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An improved method for performing immunoassays whereby specific binding proteins for vitamin B12, folate and other target analytes are utilized with antibodies with different specificities for the binding proteins. Antibodies bridge the specific binding protein directly or indirectly to a capturable material.
1. A method for determining the presence or amount of folate in a test sample comprising: linking folate binding protein which specifically binds folate to a capturable material utilizing a mix of at least two different monoclonal antibodies, each antibody of said mix of antibodies binding said folate binding protein without inhibiting the folate binding protein's binding activity for said folate and without interfering with the binding of the other antibody, said antibodies being coupled to said capturable material; forming a test mixture by contacting said folate binding protein with said folate present in said test sample; adding a detectable label to said test mixture, said detectable label being either directly or indirectly attached to a specific binding member, said specific binding member specifically binding either said folate in said test sample or the folate hiding site of said folate binding protein; capturing said capturable material by ionic interaction and separating it from said test mixture; and monitoring either the free or captured detectable label for presence or amount of said folate wherein said mix of antibodies coupled to said capturable material results in an enhanced coupling of said specific binding member directly or indirectly attached to said detectable label thereby resulting in a dramatically increased signal.
2. The method of claim 1 wherein said test mixture is formed by contacting said test sample sequentially with said folate binding protein linked to said capturable material and then said detectable label attached to said specific binding member.
3. The method of claim 1 wherein said test mixture is formed by contacting said test sample simultaneously with said folate binding protein linked to said capturable material and said detectable label attached to said specific binding member.
4. The method of claim 1 wherein said mix of antibodies is provided by mixing several capturable materials, each of which has been coupled to a different antibody.
5. The method of claim 1 wherein said test sample is treated with a reducing agent followed by treatment with an alkaline agent prior to combination with said folate binding protein.
6. The method of claim 5 wherein said reducing agent is dithiothreitol.
7. The method of claim 5 wherein said alkaline agent is potassium hydroxide, sodium hydroxide, lithium hydroxide or ammonium hydroxide.
8. The method of claim 1 wherein pteroylglutamic acid (PGA) is used as a standard.
9. A method for determining the presence or amount of folate in a test sample comprising: forming a test mixture by contacting said test sample with an indicator reagent and a capture reagent, said capture reagent comprising folate binding protein linked to a polyanion polymer by a mix of at least two different monoclonal antibodies which specifically bind said folate binding protein, each antibody of said mix of antibodies binding said folate binding protein without inhibiting the folate binding protein's binding activity and without interfering with the binding of the other antibody, said antibodies being coupled to said polyanion polymer; said indicator reagent comprising a detectable label either directly or indirectly attached to a specific binding member, said specific binding member specifically binding either said folate in said test sample or the folate binding site of said folate binding protein; separating said polyanion polymer from said test mixture by ionic adherence to a matrix and monitoring either the free or captured detectable label to determine the presence or amount of said folate in said test sample wherein said mix of antibodies coupled to said polyanion polymer results in an enhanced coupling of said indicator reagent thereby resulting in a dramatically increased signal.
10. The method of claim 9 wherein said test mixture is formed by contacting said test sample sequentially with said capture reagent and then said indicator reagent.
11. The method of claim 9 wherein said test mixture is formed by contacting said test sample simultaneously with said capture reagent and said indicator reagent.
12. The method of claim 9 wherein said mix of antibodies is provided by mixing several polyanion polymers, each of which has been coupled to a different antibody.
13. The method of claim 9 wherein said test sample is treated with a reducing agent followed by treatment with an alkaline agent prior to combination with said capture reagent.
14. The method of claim 13 wherein said reducing agent is dithiothreitol.
15. The method of claim 13 wherein said alkaline agent is potassium hydroxide, sodium hydroxide, lithium hydroxide or ammonium hydroxide.
16. The method of claim 9 wherein pteroylglutamic acid (PGA) is used as a standard.
17. The method of claim 16 wherein said PGA is stabilized by the addition of citrate.
18. The method of claim 9 wherein the said specific binding member which competes with said folate is pteroic acid.
19. The method of claim 9 wherein said detectable label is alkaline phosphatase.
20. The method of claim 9 wherein said capture reagent is diluted with a borate buffer containing 4% sucrose.
21. The method of claim 9 wherein said matrix is precoated with a polycationic material.
22. The method of claim 21 wherein said polycation material is simultaneously added with said test mixture to said matrix.
23. A reagent for use in an immunoassay comprising: folate binding protein, a capturable material and a mix of antibodies wherein said folate binding protein specifically binds folate and is linked to said capturable material utilizing a mix of at least two different monoclonal antibodies, each antibody of said mix of antibodies specifically binding said folate binding protein without inhibiting the folate binding protein's binding activity for said folate and without interfering with the binding of the other antibody, said mix of antibodies being coupled to said capturable material which is captured by ionic interaction, wherein said mix of antibodies results in an enhanced coupling of an indicator reagent thereby resulting in a dramatically increased signal.
