The present invention is a method for diagnosing a patient at risk to thrombocytopenia induced by administration of a GP IIb/IIIa receptor antagonist, which comprises combining

Drug-induced thrombocytopenia contributes to morbidity and, occasionally, mortality in patients treated with a wide range of medications (Karpatkin, (1971) Am. J. Med. Sci. 262, p. 68). More than 100 different medications have been implicated in drug-induced thrombocytopenia, including heparin, quinine, quinidine and sulfonamide antibiotics (Shulman et al. xe2x80x9cHemostasis and Thrombosisxe2x80x9d (ed 2) Philadelphia, Pa., Lippincott (1987) p.452, and Kracke et al. (1943) JAMA 122, p. 168.
Drug-dependent antibodies reactive with platelets have been identified in only a few instances. Curtis et al., (1984) Blood v. 84, n.1, 176-183, applied flow cytometry to the detection of such antibodies induced by sulfonamide antibiotics. Visentin et al. (1990) Transfusion Oct. v. 30 n. 8, 694-700 describes detection of drug-dependent, platelet-reactive antibodies by antigen-capture ELISA and flow cytometry.
The occurrence of fibrinogen receptor antagonist induced-thrombocytopenia has prompted a search for monoclonal antibodies (mAbs) specific for GP IIb/IIIa receptor:receptor antagonist complexes. Monoclonal antibodies capable of detecting alterations in glycoprotein IIb/IIIa upon binding of fibrinogen receptor antagonists to glycoprotein IIb/IIIa are useful in diagnosing patients at risk to developing fibrinogen receptor antagonist-induced thrombocytopenia. The monoclonals can be tagged with fluorescent or radioactive tracers for ease in diagnosis.
The present invention includes monoclonal antibodies which recognize induced binding sites formed on GPIIb/IIIa receptor following association of a fibrinogen receptor antagonist with the GPIIb/IIIa receptor, and a means for identifying, in a patient, the presence of one or more antibodies in GP IIb/IIIa receptor (fibrinogen receptor) antagonist-induced thrombocytopenia which recognize GPIIb/IIIa receptor:GPIIb/IIIa receptor antagonist complexes formed with purified platelets or purified GPIIb/IIIa receptor and a selected GPIIb/IIIa receptor antagonist. Identification of such antibodies identifies the patient as being at risk to development of thrombocytopenia resulting from administration to the patient of the selected GP IIb/IIIa receptor antagonist.
Diagnosis of patients at risk to developing fibrinogen receptor antagonist induced-thrombocytopenia can be implemented using monoclonal antibodies, having fluorescent, nuclear magnetic or radioactive tags, which are specific for GP IIb/IIIa receptor:receptor antagonist complexes. These monoclonal antibodies will associate with the complex unless a patient""s plasma sample, when mixed with the complex and the monoclonal antibody, prevents association of the complex with the monoclonal antibody.
The invention includes isolated monoclonal antibodies or detectable monoclonal antibodies which recognize induced binding sites formed on the GP IIb/IIIa receptor following association of a fibrinogen receptor antagonist with the GP IIb/IIIa receptor. Exemplary monoclonal antibodies of the invention include mAb10-758 and mAb15-758. The invention also includes humanized forms of the monoclonal antibodies.
The invention is also a hybridoma cell line selected from the group consisting of DD6 and FB3, which cell lines produce monoclonal antibodies of the invention, e.g. mAb10-758, and mAb15-758.
The invention is also a method for diagnosing a patient at risk to thrombocytopenia induced by administration of a GP IIb/IIIa receptor antagonist, which comprises combining
i) a plasma sample of the patient;
ii) detectable monoclonal antibody which recognizes induced binding sites formed on the GP IIb/IIIa receptor following association of a fibrinogen receptor antagonist with the GP IIb/IIIa receptor; and
iii) GP IIb/IIIa receptor:GP IIb/III receptor antagonist complex,
and determining association of the detectable monoclonal antibody with the complex in the presence of the plasma. The monoclonal antibodies, e.g. detectable mAb10-758 and detectable mAb15-758, are made detectable by labeling, e.g., with a fluorescence label.
In one class of the method for diagnosing a patient at risk to thrombocytopenia induced by administration of a GP IIb/IIIa receptor antagonist, the fibrinogen receptor antagonist is selected from the group consisting of
Mpr-(Acetimidyl-Lys)-Gly-Asp-Trp-Phe-Cys-NH2,
Mpr-(Acetimidyl-Lys)-Gly-Asp-Trp-Phe-Pen-NH2,
Mpr-(Phenylimidyl-Lys)-Gly-Asp-Trp-Phe-Pen-NH2,
Mpr-(Phenylimidyl-Lys)-Gly-Asp-Trp-Phe-Cys-NH2,
N-Methyl-D-phenylalanyl-N- [(1S)-1-formyl-4-guanidinobutyl]-L-prolinamide,
((1-(2-((4-(aminoiminomethyl)benzoyl)amino)-3-(4-hydroxyphenyl)-1-oxopropyl)-4-piperidinyl)oxy)-(S)-acetic acid,
Ethyl 3-[[4-[[4-(aminoiminomethyl)phenyl]amino]-1,4-dioxobutyl]amino]-4-pentynoate,
N-(2-(2-(((3-((aminoiminomethyl)amino)propyl)amino)carbonyl)-1-piperidnyl)-1-(cyclohexylmethyl)-2-oxoethyl)-(R,S)-glycine,
(2-S-(n-Butylsulfonylamino)-3[4-(piperidin-4-yl)butyloxyphenyl]propionic acid hydrochloride,
Ethyl 3-[[4-[[4-(aminoiminomethyl)phenyl]amino]-1,4-dioxobutyl]amino]-4-pentynoate,
2(S)-[(p-Toluenesulfonyl)amino]-3-[[[5,6,7,8-tetrahydro-4-oxo-5-[2-(piperidin-4-yl)ethyl]-4H-pyrazolo-[1,5-a][1,4]diazepin-2-yl]carbonyl]-amino]propionic acid,
(R)-methyl-3-[[[3-[4-(aminoiminomethyl)phenyl]-4,5-dihydro-5-isoxazolyl]acetyl]amino]-N-(butoxycarbonyl)-L-alanine monoacetate, and
[3(R)-[2-Piperidin-4-yl)ethyl]-2-piperidone-1]acetyl-3(R)-methyl-xcex2-alanine,
and pharmaceutically acceptable salts thereof.
The invention is also a test kit for diagnosing a patient at risk to thrombocytopenia induced by administration of a GP IIb/IIIa receptor antagonist, which comprises a GP IIb/IIIa receptor:GP IIb/III receptor antagonist complex, and a detectable monoclonal antibody which recognizes induced binding sites formed on the GP IIb/IIIa receptor following association of a fibrinogen receptor antagonist with the GP IIb/IIIa receptor.
The invention is also a method for identifying a patient not at risk to developing fibrinogen receptor antagonist-induced thrombocytopenia which comprises combining
i) patient plasma;
ii) a detectable monoclonal antibody which recognizes induced binding sites formed on the GP IIb/IIIa receptor following association of a fibrinogen receptor antagonist with the GP IIb/IIIa receptor; and
iii) a GPIIb/IIIa receptor:GPIIb/IIIa receptor antagonist complex,
to form a GPIIb/IIIa receptor:GPIIb/IIIa receptor antagonist:detectable monoclonal antibody complex, and detecting the presence of the detectable monoclonal antibody in the GPIIb/IIIa receptor:GPIIb/IIIa receptor antagonist:detectable monoclonal antibody complex.
The invention is also a method for identifying a patient at risk to developing fibrinogen receptor antagonist-induced thrombocytopenia which comprises combining
i) patient plasma;
ii) a detectable monoclonal antibody which recognizes induced binding sites formed on the GP IIb/IIIa receptor following association of a fibrinogen receptor antagonist with the GP IIb/IIIa receptor; and
iii) a GPIIb/IIIa receptor:GPIIb/IIIa receptor antagonist complex,
to form a reaction product, and detecting the absence of the detectable monoclonal antibody in the reaction product.
The anti-GPIIb/IIIa antibodies against purified GPIIb/IIIa associated with a potent fibrinogen receptor antagonist are specific to the xcex23 subunit and are able to recognize this subunit on endogenous xcex1IIxcex23 and xcex1vxcex23 integrins as shown by immunoprecipitation.
These antibodies recognize ligand-induced binding sites formed on the receptor following association of a fibrinogen receptor antagonist with the GP IIb/IIIa receptor. Specific examples of such monoclonal antibodies include mAb10-758 and mAb15-758.
mAb10-758 binding affinity to resting platelets is increased in the presence of ligand and its binding induces platelet aggregation and PAC-1 (Platelet Activation Complex-1) binding to resting platelets in the absence of platelet activation. Therefore, mAb10-758 is an anti-LIBS antibody. The term xe2x80x9cLIBSxe2x80x9d means xe2x80x9cligand induced binding sites.xe2x80x9d
mAb15-758 represents a potent anti-LIBS antibody. The binding affinity of this antibody to GPIIb/IIIa is very weak on resting and ADP activated platelets. mAb15-758 binding can be increased by the disassociation of the GPIIb/IIIa complex or by occupying the receptor with RGDS containing ligands. Like mAb10-758, mnAb15-758 is able to activate the GPIIb/IIIa complex in the absence of platelet activation.
Our results demonstrate that potent fibrinogen receptor antagonists tested, including RO-44-9883 ({1-[2-(4-carbamimidoyl-benzylamino)-3-(4-hydroxyphenyl)-propionyl]-piperidin-4-yloxy}-acetic acid), induce the neo-epitope which is recognized by mAb15-758.
The selected GP IIb/IIIa receptor antagonist suitable for the methods of the invention is any antagonist which is useful for inhibiting fibrinogen binding to the GP IIb/IIIa platelet receptor. Such antagonists are well known in the art.
The following abbreviations are used in the description: LIBS (ligand induced binding sites); RO-44-9883 ({1-[2-(4-carbamimidoyl-benzoylamino)3-(4-hydroxy-phenyl)propionyl]-piperidin-4-yloxy}-acetic acid); PAC-1 (Platelet Activation Complex-1); and GFP (gel filtered platelets).
