its treatment. Herein, we report the generation and characterization of a monoclonal antibody, YX1-40H10, that catalyzes the photooxidation of (+)-2 into the nonpsychoactive compound benzaldehyde (14) under anaerobic conditions in the presence of riboflavin (6). Studies have revealed that the antibody facilitates the conversion of (+)-2 into 14 by binding the triplet photoexcited state of 6 in proximity to (+)-2. The antibody binds riboflavin (Kd = 180 ?M), although this was not programmed into hapten design, and the YX1-40H10-catalyzed reaction is inhibited by molecular oxygen via the presumed quenching of the photoexcited triplet state of 6. Given that this reaction is another highlight in the processing of reactive intermediates by antibodies, we speculate that this process may have future significance in vivo with programmed immunoglobulins that use flavins as cofactors to destroy selectable molecular targets under hypoxic or even anoxic conditions.
Xu, Yang; Hixon, Mark S.; Yamamoto, Noboru; McAllister, Laura A.; Wentworth, Anita D.; Wentworth, Paul; Janda, Kim D.
Subcutaneous Administration of Monoclonal Antibodies in Oncology
Treatment with monoclonal antibodies (mabs) has become an established component of oncological therapy. The monoclonal antibodies available for this purpose are mainly administered intravenously in individually adapted doses according to body weight over longer treatment times. For other chronic diseases such as, for example, diabetes mellitus, the subcutaneous administration of drugs is an established therapy option. For the subcutaneous administration of larger volumes as needed for mab solutions the extracellular matrix of the subcutaneous tissue represents a problem. The co-formulation with recombinant human hyaluronidase makes the relatively pain-free administration of larger fluid volumes and thus the subcutaneous administration of monoclonal antibodies possible, as illustrated by the development of a subcutaneous formulation of trastuzumab. This constitutes a less invasive, time-optimised and flexible form of administration for patients with HER2-positive breast cancer that, with its fixed dosing possibilities, contributes to therapeutic safety. The example of trastuzumab shows that the subcutaneous administration of monoclonal antibodies can simplify oncological long-term therapy not only for the patients but also for the medical personnel.
Jackisch, C.; Muller, V.; Maintz, C.; Hell, S.; Ataseven, B.
Rapid antibody test for fragile X syndrome
Fragile X syndrome is the most common known cause of inherited mental retardation. Identification of patients and carriers of fragile X syndrome is usually done with a DNA test system but we have developed a rapid antibody to F identify fragile X patients. This non-invasive test requires only 1 or 2 drops of blood and can be used for screening
R. Willemsen; S. Mohkamsing; B. de Vries; A. van den Ouweland; H. Galjaard; B. Oostra; D. Devys; J. L. Mandel
Fluoroimmunoassays using antibody-conjugated quantum dots.
Luminescent colloidal semiconductor nanocrystals (quantum dots) are robust inorganic fluoro phores that have the potential to circumvent some of the functional limitations encountered by organic dyes in sensing and biotechnological applications. Quantum dots exhibit size-dependent tunable, narrow fluorescence emission spectra that span the visible spectrum and have broad absorption spectra. This allows simultaneous excitation of several particle sizes at a single wavelength with emission at multiple wavelengths. Quantum dots also provide a high-resistance threshold to chemical degradation and photodegradation. We have developed a conjugation strategy for the attachment of antibodies to quantum dots based on electrostatic interactions between negatively charged dihydrolipoic acid (DHLA)-capped CdSe-ZnS core-shell quantum dots and positively charged proteins (natural or engineered) that serve to bridge the quantum dot and antibody. This chapter details the materials and methods for synthesis of the DHLA-capped CdSe-ZnS core-shell quantum dots, the construction and preparation of recombinant proteins, the conjugation of antibodies to quantum dots, and the use of antibody-coated quantum dots in a fluoroimmunoassay. PMID:15923672
Goldman, Ellen R; Mattoussi, Hedi; Anderson, George P; Medintz, Igor L; Mauro, J Matthew
World Antibody Drug Conjugate Summit Europe
The World Antibody Drug Conjugate Summit Europe, organized by Biorbis/Hanson Wade was held in Frankfurt, Germany February 21–23, 2011. Antibody drug conjugates (ADCs), also called immunoconjugates, are becoming an increasingly important class of therapeutics as demonstrated by the attendance of nearly 100 delegates at this highly focused meeting. Updates on three ADCs that are in late-stage clinical development, trastuzumab emtansine (T-DM1), brentuximab vedotin (SGN-35) and inotuzumab ozogamicin (CMC-544), were presented by speakers from ImmunoGen, Genentech, Roche, Seattle Genetics and Pfizer. These ADCs have shown encouraging therapeutic effects against solid tumors (T-DM1) and hematological malignancies (SGN-35, CMC-544). The key feature of the new generation of ADCs is the effective combination of the cytotoxicity of natural or synthetic highly potent antineoplastic agents, tumor selective monoclonal antibodies and blood-stable optimized linkers. Early clinical data for ADCs were showcased by Progenics Pharmaceuticals (PSMA ADC), Celldex (CDX-011) and Biotest (BT-062). Takeda, MedImmune and sanofi-aventis outlined their strategies for process development and analytical characterization. In addition, presentations on duocarmycin based-ADCs, ? emitting immunoconjugates and antibody-conjugated nanoparticles were given by representatives from Syntarga, Algeta and the University of Stuttgart, respectively.