24. The reagent of claim 23 wherein said capturable material is a polyanionic material.
25. The reagent of claim 24 wherein said polyanionic material is carboxymethylamylose.
1. Field of the Invention
This invention relates to specific binding assays in which binding proteins specific for a sample analyte are bound by antibodies. The antibodies are specific for different epitopes of the binding protein and can be attached to either soluble or insoluble materials which facilitate separation procedures.
2. Description of the Prior Art
This invention relates to methods and means for determining the presence of a ligand in a liquid medium based on the affinity of the ligand for a specific binding partner. In particular, this invention relates to methods and means for use in specific binding assays which do not employ radioactive materials.
Folate deficiences in the human body are a common cause of megaloblastic anemia. In humans, folic acid is metabolized to tetrahydrofolic acid and subsequently to 5-methyltetrahydrofolic acid (5'-mTHF). Often, the concentration of 5'-mTHF is measured using a competitive binding assay. It would be useful to include 5'-mTHF used in the standard reagents as a calibrator. Unfortunately, 5'-mTHF is very unstable and its use can require sealing the material in lyopholized form.
U.S. Pat. No. 4,350,659 to Riceberg of Corning Glass Works disclosed a process for stabilizing 5'-mTHF by complexing it with a binding protein such as folate binding protein (FBP). The complex is then frozen and lyophilized to yield a dry powder. Recommended storage of the powder includes air tight and light resistant containers. Lypholization permits storage of 5'-mTHF in a stable form until it is needed. Such techniques are impractical in the manufacture of assay kits and make it difficult to use in clinical laboratory settings. Moreover, after reconstituting the lypholized material, instability problems can reappear.
Deficiencies in vitamin B12 may result in neurological damage. Futhermore, as this vitamin is necessary for proper folic acid metabolism, its absence also results in megaloblastic anemias. Since megaloblastosis may also be produced by folate deficiency due to other causes, it is necessary to determine if the megaloblastosis is caused by a deficiency of either or both vitamins.
U.S. Pat. No. 4,399,228 to Riceberg of Corning Glass Works discloses a folate and vitamin B2 competitive protein binding assay. Radioactive 57 Co or 125 I tracer is added to patient samples and counted with a gamma counter. In the assay, the binding protein is covalently bound to porous glass. The endogenous binding proteins in the patient sample are destroyed by boiling the reaction tube. Because of the hazard and difficulty of handling radioactive materials, there have been many attempts to devise convenient specific binding assay systems which are as sensitive and rapid as radioimmunoassays but which utilize features other than radioactivity as the means for monitoring the binding reaction.
U.S. Pat. No. 4,028,465 to Lewin et al., of Bio-Rad Laboratories discloses a radioactive competitive assay procedure where sample serum folate is measured. The serum folate binding proteins are inactivated by heat. The invention discloses the use of a sulfhydryl such as dithiothreitol in buffer which can be used to stabilize folate prior to the heating process. The use of this stabilizer was advantageous over methods utilizing mercaptoethanol because it is an easily weighed solid with only a mild odor.
Folate and vitamin B12 assays typically employed heating or boiling steps prior to testing in order to liberate folate in the sample from endogenous binding proteins. The heating or boiling steps are difficult to accurately control and are time consuming. More recent assays denature samples by chemical means without boiling. One can achieve denaturation by using a strong base with or without other chemicals.
U.S. Pat. No. 4,418,151 to Forand et al., of Rohm and Haas Company also relates to a radioassay for serum folate. A measured amount of serum is mixed with a constant amount of radioactively tagged vitamin B12 and/or folate tracer. The solution is exposed to a mercaptan denaturing agent in the presence of a conversion agent such as potassium cyanide in a highly alkaline environment. The use of mercaptan solutions allows stabilization in the protecting buffer while high pH causes inactivation of the endogenous binding proteins.
U.S. Pat. No. 4,451,571 to Allen of University Patents teaches the use of strong base with a sulfhydral compound, such as betamercaptoethanol (BME), thioglycolate, thioglycerol or dithiothreitol (DTT). The sulfhydral compounds destroy endogenous binding proteins thereby liberating the sample vitamin B12 or folate to be measured. Although the strong base releases analyte from its binding protein, it does not substantially denature all endogenous binding protein. Therefore it is helpful to have another compound such as a sulfhydral to help liberate analyte from binding protein and also eliminate blocking antibodies which may interfere in the assay. The blocking antibodies can be troublesome to the assay because they react with binding factors.