Antagonists for the GP IIb/IIIa receptor have been described in, for example, U.S. Pat. Nos. 5,470,849, 5,463,011, 5,455,243, 5,451,578, 5,446,056, 5,441,952, 5,422,249, 5,416,099, 5,405,854, 5,397,791, 5,393,760, 5,389,631, 5,380,713, 5,374,622, 5,358,956, 5,344,783, 5,340,798, 5,338,7235,334,596, 5,321,034, 5,318,899 (e.g. cyclic heptapeptides Mpr-(Acetimidyl-Lys)-Gly-Asp-Trp-Phe-Cys-NH2, Mpr-(Acetimidyl-Lys)-Gly-Asp-Trp-Phe-Pen-NH2, Mpr-(Phenylimidyl-Lys)-Gly-Asp-Trp-Phe-Pen-NH2, and Mpr-(Phenylimidyl-Lys)-Gly-Asp-Trp-Phe-Cys-NH2, wherein Mpr is mercapto propionyl), U.S. Pat. Nos. 5,312,923, 5,294,616, 5,292,756 (e.g. 2-S-(n-Butylsulfonylamino)-3[4-piperidin-4-yl)butyloxyphenyl]propionic acid hydrochloride), U.S. Pat. No. 5,281,585 (e.g. [3(R)-[2-Piperidin-4-yl)ethyl]-2-piperidone-1]acetyl-3(R)-methyl-xcex2-alanine), U.S. Pat. Nos. 5,272,158, 5,264,420, 5,260,307, 5,239,113 (e.g. Ethyl 3-[[4-[[4-(aminoiminomethyl)-phenyl]amino]-1,4-dioxobutyl]amino]-4-pentynoate), U.S. Pat. Nos. 5,227,490, 5,206,373, 4,703,036 (e.g. N-Methyl-D-phenylalanyl-N-[(1S)-1-formyl-4-guanidinobutyl]-L-prolinamide), EP 505 868 (e.g. ((1-(2-((4(aminoiminomethyl)benzoyl)amino)-3-(4-hydroxyphenyl)-1-oxopropyl)-4-piperidinyl)oxy)-(S)-acetic acid), WO 9311152 (e.g. N-(2-(2-(((3-((aminoiminomethyl)amino)propyl)amino)-carbonyl)-1-piperidnyl)-1-(cyclohexylmethyl)-2-oxoethyl)-(R,S)-glycine), WO 9418981 (e.g. 2(S)-[(p-Toluenesulfonyl)amino]-3-[[[5,6,7,8-tetrahydro-4-oxo-5-[2-(piperidin-4-yl)ethyl]-4H-pyrazolo-[1,5-a][1,4]diazepin-2-yl]carbonyl]-amino]propionic acid), WO 9514683 (e.g. methyl-N3-[2-{3-(4-formamidinophenyl)-isoxazolin-5(R)-yl}-acetyl]-N2-(n-butyloxycarbonyl)-2,3-(S)-diaminopropionate acetate salt), EP 333 356 and WO 9422820. They are described as useful for inhibiting fibrinogen binding and inhibiting clot formation.
GP IIb/IIIa (xcex1IIbxcex23) is a member of the integrin family of adhesive receptors and plays a vital role in thrombosis and hemostatis by serving as the receptor for the adhesive proteins fibrinogen, vitronectin, Von Willebrand factor, and fibronectin on platelets (Haverstick et al. (1995) Blood 66, 946-952; Savage et al. (1991) J. Biol. Chem. 266, 11227-11233; Phillips et al. (1991) Cell 65, 359-362). During the processes of cell attachment, spreading and platelet aggregation xcex1IIbxcex23 progresses through several functional states which reflect conformational changes in the receptor (Plow et al. (1991) Prog. Hemastasis Thrombosis 9, 117-156). On unactivated platelets, xcex1IIbxcex23 cannot bind soluble fibrinogen (Peerschke (1985) Seminars Heamatol 22, 241-259). However, upon platelet activation, xcex1IIbxcex23 undergoes a conformational change exposing a common ligand binding site for fibrinogen, fibronectin and Von Willebrand factor (Lam et al. (1987) J. Biol. Chem. 262, 947-950) that recognizes a small peptide sequence on the peptide (Loftus et al. (1990) Science 249, 915-918).
The binding of ligands to GP IIb/IIIa induces additional conformational changes of the extracellular domains of the receptor (Parise et al. (1987) J. Biol. Chem. 262, 12597-12602) leading to post receptor occupancy events and the exposure of neo-epitopes (LIBS) (Frelinger et al. (1988) J. Biol. Chem. 263, 12397-12402). Anti-LIBS antibodies have been generated against LIBS sites on both the xcex1lllb and xcex23 subunits (Frelinger et al. (1988) J. Biol. Chem., 263, 12397-12402; Frelinger et al. (1990) J. Biol. Chem., 265, 6346-6352; Kouns et al. (1990) J. Biol. Chem. 265, 20594-20604; Honda et al. (1995) J. Biol. Chem. 270, 11947-11954; Satoh et al. (1991) Biochem. J. 301, 785-791; Frelinger et al. (1991) J. Biol. Chem. 266, 17106-17111). The epitopes exposed by ligand binding have functional importance in ligand affinity (Frelinger et al. (1990) J. Biol. Chem. 265, 6346-6352; Frelinger et al. (1991) J. Biol. Chem. 266, 17106-17111; Moderna et al. (1994) Clinical Research 42, 131a); platelet aggregation (Kouns et al. (1990) J. Biol. Chem. 265, 20594-20604; Frelinger et al. (1991) J. Biol. Chem. 266, 17106-17111); clot retraction (Frelinger et al. (1990) J. Biol. Chem. 265, 6346-6352), and cellular adhesion (Frelinger et al. (1988) J. Biol. Chem. 263, 12397-12402; Frelinger et al. (1990) J. Biol. Chem. 265, 6346-6352; Shadle et al. (1984) J. Cell. Bio. 99, 2056-2060).
The conformationally restrained LIBS epitope is modulated by ligand binding to xcex1IIbxcex23. Binding of natural ligands (Heath et al. (1994) Annals of The New York Academy of Sciences 714, 300-302; Mondoro et al. (1996) Blood 88, 3824-3830), non peptide ligands (Satoh et al. (1991) Biochem J. 301, 785-791; Kouns et al. (1993) Thrombosis and Haemostasis 785), ligand mimetic peptides (Parise et al. (1987) J. Biol. Chem. 262, 12597-12602) and antibodies (Tomiyama et al. (1992) J. Biol. Chem. 267, 18085-18092) have been shown to induce conformational changes in the extracellular domain of GPIIb/IIIa that are recognized by conformation-dependent anti-LIBS antibodies. Small molecules which inhibit platelet aggregation may be an effective antithrombotic treatment (Gould (1994) xe2x80x9cPerspectives in Drug Discovery and Design 1xe2x80x9d537-548). Alloantibodies against platelet proteins, including epitopes on GPIIb/IIIa, have been identified in cases of autoimmune thrombocytopenic purpura (Woods et al. (1984) Blood 63, 368-375; Bierling et al. (1994) British Journal of Haematology 87, 631-633). The conformational changes induced by receptor occupancy of one integrin can influence the function of another distinct integrin within the same cell (Diaz-Gonzalez, et al. (1996) Molecular Biology of the Cell 7, 1929-1951). Therefore, it is of interest to examine the capability of various potent peptidometitic inhibitors of platelet aggregation to induce neo-epitopes. Steiner et al. (1993) Thrombosis and Haemostasis, 782, suggests that one such inhibitor, RO 44-9883, does not change the conformation of GPIIb/IIIa upon binding to the receptor. In the present invention, a series of monoclonal antibodies was generated against purified xcex1IIbxcex23 in the presence of a potent RGD-mimetic.
Three antibodies and their binding affinities to GPIIb/IIIa in the presence of various GPIIb/IIIa antagonist were characterized. In contrast to the previous study, we have found that all potent GPIIb/IIIa antagonists induce the neo-epitope which is recognized by anti-LIBS monoclonal antibodies, e.g., mAb10-758 and mAb15-758.
Purification of Proteins
GPIIb/IIIa was purified from outdated human plasma using a slight modification of the procedure described by Kouns et al. (Kouns et al. (1992) J. Biol. Chem. 267, 18844-18851). Briefly, detergent solubilized platelet lysates were passed over a Concanavalin A-Sepharose 4B column (Sigma Chemicals, St. Louis, Mo.), followed by elution with methyl-xcex1-D mannopyranozide (Sigma Chemical, St. Louis, Mo.). The eluate from the Concanavalin A column was passed through an RGDS-Sepharose affinity chromatography to purify the active form of the protein. Flow-through from the RGDS column was passed through a Sephacyl S-300 size exclusion column (Sigma Chemical, St. Louis, Mo.) to isolate the inactive form of the protein. All fractions were analyzed for purity by SDS-PAGE electrophoresis.
Antibody Production
The Kxc3x6hler and Milstein technique introduced in 1975 allows production of unlimited quantities of antibody of precise and reproducible specificity. Conventional polyclonal antisera contain a myriad of different antibodies differing in their specificity and properties, whereas a hybridoma produces a single monoclonal antibody with uniform characteristics. The Kxc3x6hler-Milstein technique starts with the immunization of an animal with subsequent fusion of spleen cells isolated from the immunized animal with an immortal myeloma cell line. These fused cells (hybridomas) grow in culture, and specific clones can be selected producing the mAb of the desired specificity. Each clone produces one specific monoclonal antibody. These hybridomas can be cultured and/or stored in liquid nitrogen. Thus a constant supply of pure antibody, i.e., monoclonal antibody, is assured.
Antibody proteins recognize specific antigens in an immunological response. Antibody molecules usually recognize a specific region of the antigen known as an epitope or determinant.
Monoclonal antibodies to different cell types have been developed. See for example, Old, Lloyd J. et al. (1981) Cancer Res. 41: 361-375, and Eisinger et al. (1982) Proc. Nat""l. Acad. Sci. USA 79: 2018.
Gel Filtration of Platelets and Cell Lines:
Gel filtered platelets (GFP) were prepared from blood anticoagulated with 13 mM trisodium citrate. Platelet rich plasma was generated by centrifigation at 180xc3x97G for 20 minutes in the presence of 1 xcexcM PGE1. Platelets were separated from plasma by gel filtration through a Sepharose 2B column (Pharmacia Biotech, Sweden) equilibarated in platelet buffer (NaCl (140 mM), KCL (60 mM), Na2HPO4 (1.9 mM). HEPES (6.7 mM), dextrose (1 mg/ml), and BSA (2%) pH 7.4). The platelets were diluted to 2xc3x97108 cells/mL in platelet buffer before use.
Human erythroleukemia (HEL) cells and Human embryonic kidney (HEK) 293 cells (Abraham et al. (1997) Molecular Pharmacology 52, 227-236) were obtained from American Type Collection (ATCC, Rockville, Md.). Human venous endothelial cells (HUVEC) were purchased from Cell Systems (Kirkland, Wash.). Recombinant HEK-xcex1IIbxcex23 cells are described in Abraham et al. (1997) Molecular Pharmacologv 52, 227-236. Recombinant HEK-xcex1vxcex23 cells are also described in (Abraham et al. (1997) Molecular Pharmacology 52, 227-236).