Production of Human Monoclonal Antibodies by Heterohybridomas.
National Technical Information Service (NTIS)
The goal is to establish an industrial process for producing human monoclonal antibodies (h-MoAb). The authors have already established a hybridoma system between human-mouse hybridomas and human lymphocytes (m.h-h) for producing h-MoAb (presented at the ...
K. Kitano Y. Shintani S. Sasai K. Tsukamoto
Antiphospholipid Antibody Syndrome Presenting with Hemichorea
A 25-year-old Bangladeshi lady presented to neurology with a three-month history of involuntary movements of her right arm, associated with loss of power. There was progression to the right leg, and she subsequently developed episodes of slurred speech and blurred vision. At the time of presentation, she was 12 weeks pregnant and the symptoms were reported to have started at conception. Past medical history was unremarkable apart from one first trimester miscarriage and there was no significant family history suggestive of a hereditary neurological condition. MRI of the head revealed no abnormalities but serology showed positive antinuclear antibodies (ANAs) at a titre of 1/400. Further investigations revealed strongly positive anticardiolipin antibodies (>120) and positive lupus anticoagulant antibodies. The patient had a second miscarriage at 19 weeks gestation strengthening the possibility that the chorea was related to antiphospholipid antibody syndrome and she was started on a reducing dose of Prednisolone 40?mg daily and aspirin 300?mg daily. Six months later, she had complete resolution of neurological symptoms. There are several reports of chorea as a feature of antiphospholipid syndrome, but no clear consensus on underlying pathophysiology.
Ayalew, Yezenash; Khattak, Fazlihakim
Antiphospholipid antibody syndrome presenting with hemichorea.
A 25-year-old Bangladeshi lady presented to neurology with a three-month history of involuntary movements of her right arm, associated with loss of power. There was progression to the right leg, and she subsequently developed episodes of slurred speech and blurred vision. At the time of presentation, she was 12 weeks pregnant and the symptoms were reported to have started at conception. Past medical history was unremarkable apart from one first trimester miscarriage and there was no significant family history suggestive of a hereditary neurological condition. MRI of the head revealed no abnormalities but serology showed positive antinuclear antibodies (ANAs) at a titre of 1/400. Further investigations revealed strongly positive anticardiolipin antibodies (>120) and positive lupus anticoagulant antibodies. The patient had a second miscarriage at 19 weeks gestation strengthening the possibility that the chorea was related to antiphospholipid antibody syndrome and she was started on a reducing dose of Prednisolone 40?mg daily and aspirin 300?mg daily. Six months later, she had complete resolution of neurological symptoms. There are several reports of chorea as a feature of antiphospholipid syndrome, but no clear consensus on underlying pathophysiology. PMID:22937452
Ayalew, Yezenash; Khattak, Fazlihakim
Single-Domain Antibodies: Rugged Recognition Element.
National Technical Information Service (NTIS)
The ability to quickly and accurately detect potential bio-threat agents is a priority for the Department of Defense and for homeland security. Most rapid diagnostic and detection immunoassays rely on monoclonal or polyclonal antibodies (IgG) as the recog...
A. Hayhurst E. R. Goldman G. P. Anderson J. B. Delehanty J. L. Liu
Immunostimulatory monoclonal antibodies for cancer therapy
Increasing immune responses with immunostimulatory monoclonal antibodies (mAbs) directed to immune-receptor molecules is a new and exciting strategy in cancer therapy. This expanding class of agents functions on crucial receptors, either antagonizing those that suppress immune responses or activating others that amplify immune responses. Complications such as autoimmunity and systemic inflammation are problematic side effects associated with these agents. However,
Sandra Hervas-Stubbs; Martin Glennie; Drew M. Pardoll; Ignacio Melero; Lieping Chen
Neutralizing antibodies in hepatitis C virus infection.
Hepatitis C virus (HCV) is a major cause of hepatitis world-wide. The majority of infected individuals develop chronic hepatitis which can then progress to liver cirrhosis and hepatocellular carcinoma. Spontaneous viral clearance occurs in about 20%-30% of acutely infected individuals and results in resolution of infection without sequaelae. Both viral and host factors appear to play an important role for resolution of acute infection. A large body of evidence suggests that a strong, multispecific and long-lasting cellular immune response appears to be important for control of viral infection in acute hepatitis C. Due too the lack of convenient neutralization assays