U.S. Pat. No. 4,828,985 to Self of Cambridge Patent Developments teaches a method where secondary antibodies are raised against complexes of nonimmunogenic materials and primary antibodies against the nonimmunogenic materials. The secondary antibodies are not antibodies against either the nonimmunogenic materials nor the primary antibodies. Detection is accomplished by labelling the secondary antibodies with enzyme or some other detectable means.
The present invention is an improvement over existing technology in that it discloses a method that enables more coupling of specific binding proteins. This invention discloses a method whereby a mixture of two monoclonal or polyclonal antibodies or a mixture thereof against different epitopes of a binding protein gives increased coupling. This method can be utilized for several different assays in which analyte is detected. Another advantage of the present invention is that this multiclonal format allows the use of pteryolglutamic acid (PGA) as a calibrator in a folate assay instead of the unstable 5'-mTHF.
The present invention relates to a method of a heterogenous assay whereby the ability to bind specific binding proteins is enhanced with a multiclonal antibody format. The multiclonal format couples binding proteins up to ten times the efficiency of a singular antibody format. The improved method generally comprises directly or indirectly binding a specific binding pair member to a capturable material by utilizing a mixture of antibodies. The specific binding pair member contains binding sites which will then be occupied by either the sample analyte of interest or a labelled analyte analog. A capturable material, one in which the antibodies, specific binding pair members and sample analyte or analyte analog are attached, can then be isolated by ionic interactions with a matrix material where detection can take place.
This invention can be utilized for any assay employing binding proteins which can couple specific ligands. Another advantage of the present invention is the use of PGA as a calibrator in a folate assay instead of 5'-mTHF. Traditional use of 5'-mTHF in folate assays has proved it to be an unstable compound. The use of the present invention allows PGA to be used as a calibrator whereas a monoclonal or polyclonal format alone exhibited differential performance between PGA and 5'-mTHF.
FIG. 1 shows folate binding protein binding PGA similarly to 5'-mTHF in the multiclonal format.
FIG. 2A shows the effects of not adding citrate
FIG. 2B shows the effects of adding citrate for increased PGA stability.
This invention relates to methods and means for determining the presence of a ligand in a liquid medium based on the affinity of the ligand for a specific binding protein.
The present invention discloses a method of a heterogenous assay for measuring analytes in patient samples. There are two separate phases to this procedure. The first phase is the coupling of a polyanionic substance to antibodies with different binding specificities for a particular binding protein. The binding protein is then added to the antibodies creating a polyanion:anti-binding protein antibody:binding protein complex. This complex is referred to as a capture reagent. The second phase is the reaction of particular reagents with the patient sample. The binding protein in the complex captures its particular analyte in the patient sample. The reaction mixture is transferred to a polycation matrix where the polyanion is caught. A reagent containing an analyte analog coupled to an enzyme is added to bind to any unoccupied binding proteins. Non-bound materials are washed away from the matrix and after addition of a standard fluorescent substrate for the enzyme, analyte concentration can be determined from fluorescent intensity.
The present invention also employs a method in which a mixture of two or more anti-binding protein monoclonal or a mixture of monoclonal and polyclonal antibodies works better than a single antibody alone. One example of how this method can work is folate binding protein (FBP). The two or more antibodies (two or more monclonal or a mixture of monoclonal and polyclonal antibodies) are attached to a polyanion such as carboxymethylamylose (CMA) through covalent linkage. FBP is then added to the mixture and the FBP is coupled non-covalently to the antibodies. In effect, the antibodies act as a linker between the FBP and the polyanion. This method prevents having to directly link the binding protein to the polyanion. Direct linkage of the binding protein to the polyanion could conformationally change the binding protein thereby affecting its ability to bind test sample analyte. To show the marked difference when two antibodies are used as opposed to a single monoclonal, two individual monoclonal antibodies were tested against a 1:1 mixture of the two antibodies. In the competitive assay described above, the FBP was linked to the CMA by one or both monoclonal antibodies. The 1:1 mixture of the two anti-FBP monoclonals works better than either monoclonal alone. Table 1 gives an indication as to how effective the multiclonal format is. The numbers in Table 1 refer to the substrate turnover rate. It is important to note that this format uses antibodies that have different affinities to the same protein.
TABLE 1______________________________________ CLONE CLONE 1:1[FLOATE] A (c/s/s) B (c/s/s) MIXTURE______________________________________ 0 ng/ml 370 687 2342 4 ng/ml 206 337 141230 ng/ml 32 48 141______________________________________
The 1:1 mixture of the two antibodies gave a dramatically increased signal. This indicates that there is more folate binding protein bound by the antibodies in the 1:1 mixture. This in turn means more coupling of analyte analog and hence more signal.