Synthetic Peptides
Non-potent GPIIb/IIIa ligands 4-(N-piperazine)benzoyl-(N-1,2,3,4-tetrahydroisoquinoline-7-yl)oxy-acetic acid (compound A); and potent fibrinogen receptor antagonists, tirofiban (compound 1-9 in U.S. Pat. No. 5,292,756, the name of which is 2-S-(n-Butylsulfonylamino)-3[4-(piperidin-4-yl)butyloxyphenyl]-propionic acid hydrochloride); cyclo-4-I-benzoyl-(Cys-Asn-Pro-Arg-Gly-Asp-Cys)-OH HOAc salt (compound B), N-[3(R)-[2-(Piperidin-4-yl)ethyl]-2-piperidon-1-yl] acetyl-3(R)-methyl-xcex2-alanine (compound C), and 2-(S)-(3-pyridylsulfonyl)amino-3-N-[5-[2-(4-piperidinyl)ethyl]-thienothiophene-2-carboxamide]propionic acid (compound D) were evaluated for binding affinity. The binding affinities of these compounds to purified human platelet xcex1IIbxcex23 is summarized in Table 1.
RO044-9883 ({1-[2-(4-carbamimidoyl-benzoylamino)3-(4-hydroxy-phenyl)propionyl]-piperidin-4-yloxy}-acetic acid) is described in Kouns et al. (1992) J. Bio. Chem. 267, 18844-18851); echistatin is described in Gan et al. (1988) J. Biol. Chem. 263, 19827-19827 and Garsky et al. (1989) Proc. Nat""l. Acad. Sci./USA 86, 4022-4026. RGDS was purchased from Sigma Chemical Company (St. Louis, Mo.).
Production and Purification of Monoclonal Antibodies
Purified human GPIIb/IIIa was purchased from Enzyme Research Laboratories (South Bend, Ind.). Monoclonal antibodies were generated using approximately 1 xcexcM of purified GPIIb/IIIa in the presence of 10 xcexcM of 2-(S)-(3-pyridylsulfonyl)amino-3-N-[5-[2-(4-piperidinyl)ethyl]-thienothiophene-2-carboxamide]propionic acid as the immunogen for BALB/c mice. Sera samples from the mice were screened using ELISA for antibodies against immobilized GPIIb/IIIa. Hybridomas were generated using the mouse with the highest titer. Supernates from the hybridoma fusion were screened for antibodies using ELISA against immobilized GPIIb/IIIa. Hybridoma supernates with the highest titer were further testing for binding to gel filtered platelets using flow cytometry. Briefly, 100 xcexcl of GFP (2xc3x97108 cells/mL) were incubated with flow cytometry buffer (10 mM Hepes, 5 mM KCl, 145 mM NaCl and 1 nM MgCl) or 20 xcexcM 2-(S)-(3-pyridylsulfonyl)amino-3-N-[5-[2-(4-piperidinyl)ethyl]-thienothiophene-2-carboxamide]propionic acid for 15 minutes, followed by the addition of 5 xcexcL of hybridoma supernate. Samples were incubation at room temperature for 1 hour. Bound antibodies were separated from free antibodies by centrifugation in the presence of 100 xcexcM PGE1 (Sigma Chemical, St. Louis, Mo.) followed by resuspension in flow cytometry buffer containing 100 xcexcM FITC conjugated goat anti-mouse IgG (Jackson Immunoresearch Laboratories, West Grove, Pa.). Following incubation for 1 hr at room temperature, the sample was analyzed by flow cytometry using the FACSCaliber system, Becton Dickinson (Franklin Lakes, N.J.). The light scatter and fluorescent intensity of 20000 cells was collected using logarithmic gain.
Several hybridomas which showed an increased fluorescence in the presence of ligand, and one which did not show such increase, were subcloned. The single cell clones were tested using the flow cytometry assay described above. Monoclonal antibodies of interest were purified from ascites fluid by passage through a Protein G-Sepharose column (Pierce, Rockford, Ill.). Purified antibodies were quantitated by amino acid analysis and isotyped using the Isostrips test kit purchased from Boehringer Mannheim Corporation (Indianapolis, Ind.).
Gel Electrophoresis and Western Blotting
Purified GPIIb/IIIa (10 xcexcg) was subject to electrophoresis through an 8% SDS-PAGE (Novex (San Diego, Calif.)) under nonreducing conditions. The proteins were transferred to nitrocellulose membranes and reacted with purified antibody. Bound antibody was detected by sheep anti-mouse, Ig horse radish peroxidase conjugate (Amersham (Arlington Heights, Ill.)). Proteins were visualized using the Renaissance chemiluminescence system (Dupont-NEN (Boston, Mass.)).
Surface Labeling and Immunoprecipitation
2xc3x97106 cells or 2xc3x97107 platelets were surfaced labeled with 5 mM Immunopure-Sulfo-NHS-LC-Biotin (Pierce, Rockford, Ill.) and then solubilized in RIPA buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM CaCl, 1% NP-40, 0.5% Deoxycholate, 0.1% Sodium Dodecyl Sulfate) containing 1 mM phenylmethylsulfonyl fluoride, 10 xcexcg/ml aprotinin, and 100 xcexcg/mL leupeptin. Cell extracts were precleared with Pansorbin (CalBiochem, (La Jolla, Calif.)) and immunoprecipitated overnight with 5 xcexcg of antibody followed by the addition of goat anti-mouse IgG-sepharose-4B beads (Organon Teknika Corporation (Durham, N.C.)). The anti mouse-sepharose beads were pelleted and washed in RIPA buffer, resuspended in reducing sample buffer containing 6 mM 2-mercaptoethanol and boiled for 5 minutes. The proteins were resolved on an 8% SDS-PAGE and transferred to nitrocellulose. Following transfer the proteins were stained with stepavidin-horseradish peroxidase conjugate (Amersham (Arlington Heights, Ill.)) and developed using the Renaissance chemiluminescence system.
Determination of Antibody Binding Affinity
The binding affinities of the antibodies to resting, ligand occupied and activated gel filtered platelets were determined using a flow cytometric method developed for measuring the kinetics of binding parameters to GPII/IIIa on platelets. Prior to the Kd determination of mAb10-758 and mAb15-758, the Kd of the FITC labeled goat anti-mouse secondary antibody used in the flow cytometry method had to be determined. Briefly, gel filtered platelets were diluted in flow cytometry buffer (10 mM Hepes, 5 mM KCl, 145 mM NaCl and 1 mM MgCl) to a final concentration of 2xc3x97107 cells/mL, which gives a range of receptor concentration from 2.0-5.0 nM. The platelets were then incubated with saturating concentrations (10 nM) of the anti GP IIb/IIIa monoclonal antibody CD61 (Immunotech (Westbrook, Me.)) for 1 hour at room temperature. Increasing amounts of FITC conjugated goat anti-mouse were added to 200 xcexcl of platelet suspension and incubated for 1 hour at room temperature. Following incubation, the samples were analyzed using FACScaliber flow cytometry system measuring fluorescent intensity of 20000 cells. Kenetic determinations were done using Sigma Plot software from Jendel Scientific. A value of 32 nM was determined for the FITC anti-mouse secondary antibody. Based on this result 150 nM of FITC conjugated goat anti-mouse antibody was used in all subsequent Kd determinations.
The binding affinities of mAb10-758 and mAb15-758 were determined by incubating 200 xcexcL of 2xc3x97106 GFP/mL (gel filtered platelets/mL) with increasing concentrations of antibody for 1 hour at room temperature. For the ligand treated samples the platelets were incubated with 1 xcexcM of 2-(S)-(3-pyridylsulfonyl)amino-3-N-[5-[2-(4-piperidinyl)ethyl]-thienothiophene-2-carboxamide]propionic acid for 15 minutes prior to antibody addition. Incubation of the GFP with 20 xcexcM ADP (Chrono-log Corporation (Havertown, Pa.)) for 5 minutes prior to antibody addition provided the activated platelet sample. 150 nM of FITC conjugated goat anti-mouse secondary antibody was added to the sample and incubated for 1 hour followed by analysis using flow cytometry. The light scatter and fluorescent intensity of 20000 cells was collected using logarithmic gain. Nonspecific secondary antibody binding was corrected for by subtracting the fluorescent mean/platelet value of secondary antibody binding to platelets in the absence of primary antibody.
The binding affinities of FITC-labelled CD61 as well as CD41a (Dako Corporation (Carpinteria, Calif.)) to resting gel filtered platelets were determined in a similar manner.
Flow Cytometry Binding Assays on Platelets
To study the effect of receptor occupation with various fibrinogen receptor antagonist on antibody binding, 100 xcexcL of 2xc3x97108 GFP/mL in platelet buffer, were pretreated for 15 minutes with 1 xcexcM of ligand: previously defined compound A, tirofiban, compound B, compound C, compound D, RO44-9883 and echistatin, or 1 mM RGDS diluted in flow cytometry buffer. 100 nM of purified antibody was added and incubated for 1 hour at room temperature. The platelet bound antibodies were separated from free antibodies by centrifugation in the presence of 100 xcexcM PGE1 followed by resuspension in flow cytometry buffer containing 100 nM of FITC conjugated goat anti-mouse antibody, incubated for 1 hour at room temperature and analyzed using flow cytometry. The light scatter and fluorescence intensity of 20000 cells was collected using logarithmic gain. For analysis of binding to activated platelets the platelets were activated with 20 xcexcM of ADP for 5 minutes at room temperature prior to antibody addition.
To study the influence of complex denaturization on antibody binding the platelets were treated with of 5 mM EGTA pH 8.0 at 37xc2x0 C. (Shattil et al. (1985) Blood 66, 92-98) prior to the addition of 100 nM purified antibody addition and further incubation for 1 hour at 37xc2x0 C.
PAC-1 (Shattil et al. (1985) J. Biol. Chem. 260, 11107-11114) binding studies were done using 20 xcexcg/mL FITC conjugated PAC-1 (University of Pennsylvania Cell Center (Philadelphia, Pa.)) diluted in PBS. 25 xcexcL of resting 1xc3x97108 GFP/mL, in flow cytometry buffer containing 1 mM CaCl, was combined with 100 nM of antibody and FITC conjugated PAC-1 to a final volume of 100 xcexcL. The sample was incubated for 1 hour at room temperature. Following incubation the cells were diluted to 300 xcexcL in flow cytometry buffer immediately before flow cytometry analysis. Contol PAC-1 binding to activated platelets was tested by first activating the receptors with either 1 nM thrombin or 20 xcexcM ADP. Some samples were pretreated with activation inhibitors 100 xcexcM PGE1, 20 xcexcg/ml apyrase (Sigma Chemical Company (St. Louis, Mo.)) and 10 xcexcM PGI2 (BioMol (Plymouth Meeting, Pa.)) prior to antibody addition or platelet activation with ADP or thrombin.
Cell Binding Assays
Binding of the antibodies to various cell lines was analyzed by single-color flow cytometry. Cells were harvested with trypsin/EDTA, washed three times in Dulbecco""s phosphate buffer saline and diluted to 2xc3x97105 in flow cytometry buffer containing 1 mM CaCl. For compound treated sample, 1 xcexcM of compound was added 15 minutes prior to the addition of 100 nM of mAb3-758, mAb10-758 or mAb15-758. Samples were incubated for 40 minutes at room temperature. 100 nM of FITC conjugated goat anti-mouse was added directly to the sample and incubated for 1 hour at room temperature followed by flow cytometry analysis.
Clot Retraction
Inhibition of clot retraction by the monoclonal antibodies was done essentially as described by Chen et al. (Chen et al. (1995) Blood 86, 2606-2615). Briefly, 100 xcexcL of 2xc3x97108 GFP/mL were incubated with 100 nM of the test monoclonal antibody for 30 minutes followed by the addition of DMEM containing HEPES and calcium and human platelet poor fibronectin-depleted plasma. Clot retraction was initiated by the addition of 1 nM thrombin. Sample were incubated for 2 hours at 37xc2x0 C. and clot retraction was estimated visually.
Platelet Aggregation
Aggregation by platelets stir red (1000 rpm) at 37xc2x0 C. was assayed using the Chrono-Log Lumiaggregometer. Antibody mAb3-758, mAb10-758 or mAb15-758 were added to 250 xcexcL of 2xc3x97108 GFP/mL, in platelet buffer containing 2 mM CaCl, in the absence or presence of 20 xcexcM ADP followed by the addition of 200 xcexcg/mL fibrinogen. In some experiments PGE1, apyrase and PGI2 were included as activation inhibitors. Changes in light transmittance were recorded and transferred to Microsoft Excel for data plotting. For the determination of EC50 and IC50 of mAb3-758 and mAb10-758, respectively, control aggregations Pusing ADP alone were performed with each set of concentrations tested.
Fibrinogen Binding
Fibrinogen (Sigma Chemical Company (St. Louis, Mo.)) was labeled with a fluorescence moiety as described in Xia et al. (1996) Br. J. Haematol. 93, 204-214). 100 xcexcL of 1xc3x97108 GFP/mL in flow cytometry buffer containing 2 mM CaCl2 w ere preincubated with 100 nM of antibody for 30 minutes prior to the simultaneous addition of 40 xcexcM ADP and 50 xcexcg/ml FITC fibrinogen. Following incubation for 15 minutes the platelet suspension was diluted 1:10 in flow cytometry buffer and measured on the flow cytometer.
Antibody Competition
The Kd of all antibodies to resting gel filtered platelets was determined using the flow cytometry method previously described. All antibodies were used at saturating concentrations based on their Kd. 2xc3x97107 GFP/mL were either pre-incubated with 100 nM of mAb3-758, mAb10-758 or mAb15-758 prior to the addition of 100 nM FITC conjugated CD61 (Dako (Carpinteria, Calif.)); or 50 nM of FITC conjugated CD41a (Dako (Carpinteria, Calif.)); or the antibodies were co-incubated. Following incubation for 1 hour at room temperature the sample were analyzed by flow cytometry. A decrease in the fluorescent intensity was used as a measure of antibody competition.
Characterization of Anti-LIBS Antibodies
Monoclonal antibodies were generated by immunizing BALB/c mice with purified xcex1IIbxcex23 in the presence of the RGD-mimetic 2-(S)-(3-pyridylsulfonyl)amino-3-N-[5-[2-(4-piperidinyl)ethyl]-thienothiophene-2-carboxamide]propionic acid. The resulting 33 hybridomas yielded 18 supernates which contained antibodies reactive against immobilized purified GPIIb/IIIa in an ELISA screen (absorbance 405 nm greater then 0.5). Flow cytometry analysis for antibodies that had preferential binding to ligand occupied receptors revealed that seven of the hybridomas showed at least a 20% increase in binding in the presence of 20 xcexcM 2-(S)-(3-pyridylsulfonyl)amino-3-N-[5-[2-(4-piperidinyl)ethyl]-thienothiophene-2-carboxamide]propionic acid. Five of these were chosen for single cell cloning (Table 2).
In Table 2, percent increase in binding was calculated following flow cytometry analysis on 2xc3x97108 GFP/mL in the presence or absence of 20 mM 2-(S)-(3-pyridylsulfonyl)amino-3-N-[5-[2-(4-piperidinyl)ethyl]-thienothiophene-2-carboxamide]propionic acid and 5 uL of hybridoma supernate. Values were calculated as             treatment      -      control        control    xc3x97  100    %
Relative fluorescence was determined following cytometry analysis on 2xc3x97108 GFP/ml in the presence or absence of 20 mM 2-(S)-(3-pyridylsulfonyl)amino-3-N-[5-[2-(4-piperidinyl)ethyl]-thienothiophene-2-carboxamide]propionic acid and 5 uL of supernate. Values were calculated as       treatment  fluorescence        control  fluorescence
Single cell cloning yielding 20 antibodies, 9 of which showed at least a two fold increase in binding to ligand occupied receptors and could be potential anti-LIBS antibodies (Table 2). Two of these antibodies designated mAb10-758 and mAb15-758, and one antibody with less then a two fold increase, mAb3-758, were chosen for further examination (Table 3).
Relative fluorescence increase in binding to 2xc3x97108 GFP/mL is calculated as described above. Subunit specificity was determined by western analysis as described above. KD values were determined as described above.
Subunit and Binding Specificity
The binding specificity of the antibodies was examined by Western blotting against purified GPIIb/IIIa and immunoprecipiation of surface biotinylated receptors.
mAb10-758 and mAb15-758 recognized a band that migrated to the position of xcex23 subunit under nonreducing conditions. In contrast, mAb3-758 showed no band recognition in Western blots. These results suggest that mAb3-758 may require an integrin complex or that the epitope for the antibody is not present under these conditions.
To further examine the heterodimer requirements as well as integrin specificity of the antibodies, immunoprecipitation of biotinylated surface proteins from the cell lysates of gel filtered platelets, HEL cells (Tabilio et al. (1984) The EMBO J. 3, 453-459), HUVEC cells (Ginsberg et al. (1987) J. Biol. Chem. 262, 5437-5440), HEK-293 cells; and two recombinant cell lines HEK-xcex1vxcex23 and HEK-xcex1IIbxcex23 cells was performed. Table 4 lists the major integrins contained in each cell line.
Integrins on platelets, HEL, HUVEC and 293 cell lines are endogenous. Integrins on HEK-293 and IIbxcex23 cell lines are recombinant.
All three antibodies immunoprecipitated two proteins from HEL cells with mobilities identical to surface labeled proteins from platelets and immunoprecipitated two proteins from HUVEC cells with mobilities corresponding to the xcex1v and xcex23 proteins. None of the antibodies immunoprecipitated proteins from the HEK-293 cells. Taken together these results indicated that these antibodies recognize the xcex23 subunit of xcex1IIbxcex23 and/or xcex1vxcex23 when the two subunits are in an endogenous cell system. However, in the recombinant cell lines HEK-IIbxcex23 and HEK-xcex1vxcex23 only mAb10-758 was able to immunoprecipitate recombinant xcex1IIb and xcex23 proteins from HEK-IIbxcex23 cells and the xcex1v or xcex23 proteins from HEK-xcex1vxcex23 cells and indicate that the antibody does not require the xcex1IIbxcex23 heterodimer for antibody binding. mAb3-758 did not immunoprecipitate any proteins from the HEK-xcex1vxcex23, and mAb15-758 was only able to immunoprecipitate the xcex23 subunit from HEK-xcex1vxcex23. Unpublished results indicate that the receptors in the recombinant HEK-IIbxcex23 and HEK-xcex1vxcex23 cells are conformationally distinct from their endogenous counterparts in platelets or HEL cells and HUVEC cells respectively. These conformational differences could indicate that mAb3-758 and mAb15-759 recognize the xcex23 subunit, and that the subunit has to be in a specific conformation in order for the antibody to bind.
Binding of the Antibodies to Resting Platelets and the Influence of Receptor Occupancy
The binding affinity of the monoclonal antibodies to resting and unoccupied receptors, resting ligand occupied receptors and ADP activated platelets was determined using a flow cytometry method previously described. Examination of the Kd values (Table 3) and histograms of cell count versus fluorescence reveal that monoclonal antibody mAb3-758 has a high binding affinity to resting platelets that is not influenced by the presence of ligand, whereas mAb10-758 has a high binding affinity to resting platelets which is slightly increased on ligand occupied receptors, and mAb15-758 binding to resting platelets is very weak, and is greatly increased in the presence of 1 xcexcM 2-(S)-(3-pyridylsulfonyl)amino-3-N-[5-[2-(4-piperidinyl)ethyl]-thienothiophene-2-carboxamide]propionic acid.
The binding characteristics of mAb3-758 and mAb10-758 to resting platelets and ligand occupied platelets indicate that the epitope for these antibodies is fully exposed on resting platelets. However, the binding characteristics of mAb15-758 to resting unoccupied and occupied receptors raises the possibility that the epitope for mAb15-758 is cryptic in resting platelets and is exposed by ligand induced conformational changes.
To test the hypothesis the GPIIb/IIIa complex was irreversibly denatured by the addition of 5mM EGTA at 37xc2x0 C. Irreversible disassociation of the GPIIb/IIIa complex with 5 mM EGTA abolished mAb3-758 binding to resting platelet. These results support the immunoprecipitation data that indicate that the epitope for mAb3-758 is exposed on the xcex23 subunit but requires a particular conformation of the subunit. Disassociation of the GPIIb/IIIa complex with EGTA did not abolish mAb10-758 binding again confirming that the epitope for this antibody is exposed on resting platelets. In contrast, mAb15-758 binding was enhanced under these conditions. These data support the previous findings that indicate that the epitope for mAb15-758 is not exposed on resting unoccupied receptors, but is exposed due to a conformational change in the receptor. Complete disassociation of the complex under EDTA treatment was confirmed by diminished binding of the complex dependent antibody CD41a to background levels.
Platelet activation by agonists also cause conformational changes of the surface exposed domains of the GPII/IIIa receptor (Kieffer et al. (1991) J. Cell. Bio. 113, 451-461). Binding of all three antibodies was not enhanced by activation of resting platelets by 20 xcexcM ADP. Therefore, the conformational changes in the GPIIb/IIIa complex due platelet activation are different then the conformational changes caused by ligand binding and are not sufficient to enhance antibody binding. Similar results were shown for other anti-LIBS antibodies (Kouns et al. (1990) J. Biol. Chem. 265, 20594-20604).
Effects of Receptor Occupancy by Various Fibrinogen Receptor Antagonists on Antibody Binding
One characteristic of anti-LIBS antibodies is increased affinity for ligand occupied GPIIb/IIIa. In order to determine if various ligands that can bind to GPIIb/IIIa induce a universal conformational change which can be recognized by an anti-LIBS antibody, a series of GPIIb/IIIa antagonist, ranging in structure and affinities (Table 1), were tested for their influence on mAb3-758, mAb10-758 and mAb15-758 binding. Flow cytometric binding measurements showed that all potent GPIIb/IIIa ligands were able to induce the expose of mAb10-758 and mAb15-758. None of the ligands enhanced mAb3-758 binding. These finding reveal that the neo-epitope is induced by multiple ligands including RO-44-9883, a compound which had previously been reported to not induce a LIBS binding site for mAbD3 (Steiner et al. (1993) Thrombosis and Haemostasis 782). These results show that potentially any ligand upon bind to GPIIb/IIIa has the ability to induce conformational changes in the receptor. Similar results were seen in binding studies on platelet rich plasma.
Binding of the Antibodies to Integrins in Cell Lines
The GPIIb/IIIa complex expressed on HEL cell is functionally distinct from the receptor expressed in platelets in terms of ligand binding (Kieffer et al. (1991) J. Cell. Bio. 113, 451-461). These functional differences are partially due to differences in the conformation of xcex1IIbxcex23 is HEL cells versus platelets (Jennings et al. (1996) Blood Cells Molecules and Diseases 22, 23-35). To determine if the antibody binding properties are influence by receptor conformation cell binding studies were performed. Histograms of antibody binding to HEL cells indicate that all three antibodies bind to HEL cells, but that the addition of ligand no longer increased the affinity of mAb15-758 for the receptors. Therefore, even though the antibodies are able to recognize the GPIIb/IIIa receptor in various microenvironments ligand binding does not alter the conformation of xcex1IIbxcex23 in HEL cells in the same was as ligand binding in platelets. Thus supporting the previous findings that the GPIIb/IIIa receptor expressed in HEL cells is functionally and conformationally distinct from the GPIIb/IIIa receptor in platelets.
RGD-containing ligands are also recognized by other integrins including the vitronectin receptor (Pytela et al. (1986) Science 263, 12397-12402). To determine if the occupancy of the vitronectin receptor by ligand also induced LIBS expression, the binding of the antibodies to xcex1vxcex23 expressed in recombinant HEK-xcex1vxcex23 cells and Huvec cells was examined. Both cell lines have minimal expression of xcex1IIbxcex23. Histogram analysis of cell number versus fluorescence intensity showed the addition of ligand (1 xcexcM echistatin) increased the binding of the potential anti-LIBS antibodies mAb15-758 and mAb10-758 to xcex1vxcex23 in HEK-xcex1vxcex23, but not the control antibody, mAb3-758. However, this was not observed when for the endogenous receptors in Huvec cells. Only mAb10-758 bound to xcex1vxcex23 in this cells. The binding differences between the endogenous and recombinant cell lines again supports the idea that the receptors in these two cell lines are conformationally distinct.
Influence of Antibodies on Platelet Aggregation and Clot Retraction
Several anti-LIBS antibodies have been shown to influence platelet aggregation and clot retraction (Kouns et al. (1990) J. Biol. Chem. 265, 20594-20604; Frelinger et al. (1991) J. Biol. Chem. 266, 17106-17111; and Frelinger et al. (1990) J. Biol. Chem. 265, 6346-6352). The ability of mAb3-758, mAb10-758 and mAb15-758 to influence aggregation of gel filtered platelets was assayed by luminaggregometry. 50 nM mAb3-758 added to stirring gel filtered platelets preincubated with 200 xcexcg/mL fibrinogen inhibited ADP stimulated platelet aggregation with an IC50 10.2 nM. Platelets treated with mAb3-758 prior to ADP addition had a 1.3% aggregation response, in contrast to control platelet that had a 69.6% aggregation response to the addition of 20 xcexcM ADP alone.
Like other anti-LIBS antibodies (Kouns et al. (1990) J. Biol. Chem. 265, 20594-20604; Frelinger et al. (1991) J. Bio. Chem. 266, 17106-17111), 100 nM mAb10-758 was able to stimulate an agonist independent platelet aggregation response of 27.1% with an EC50 35 nM compared to ADP stimulated platelet aggregation response of 69.6% with an EC50 1.7 xcexcM. 1 xcexcM mAb15-758 also stimulated an ADP independent platelet aggregation response of 25%. No EC50 value was determined for mAb15-758. Platelet aggregation stimulated by mAb10-758 and mAb15-758, like ADP induced aggregation, is dependent on the presence of fibrinogen as evident by blockage of aggregation by a potent fibrinogen receptor antagonist. Following incubation with 10 xcexcM of antagonist, mAb10-758 induced a 2.2% aggregation response, mAb15-758 a 4.8% aggregation response and ADP a 2.5% aggregation response as compared to 26.9%, 31% and 69.6% respectively in control platelets. However, mAb10-758 and mAb15-758 stimulated platelet aggregation is not dependent upon platelet activation. Platelets that were pretreated with a mixture of PGE1, PGI2, and apyrase prior to the addition of 100 nM mAb10-758 or 1 xcexcM mAb15-758 had a 19.3% and 19% response respectively, as compared to the 27.1 % and 25% aggregation response in control platelets upon antibody addition. Inhibitor treated platelets had a 3.3 % aggregation response following ADP addition, in contrast to 69.6% for control platelets.
Since mAb3-758 blocks platelet aggregation it was of interest to test if this antibody also blocks clot retraction. Clot retraction was blocked mAb 13-758. Whereas the antibodies which do not inhibit platelet aggregation namely, mAb10-758 and mAb15-758, also do not inhibit clot retraction.
Platelet function is mediated by fibrinogen binding to activated platelets (George, et al. (1984) N. Eng. J. Med. 311, 1084). Therefore it was ascertained if mAb3-758 was inhibiting the processes of platelet aggregation and clot retraction by blocking fibrinogen binding to activated platelets. Previous work has shown that fibrinogen binding can be examined using FITC labeled fibrinogen and flow cytometry (Heilmann et al. (1994) Cytometry 17, 287-293). GFP that were preincubated with 100 nM of mAb3-758 prior to activation with ADP and the addition of FITC-Fg. Decreases FITC fibrinogen binding was observed in the samples that were pretreated with mAb3-758 indicating that the antibody does block fibrinogen binding to activated platelets.
Influence of Antibodies on PAC1 Binding
PAC-1 is a murine monoclonal antibody that mimics RGD containing ligands and shows preferential binding to agonist stimulated platelets (George, et al. (1984) N. Eng. J. Med. 311, 1084; Heilmann et al. (1994) Cytometry 17, 287-293). This antibody was used to characterized the activation state of the GPIIb/IIIa complex post antibody binding. Results show that PAC-1 does not bind to unactivated platelets, however activation of the platelets with agonist or incubation with 100 nM mAb10-758 or 1 xcexcM mAb15-758 induced FITC conjugated PAC1 binding to GFP. mAb3-758 did not induce PAC-1 to bind to resting platelets. These results would suggest that mAb10-758 and mAb15-758 either alter the conformation of GPIIb/IIIa and allow PAC-1 to bind to resting platelets or that the antibodies activate the receptor. To determine if platelet activation or a conformational change is involved in the ability of mAb10-758 and mAb15-758 to induce PAC-1 binding to resting platelets the platelets were pretreated with a combination of activation inhibitors, namely PGE1, PGI2 and apyrase prior to the addition of antibody or agonist. The inhibitors completely blocked the ability of ADP to promote PAC-1 binding, however PAC-1 binding to antibody treated platelets was only partially blocked by the addition of inhibitors. These results support that the antibodies induce a conformational change in the receptor which allows PAC-1 to bind.
Competition Between Other GPIIb/IIIa Antibodies:
To elucidate the location of the epitope for the antibodies, competition analysis between the monoclonal antibodies and several other known GPIIb/IIIa antibodies was performed. Table 5 shows the results of the competition studies.
xe2x80x9cYesxe2x80x9d means the antibody competes with the monoclonal antibody. xe2x80x9cNoxe2x80x9d means the antibody does not compete with the monoclonal antibody.
Competition analysis between monoclonal antibodies and other anti-GPIIb/IIIa antibodies was performed on 2xc3x97107 GFP/ml using flow cytometry.
PAC-1 blocks fibrinogen mediated platelet aggregation. Therefore, it was of interest to test if mAb3-758 could compete with PAC-1 binding to platelets. Pre-incubation of activated platelets with 100 nM of mAb-3 reduced PAC-1 binding but did not completely block binding.
The epitopes for CD61 and CD41a are located on the xcex23 and xcex1IIb subunit of GPIIb/IIIa respectively. Competition studies between these two antibodies and all three monoclonal antibodies revealed that none of the antibodies competed with CD61 or CD41a, indicating that these antibodies recognize an epitope that does not overlap the epitope for CD61 or CD41a.
Preparation of Humanized Monoclonal Antibodies
General procedures for making of hybridoma cell lines for the purpose of producing monoclonal antibodies are well known. Procedures for humanizing monoclonal antibodies are exemplified in U.S. Pat. No. 5,565,354, which describes procedures for obtaining human monoclonal antibodies effective in particular against hepatitis B surface antigen (HBsAg), such antibodies being prepared according to a generally applicable method described by the applicant in Hybridoma (1983) v. 2 n. 4, p. 361 and British Patent Publication 2,113,715A. It has been shown, for example, that a hybridoma cell line comprising a parent rodent immortalizing cell, such as a murine myeloma cell, e.g., SP-2, fused to a human partner cell, results in an immortalizing xenogeneic hybridoma cell. This xenogeneic hybridoma cell may be fused to a cell capable of producing an anti-HBsAg human antibody, resulting in a novel trioma cell line capable of generating human antibody effective against such antigen in the human. Alternately, when greater stability is desired, a trioma cell line which preferably no longer has the capability of producing its own antibody is made and this trioma is then fused with a further cell capable of producing useful against said antigen so as to obtain a still more stable hybridoma (quadroma) which produces antibody against the antigen.
In U.S. Pat. No. 5,565,354, a xenogeneic hybridoma referred to as SPAZ 4 was prepared from drug resistant cell line SP-2 obtainable, e.g., from the NIGMS Human Genetic Mutant Cell Responsitory Ref. GM35669A (see United States DHHS 1982 Catalog of Cell Lines). Preparation of SPAZ 4 is summarized as follows. The SP-2 cell line is fused with normal human peripheral lymphocytes by conventional techniques. A large number of hybrids is obtained and, after approximately five weeks, five clones are selected which show fast growth and no antibody production. These cells are selected for resistance to 8-azaguanine and with three of these lines it is possible to obtain mutants which are resistant to 20 xcexcg/ml of 8-azaguanine. These cells are at the same time sensitive to Hypoxanthine-Aminopterin-Thymidine (HAT) medium which showed that they had lost their ability to produce hypoxanthine phosphoribosyl transferase. One of these cell lines is SPAZ 4.
Cell line SPAZ 4 may be fused with cells obtained from the blood of persons immunized with hepatitis B vaccine to obtain hybridoma cell lines which provide positive cultures when standard selection procedures are used involving binding of antibodies to relevant viral antigens. Positive cultures may be placed through a second selection process in which different subtypes of the virus are used for antigen preparation. This provides us an opportunity to pinpoint the exact antigenic determinant recognized by the antibody.
The cell lines resulting from the fusion of a xenogeneic hybridoma and the human monoclonal antibody producing cell (trioma) are therefore useful in providing monoclonal antibodies capable of effective activity in neutralizing a virus causing hepatitis, and the antibodies can therefore prevent the spread of hepatitis through, e.g., blood transfusion. They can also be used to give initial protection to newborn babies or exposed individuals earlier than a vaccine could be effective. Anti-hepatitis antibodies may be used to protect immunosuppressed patients, including transplantation patients, from recurrent hepatitis. This is most significant in cases of hepatitis B positive liver recipients. Further, the antibodies can be used in diagnostic assays. The antibodies can be humanized using humanizing techniques generally known in the art.
The cell line DD6, which produces mAb10-758, has been placed on deposit at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md., 20852 on Aug. 27, 1997 under the ATCC designation HB-12393. The cell line FB3, which produces mAb15-758, has been placed on deposit at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md., 20852 on Aug. 27, 1997 under the ATCC designation HB-12394. These deposits were made under the provisions of the Budapest Treaty of the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure and the Regulations thereunder (Budapest Treaty). Maintenance of a viable culture is assured for 30 years from date of deposit. The organisms will be made available by ATCC under the terms of the Budapest Treaty, and subject to an agreement between Applicants and ATCC which assures unrestricted availability upon issuance of the pertinent U.S. patent. Availability of the deposited strains is not to be construed as a license to practice the invention in contravention rights granted under the authority of any government in accordance with its patent laws.

Hybridomas and monoclonal antibodies for an anticoagulant test

Abstract: i) a plasma sample of the patient;


Systems and methods of determining motion vectors, such as for video encoding, are disclosed. In one example, motion vectors are determined for a current frame, using sampled pixel information from a reference frame. Sampled pixel information is obtained using a sampling pattern. The sampling pattern, in one example, includes subsampling pixels at different rates for horizontal and vertical directions. The subsampling rate can differ, based on an amount of motion represented by a matching block (e.g., the farther a match is found away from an origin of the block, the more subsampling can be done). In another example, a full pixel resolution is maintained proximal an original location of the block; as distance increases in one or more directions, subsampling can begin and/or increase. Sampled pixels can be stored. Interpolation of the sampled pixels can be performed and the sampled and resulting interpolated pixels can be used for comparison.

The present invention relates to a method and apparatus for determining motion between a first and second video image and, in particular, to reducing the number of pixels which are required to be used in order to determine the motion.
Motion video consists of a sequence of image frames. Motion estimation algorithms exploit the fact that these frames do not change significantly in time. Motion vector fields are calculated that describe the displacement of all the pixels in a frame to an earlier frame. The image is usually divided into a grid so that a single motion vector is associated with a group (block) of pixels to reduce computational complexity.
Motion estimation can be performed using a number of different methods, including, for example:                Block-matching techniques as described in section II of De Haan, G.; Biezen, P. W. A. C. "An efficient true-motion estimator using candidate vectors from a parametric motion model" Circuits and Systems for Video Technology, IEEE Transactions on Vol. 8, Issue 1, February 1998, pp. 85-91.        Gradient based techniques as described in Horn, B. K. P. & B. G. Schunck, 'Determining Optical Flow,' Artificial Intelligence, Vol. 16, No. 1-3, August 1981, pp. 185-203.
FIG. 1 illustrates the known block matching technique between a sequence of two images and demonstrates that block-matching motion estimators typically attempt to find a block of pixels (30) in an earlier frame that has a high correlation to an equally sized block of pixels (20) in the current frame. The correlation is usually measured by a form of error function such as the sum of absolute differences (SAD) or the mean squared error (MSE). The displacement between the best matching blocks represents the motion vector (40). 'Full search' motion estimators choose to search for a matching block across the full range of the previous image. However, to reduce computation, most motion estimators reduce the search window for matching. blocks by defining a maximum search area (10) and either perform a full-search within the window or evaluate only a number of 'candidate' blocks. This search area effectively limits the maximum motion vector size that is supported, and hence the fastest motion that can successfully be estimated as shown in FIG. 2.
Motion estimation is useful for several applications including, for example, motion compensated interpolation, where a number of interpolated frames are reconstructed and displayed between original Images in a sequence. This reduces the motion judder of moving objects in the image sequence. The motion vectors here can be defined at any point in time between the two images, for example the midpoint, and are also used to reconstruct the interpolated frame using motion compensation.•
A further application is video compression, for example, where spatial and/or temporal redundancy is removed. Motion vectors are again used to describe the displacement of Image pixels between successive frames and, in this application, are transmitted in place of image data, thereby reducing the amount of data needed to represent the image sequence.
Other applications that use motion estimation to enhance video sequences include de-interlacing and noise reduction.
Detection and measurement of motion blur is also relevant to the derivation of motion vectors. Motion blur is an effect that occurs in natural image sequences as cameras attempt to capture moving objects. If an object moves while the camera shutter is open, the film acts as an integrating medium and the motion that has occurred is visible as a blur along the trajectory of motion. This known effect' is shown in FIG. 3 and is effectively a low pass filtering operation on the moving image pixels.
The faster object is moving relative to the camera, the greater the motion blur. Since motion blur occurs only along the trajectory, detail present on the moving objects is low-pass filtered in this direction only. This can be seen further in FIG. 3 where detail of the front and rear edges of moving vehicles are not visible, whereas the roof and base edges can still be distinguished. The level of sharpness of edges in an area of the image can be used as an indication of the level of motion. If sharp edges are detected in a particular direction, it can be deduced that in a perpendicular direction, fast motion is unlikely.
In order to successfully estimate motion in a wide variety of image sequences, large vector ranges are required. This increases the search area, hence the number of pixels that are required to be available during the calculation and evaluation of motion vectors within this region.
In order to perform motion estimation with large search areas, the transfer of large amounts of pixel information between processing blocks is necessary which demands large bandwidth.
Pixel storage, when incorporated into a hardware implementation, is expensive and bulky therefore it is desirable to reduce the number of available pixels required in the motion estimation and compensation stages.
Some schemes have previously been proposed to reduce the cost of implementing a large search area and these techniques will be known to those skilled in the art. For example U.S. Pat. No. 6,687,303 describes a scheme where neighbouring pixels in blocks to be evaluated are sub-sampled by 4:1 or 2:1. A similar scheme is described in U.S. Pat. No. 6,317,136 and also U.S. Pat. No. 5,982,910 in which 2:1 sub-sampling is performed on incoming pixel data. Simple sub-sampling such as this is successful in reducing complexity, however useful image information is discarded in the process. This can lead to a reduction in the quality of motion' estimation as details in the image sequence, that aid the estimation and convergence of vectors, may be lost.
We have appreciated that as the speed of motion of an object in an image increases, the degree of low-pass filtering on the object in the image increases due to the motion blur and less detail is visible along the motion trajectory. Therefore, the faster the motion, the less high frequencies are present in the image at this location. It is known from the sampling theorem that the higher the frequency content in a signal, the higher the sampling rate must be in order to fully reconstruct the signal. It therefore follows that as the speed of motion increases, the lower the required sampling rate becomes.
As an extension of this, objects that are moving in a horizontal direction become blurred along the horizontal only and, similarly, vertically moving objects lose detail in this direction but retain horizontal detail. Therefore, for pixels undergoing motion in a certain direction, there will be relatively low frequency content along this trajectory, while frequency content perpendicular to this direction is maintained. This implies that the sampling frequency required along the trajectory of motion is lower than that required in the perpendicular direction. Therefore, in order to minimise the loss of important image details, pixels undergoing horizontal motion can be sub-sampled horizontally and pixels undergoing vertical motion can be sub-sampled vertically.
We have appreciated that in order to retain maximum detail in the search area, all pixels should be maintained. However, pixel storage is expensive and the transfer of large amounts of pixel information requires large bandwidth. Images which include objects in motion include blurring and we have appreciated that in such cases the sampling frequency along the direction of motion can be decreased without experiencing a significant loss of image detail.
Preferred embodiments of the invention perform a sub-sampling of the pixels within a search area in a predefined pattern. The pattern of sub sampled pixels vanes throughout the search area in order to reduce the number of pixels that are stored, and, thus, to reduce the processing and memory requirements of the system, while maintaining important image details.
Preferred embodiments of the invention increase the degree of sub-sampling of image pixels as the distance from the centre of the search area increases since pixels of the block which appear furthest from the centre are due to the fastest motion and can be sub sampled without a loss in image detail.
Preferred embodiments of the invention maintain high pixel resolution close to the centre of the search area in order to maintain detail of static or slow moving blocks. The size of this high resolution area may be influenced by the maximum available search-area memory.
The invention in its various aspects will now be defined in the claims, to which reference should now be made.
A preferred embodiment of the invention Is now described when incorporated into a television system with reference to FIG. 4. This embodiment is used to increase the frame rate of High Definition (HD) motion video sequences using motion compensated interpolation. High Definition sequences have increased vertical and horizontal resolution which requires larger vector Omits for describing the motion between frames than for Standard Definition (SD) material.
FIG. 4 shows a television system (10). An antenna (20) (or other video input such as a DVD player) supplies a tuner which in turn provides a video signal to the processor (30). A processed video signal is then supplied to a motion estimator unit (40). The motion estimator estimates the direction and degree of motion between frames and preferably provides this data in terms of vectors. The temporal frame rate of the video signal is then increased by feeding a motion compensated interpolator (50) with vectors supplied from the motion estimator in order to construct intermediate frames. The enhanced video signal is then displayed on a display device. (60).
The motion estimation unit (40) which incorporates the invention is described in detail below with reference to FIG. 5. The function of the motion estimation unit (40) is to determine the motion between consecutive frames. This motion is described in terms of a vector. The motion between frames is determined by breaking down each video frame into a series of blocks containing a predefined number of pixels. That block is then compared with blocks of pixels in an adjacent frame in order to determine the motion of the block between the frames using block matching techniques, for example SAD or MSC.
In order to reduce the area of the frame in which block matching Is executed, embodiments of the invention conduct the block comparison within a specific search area of the adjacent frame. Preferably, the search area is centred on the position of the block in the original frame. The size of the search area is selected in dependence on the memory and processing capabilities of the system. As discussed above with reference to FIG. 2, larger search areas allow greater degrees of motion to be detected and so can be used to estimate faster motion. Motion of the block is then determined using block matching techniques within the search area. Preferred embodiments of the invention process each block of the frame.
In order to provide maximum quality of motion estimation, all pixels within a search area would be used and compared with the block in motion. However, pixel storage, when incorporated into hardware implementation, is expensive and, therefore, it is desirable to reduce the number of pixels used in the search area. In order to reduce the number of pixels, embodiments of the invention sub-sample the pixels of the search area.
A number of factors may be taken into account when deciding how to subsample the pixels of the search area, namely                the direction in which motion estimation is required to be most accurate;        whether it is acceptable to lose information in order to reduce the number of pixels;        the magnitude of motion of the block; and        the location of blocks of pixels within the search area which will be used as comparison blocks.
In order to estimate motion most accurately in a particular direction, full resolution of pixels should be maintained in that direction to provide more pixels to be available for block matching calculations. Therefore, if accurate motion estimation in the horizontal direction is required, full resolution can be maintained in the horizontal direction. The number of pixels required to be stored can then be reduced by subsampling in the vertical direction. An example of subsampling pixels in the vertical direction, while maintaining a full resolution in the horizontal direction, is shown in FIG. 6. Such a subsampling pattern could be used for video sequences of natural images in which horizontal movement of objects and motion due to camera pans are more common than vertical motion. Such a pattern enables a high accuracy of motion to be determined in the horizontal direction while reducing pixel storage by subsampling in the vertical direction.
Alternatively, the choice of direction of subsampling may be made on the basis of the direction in which subsampling will have minimum effect on data loss. When blocks of pixels are moving in a particular direction detail can be lost in the direction of motion due to motion blur. Since little information can be gained from the blurred pixels, by subsampling in the direction of motion the number of stored pixels can be reduced without losing significant information. Therefore, if an object Ie moving in the horizontal direction, subsampling in the horizontal direction can be used without losing significant information. Conversely, if an object is moving in the vertical direction, vertical subsampling can be used without losing significant information. Therefore, the vertical subsampling pixel pattern of FIG. 6 can be used for vertical motion in order to reduce the number of pixels stored without losing significant detail.
A further consideration in determining the subsampling pattern is the :expected degree of motion of the block. When a block has moved between image frames, search areas which are centered on the original position of the block include the relevant pixels away from the centre of the search area. Specifically, for horizontal motion the relevant pixels are to the left and right of the centre and for vertical motion the relevant pixels are positioned above and below the centre of the search area. As the speed of motion of the object is increased, the degree of subsampling can be increased without compromising on losing image-detail. Conversely, if there is little or no motion the relevant pixels required for motion estimation will be positioned predominantly towards the centre of the search area. Since there will be no motion blur for static or slow moving blocks it is desirable not to subsample the pixel data close to the centre of the search area to avoid losing important image detail.
The final selected subsampling pattern may be chosen to allow measurement of static, medium and fast motion. The degree of sub-sampling may be chosen to remain constant outside a central full-resolution area or, preferably, can be increased as the distance increases from the centre of the search area. For example, zones of 2:1 sub-sampling can be used for moderate motion, 4:1 for fast motion and 8:1 for very fast motion. Such a pattern maintains detail when the block is stationary by maintaining a full resolution at the centre of the search area and, as speed of motion increases and the relevant blocks are further from the centre, the loss of information Is limited due to increasing degrees of motion blur.
A further reduction in the pixel storage requirements can be obtained by using a combination of horizontal and vertical subsampling in combination with regions of full resolution. Again, combination of subsampling ratios can be used in different regions of the search area in order to store the minimum number of pixels while experiencing minimum loss of useful information.
FIG. 7 is an example of a subsampling pattern which incorporates both vertical and horizontal subsampling of different degrees at different regions of the search area. The embodiment of FIG. 7 requires less pixels to be stored than FIG. 6 for an equivalent size search area.
FIG. 8 shows a further subsampled search area in which diagonal pixels are also removed in order to reduce the pixel storage requirements.
Returning to FIG. 5, for each block in the input frame, a candidate selector (42), chooses a number of candidate motion vectors,
            C      →        =          [                                    Cx                                                Cy                              ]        ,relating to blocks in the defined search area. The set of candidate vectors are supplied to the candidate evaluator (44) to discover the best match. The motion estimator can choose to use all possible motion vectors within the search area or test only a selection of the vectors. This decision is typically made during the design of the system but may be selectable during use. Described here, the candidate evaluator compares vectors to determine the most suitable candidate using the SAD criterion for simplicity, as described by Equation 1,
                              SAD                                (                                          C                _                            ,                              x                _                                      )                          =                                            ∑                              x                ∈                                  B                                                        (                                          x                      →                                        )                                                                        n                                                                                    F                                                  (                                                            x                      →                                        ,                    n                                    )                                            -                              F                                                  (                                                                                x                        →                                            -                                              C                        _                                                              ,                                          n                      -                      1                                                        )                                                                                                    Equation                                                  1
, where C represents a candidate motion vector,
      x    _    =      [                            x                                      y                      ]  represents the spatial location within the search area, B( x) represents the block of pixels over which evaluation occurs and F( x, n) represents the pixel intensity value at spatial location x and temporal location n.
The pixel information F( x, n), is supplied from the current block (48) from the input image data. In order to acquire pixel information from the previous frame, F( x, n−1), the input image data passes through a delay (41) and into the sub-sampling block (47). The process of this block is described in more detail later.
The sub-sampling block provides the search-area memory (43) with sub-sampled pixels which in-turn, via an interpolator (49), provides the candidate evaluator (44) with the necessary pixel information to assess the vector candidates. The subsampled pixels are then compared with the pixel information of the current block using the candidate motion vectors. The candidate with the lowest error function is determined to be the best match for the current block, and the associated motion vector is assigned to the block of pixels.
For this embodiment, the defined search area will allow a maximum vector size of +/1 64 pixels horizontally and +/−32 pixels vertically as shown in FIG. 9. In further embodiments different sized search areas may be used depending on the requirements and constraints of the system. With a block size of 16×16 pixels, this defines the search area as a set of pixels with dimensions 144×80. The sub-sampling block (47) allows a reduced number of pixels to be stored by directionally sub-sampling the pixels in the search area. For this example, horizontal and vertical sub-sampling are utilised and the pixels selected in the sub-sampling operation are shown in FIG. 10. For simplicity, only the right-half of the full search area (10) is shown es the pattern of pixel selection is symmetrical about the origin. The labels indicate the horizontal and vertical pixel coordinates respectively, with reference to the origin at the centre of the block.
Pixels in-between now no longer need to be stored and can instead be estimated during evaluation by the interpolator (49), as shown in FIG. 5. This interpolator performs, for example, a linear interpolation between available pixels although other interpolations could be used. Using the coordinate system. as shown in FIG. 10, if, for example, the pixel within the search area at coordinate (24,9) Is required, an interpolation is performed between pixels (23,0) and (31,0) available from the search area memory as described by Equation 2.
                                                                                F                  ^                                                                  (                                      24                    ,                    n                                    )                                            =                                                                                (                                          1                      -                                                                        31                          -                          24                                                8                                                              )                                                                          F                                                              (                                              31                        ,                                                  n                          -                          1                                                                    )                                                                      +                                                                                (                                      1                    -                                                                  24                        -                        32                                            8                                                        )                                                                  F                                                        (                                          23                      ,                                              n                        -                        1                                                              )                                                                                =                                                                                1                    8                                                                          F                                                              (                                              31                        ,                                                  n                          -                          1                                                                    )                                                                      +                                                      7                    8                                                                          F                                                              (                                              23                        ,                                                  n                          -                          1                                                                    )                                                                                Equation                                                  2
This is trivially extended into a two-stage process for pixels requiring horizontal and vertical interpolation.
Using the sub-sampled search area and the interpolator, pixel information in a large search area can be provided to the candidate evaluation stage of the motion estimator using a reduced number of stored pixels and reduced bandwidth. From the selection of candidates, the best matching vector with the lowest error function is determined and assigned to the current block in the vector memory (46), as shown in FIG. 58.
The vector field from the motion estimator (40) is then used by the motion compensated interpolator (50), as shown in FIG. 4, to reconstruct new frames to increase the frame rate of the video signal. The same search area memory can also be utilised during the motion compensation stage to provide pixels for the reconstructed frame.
It will be clear to those skilled in the art that embodiments of the invention use subsampling to reduce the pixel storage requirements for search areas while maintaining Important Image information which is used to determine the correct motion vectors within video frames. As a result of reducing the number of pixels required to be stored, the bandwidth requirements between the external pixel memory and the internal processing blocks is also reduced.

This patent is subject to a terminal disclaimer.

Method and apparatus for determining motion between video images


The invention concerns a solid or liquid fuel composition capable while burning of producing a selected and varied colored flame other than the color of a standard flame. The composition comprises triethanolamine and a color-forming agent. The invention also concerns a candle or lighting device with a colored flame prepared from the composition. The invention further concerns a method for making a candle with a colored flame comprising the steps of casting the composition into a mold wherein a wick has previously been fixed, cooling the cast composition, and removing the cooled composition from the mold.

The present invention relates to an article and method of producing a colored flame, wherein the article comprises a fuel material and a chromogenic agent.
According to a first aspect, the invention relates to a solid or liquid fuel composition capable, while burning, of producing a selected and varied colored flame other than a standard or usual flame color. The present invention also concerns a candle or a lighting device with a colored flame prepared from the fuel composition. According to a third aspect, the present invention relates to a process for the manufacture of a colored flame candle.
The word xe2x80x9ccandlexe2x80x9d is used to designate a functional or decorative lighting device comprising a wick, generally braided, embedded in a meltable fuel material capable, while burning, of producing a flame with a height generally between 2 and 4 cm. Usually, the meltable fuel material is comprised of waxes of various origins. A mixture of paraffin, stearin and, in minor amount, beeswax and mineral wax is typically used as meltable fuel material.
Commercial compositions often comprise major amounts of paraffin and stearin at 80-90 wt. % and 10-20 wt. % respectively. This composition improves the melting properties of the composition, improves compatibility between the various constituents, and avoids smoking during combustion.
During the melting of the meltable fuel material, occurring generally at a temperature between 60-80xc2x0 C., the liquid mixture rises into the wick by capillary action and catches fire at the wick end. The flame temperature of a conventional candle is about 1000-1,200xc2x0 C.
Various additives may be added to the meltable fuel material, for example, fluidity control agents to control the molten fuel material fluidity, and aromas or fragrance, such as incense.
For a long time, numerous efforts have been made to obtain a candle capable, while burning, of producing a colored flame other than a standard flame color. For example, it is known that ions of certain elements, including some metalloids having a sufficiently low excitation potential and most metals, radiate in the visible spectrum when they are in a gaseous phase and are in a sufficiently excited state. The combustion of such an element, or chromogenic agent, in a flame produces a characteristic color.
Chromogenic agents conventionally used for the preparation of a candle with a selected colored flame are, for example, derivatives of boric acid, copper or thallium compounds, etc. for a green colored flame, lithium or strontium compounds, etc. for a red colored flame, copper halides for a blue colored flame, sodium compounds for a yellow colored flame, lanthane compounds for an orange colored flame, etc.
U.S. Pat. No. 4,386,904, JP-Kokai-53-30,176, and JP-Kokoku-50-22,828 use a candle with a wick, which has been previously impregnated with a solution of an alkaline or alkaline-earth metal salt, oxide or hydroxide. Attempts have also been made to use a conventional meltable fuel material, such as wax, paraffin, fatty acid, etc., into which is added a chromogenic agent as a dust or powder of a metal, mineral salt, oxide or hydroxide (JP-Kokai-47-14,973), borate or perchlorate compounds dissolved in an ethanolamine derivative (U.S. Pat. No. 1,816,140, U.S. Pat. No. 2,551,574, U.S. Pat. No. 3,150,510 or DE-C-530,147) or, more recently, as a fatty acid salt.
Candles having a coating containing these various chromogenic agents were also disclosed. When the candle bums, the coating melts and releases theses chromogenic agents into the molten mixture in which the wick is imbedded.
None of these solutions is actually satisfactory because inhomogeneous distributions of these compositions do not allow a candle to produce a flame having a stable color. As indicated above, the temperature of a conventional candle flame is of about 1000-1200xc2x0 C.; however, the inventors have observed that this temperature range is largely insufficient to excite conventional chromogenic agents. On the other hand, with a xe2x80x9chotxe2x80x9d flame having a temperature higher than or of about 1,700xc2x0 C., it was observed that these chromogenic agents could provide a colored flame fully satisfactory as to the obtained color.
Stemming from this principle, prior art discloses introducing a fuel component into the fuel composition that is able to increase the flame temperature. For example, French utility model application 2,675,813 teaches the use of hexamethylenetetramine to this end. It is also possible to find in the literature mention of the use of methaldehyde or hydrazine for the same purpose. Practically, these solutions are not satisfactory.
An acceptable candle must comprise a mixture that melts in a controlled way under the effect of heat. In other words, a crater of molten material must form at the candle surface at the level of the wick and its diameter must remain constant when the candle bums. If the crater diameter shrinks, the candle will eventually go out. If the crater diameter increases, the whole candle softens in a totally non-aesthetic manner. The crater diameter and consequently the quantity of molten material is controlled by the flame temperature, which itself depends on the composition of the mixture constituting the candle. Further, the molten mixture must rise into the wick by capillary action and catch fire at the wick end. For the flame of a burning candle to be stable, the speed of the molten material rising into the wick must balance the combustion rate of the same material in the flame. When the speed of the molten material rising into the wick is lower than the combustion rate, the flame rapidly runs out of fuel and eventually the wick itself burns. Consequently, the wick length shortens and the candle goes out. In the opposite situation, the fuel material arrives at the flame level in an amount such that the flame grows and comes down to the molten material crater level. The molten material eventually catches fire and the entire candle ignites.
Further, the molten material crater must contain a homogeneous mixture of the fuel composition, comprising, beside the conventional candle constituents, the chromogenic agent and the compound which, when burning, increases the flame temperature. The temperature of the molten material in the crater should ideally remain stable and be sufficient to ensure the fluidity of the molten material mixture so that it can rise into the wick by capillary action. On the other hand, at this temperature, the mixture must not be able to catch fire spontaneously; otherwise, the molten material crater could catch fire directly and ignite the entire candle.
According to the present invention, it is proposed to prepare a fuel composition capable, while burning, of producing a selected colored flame, by using a fuel composition comprising a chromogenic agent and triethanolamine. Conventional fuel constituents may comprise up to 50 wt. % of the fuel composition. Within the scope of the present invention, the chromogenic agent can be a mixture of several chromogenic agents.
The inventors note that triethanolamine can be used advantageously as a fuel component and is capable of increasing the flame temperature well above 1,700xc2x0 C., which is a temperature sufficient to excite conventional chromogenic agents.
Further, and extremely advantageously, triethanolamine can entirely replace conventional waxes used as meltable fuel material, that is, unlike other above-mentioned fuel components capable of increasing the flame temperature, triethanolamine and a chromogenic agent can by themselves provide the fuel for the candle. Other conventional constituents, such as waxes, paraffin, etc., of the candle, if any, are present in a minor amount not greater than 50 wt. % and preferably below 20 wt. % of the composition. The very low cost of triethanolamine in comparison with conventional candle constituents, is not a negligible advantage.
Additionally, the inventors note that boric acid and its derivatives, the majority of the copper, thallium, tellurium, strontium, calcium, lanthane, sodium, etc. compounds or, more generally, mineral or organometallic compounds conventionally used as chromogenic agents are largely soluble in triethanolamine. Therefore, a fuel composition according to the invention is perfectly homogeneous at the time of its preparation for the manufacture of the candle, and consequently, the manufacture of the candle is simplified. Besides that, a homogeneous mixture is also obtained in the molten material crater when the candle is in use and consequently, the mixture, which rises into the wick by capillary action and which bums at the wick end, is also perfectly homogeneous. The chromogenic agent is therefore burnt at a constant rate and the produced color is stable and constant during the entire combustion.
One skilled in the art will easily determine compositions to prepare from the examples provided hereafter and from routine trials the proportions of the various constituents. Typically, a fuel composition according to the invention can comprise from 1 to 99% by weight of triethanolamine with the remainder being chromogenic agent. As indicated above, triethanolamine and a chromogenic agent alone can furnish the fuel composition according to the invention. However, it is advantageous that the composition also comprises stearin. The addition of stearin reduces smoking of the fuel while burning and improves melting of the fuel mixture.
One skilled in the art will also very easily determine the proportions of various constituents from the examples provided hereafter and from routine trials. Advantageously, a fuel composition according to the invention can comprise from 1 to 98% by weight of triethanolamine, up to 50% by weight of stearin, and the remainder of a chromogenic agent.
A further advantage of the triethanolamine is its compatibility with conventional wax constituents used in the candles. As above indicated, the fuel composition should be as homogeneous as possible. The present invention can produce a homogeneous mixture unlike solutions taught in the prior art.
Advantageously, the chromogenic agent is an ethanolamine derivative, such as triethanolamine borate if a green flame is desired. In this case, the chromogenic agent is infinitely soluble in the fuel composition and, by increasing the chromogenic agent""s concentration, color intensity is markedly improved. Further, ethanolamine derivatives can burn without forming unaesthetic, black combustion residues around the molten material crater. When the chromogenic agent is a mixture of several chromogenic agents, one, several or all of them can be an ethanolamine derivative.
The fuel composition according to the present invention may be used in numerous applications, such as burning lamps, torches, etc. or, generally, as fuel for a lighting device comprising a tank and having a wick partially dipped into the tank, however, since initially it was designed for the manufacture of candles, its main use can be as a candle.
Such colored flame candles may be used during various ceremonies, parties or events for decorative or religious purposes. Accordingly, in another aspect, the invention relates to a colored flame candle characterized in that it consists essentially of a solid solution of triethanolamine and a chromogenic agent. All the compositions according to the invention can clearly be used for the manufacture of candles, the preferred compositions furnishing the preferred candles.
According to a final aspect, the invention relates to a process for the manufacture of a candle comprising the following steps:
(a) heating triethanolamine to a temperature between 53 and 100xc2x0 C.;
(b) incorporating a chromogenic agent into the triethanolamine;
(c) optionally, incorporating stearin into the triethanolamine;
(d) pouring of the homogeneous mixture resulting from the two or three preceding steps, or more generally, of a composition according to the invention into a mold wherein a wick was previously attached;
(e) quenching of the molded composition into the mold; and
(f) removal of a candle from the mold.
Steps (b) and (c) may be inverted or carried out simultaneously. Step (a), the heating of triethanolamine, can also be carried out before, at the same time as, or after steps (b) and (c) or according to any other combination. Given the slow dissolution rate of the chromogenic agent, which can take up to 6 hours, the sequence (a), then (b), and then (c) is preferred. Steps (b) and (c) are preferably carried out under stirring and heating until complete dissolution of the chromogenic agent and, if any, of the stearin.
The wick is a conventional large-size, braided wick. The quenching of step (e) can be performed according to conventional manners known by one skilled in the art such as dipping into a water bath, cooling in air, or simply storing at ambient temperature.
Optionally, the above-described process may comprise a further step (g) of dipping the candle, once removed from the mold, into a bath of liquid coating material. Preferably, the coating material consists essentially of stearin, optionally comprising a coloring agent according to the desired appearance of the candle.
According to a particular embodiment, the candle can comprise various aromas or fragrances well known by one skilled in the art, so that these aromas and fragrances are released into the atmosphere when the candle is burning.
According to another embodiment, the candle comprise several superimposed sections of different compositions (particularly as to the chromogenic agent) so that, when the candle is burning, the flame color changes.

Fuel composition producing a colored flame