The coupling of antibody to the polyanion can be achieved in one of two ways. First, the individual monoclonals or mixture of monoclonal and polyclonals can be coupled at separate times. Therefore, each of the antibodies is coupled to the polyanion in a separate incubation step before being mixed in a 1:1 fashion. The second method is to mix both antibodies together (using appropriate ratios of antibodies) and then couple the mixture to the polyanion in a single incubation step. The second method is useful when working with large quantities of antibody because of the single incubation time.
Different ratios of monoclonal antibodies give better results with the multiclonal format than the individual clones alone. Apparently, even minor additions of a multiclonal format yield increased anti-binding protein antibody:binding protein capabilities. Results of experiments utilizing the monoclonal versus multiclonal format and varying ratios of the multiclonal format are presented in Table 2. Once again, values presented are substrate turnover rate due to detectable label binding to unoccupied binding protein sites.
TABLE 2______________________________________% of % ofClone Clone 0 ng/ml 4 ng/ml 20 ng/mlA B Folate Folate Folate______________________________________100 0 447 245 4380 20 2479 1581 17070 30 2551 1640 17850 50 2461 1571 17230 70 2403 1487 168 0 100 640 360 50______________________________________
FBP antigen was isolated from bovine whey by PGA affinity chromatography. Silver-stained polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing verified the homogeneity of the protein. Two bands visible by PAGE corresponded to the published molecular weights of bovine FBP with and without glycosylation. Cation and anion exchange chromatography, as well as reverse phase high performance chromatography methods, showed no more than two entities in the FBP preparation. Chemical deglycosylation with trifluoromethanesulfonic acid converted the higher molecular weight component into a single entity with an electrophoretic mobility identical to the lower molecular weight band. These data support the conclusion that the two components represent FBP with different degrees of glycosylation.
Additional verification that the protein was FBP was provided by the following: 1) the protein exhibited specific, high affinity binding of radioactive folate, 2) N-terminal amino acid sequence analysis of the first twenty three amino acids provided an unambiguous sequence in perfect agreement with the amino acid sequence published for bovine FBP (Svendsen, I., Hansen, S. I., Holm, J., and Lyngbye, J., Carlsberg Research Communications, 49:12-31, 1984), and 3) gas chromatography mass-spectroscopy analysis showed two components with molecular masses of 30,850 and 25,968 consistent with the expected mass of the FBP polypeptide with and without carbohydrate side chains.
Immunogen Preparation
Purified folate binding protein (FBP) was used as the immunogen for animal immunizations and the antigen for reactivity screening.
Immunization Strategy
Two female 6-8 week old BALB/c mice (Charles River, Wilmington, Mass.) were immunized with purified folate binding protein (FBP). The dose level was 200 μg in 100 μl of a 1:1 ratio of the FBP solution in Freund's Complete Adjuvant (Difco Laboratories, Detroit, Mich.). The adjuvant emulsion route of injection was equally distributed interperitoneally and subcutaneous. The animals were allowed a 3 week rest period before a 100 μg FBP intravenous prefusion boost was administered in 100 μl, 3 days prior to fusion.
On the day of the fusion, the 2 mice were sacrificed by cervical dislocation and the spleen was removed. The splenocytes were washed one time in Iscove's Modified Dulbecco's Medium (IMDM) (GIBGO, Grand Island, N.Y.) and centrifuged 1000 RPM for 10 minutes. The pelleted splenocytes were combined with SP2/O myeloma cells (from the laboratory of Dr. Milsrein, Cambridge, U. K.) at a 1:1 ratio, washed in IMDM, and centrifuged. The supernatant was removed and 1 ml of 50% polyethylene glycol (PEG) (American Type Culture Collection, Rockville, Md.) was added to the pellet for 1 minute as the pellet was gently being dispersed by tapping and swirling. Thirty mls of IMDM were added to the mixture and centrifuged as previously described. The supernate was decanted and the pellet resuspended in IMDM with HAT (hypoxanthine, aminopterin, and thymidine) (Gibco, Gaithersburg, Md.), 10% Fetal Bovine Serum (FBS) (Hyclone Laboratories, Logan, UT.) and Salmonella typhimurium mitogen (STM) (1% v/v) (RIBI Immunochem Research, Inc., Hamilton, Mont.). STM is a B-cell specific mitogen, and is used to enhance fusion frequency. The fusion cell suspension was plated into 96-well tissue culture plates.
Primary Fusion Screening
The primary screening of the fusion occurred on day 10 at which time the cultures were confluent. An enzyme immunoassay (EIA) was used to detect anti-FBP reactivity in the supernate samples. Microtiter wells were coated with 100 μl of a 5 μg/ml FBP in phosphate buffered saline (PBS) and incubated at room temperature overnight. The following day the plates were blocked for 30 minutes with 200 ILl per well of 3% bovine serum albumin (BSA) in PBS. After washing the plates 3 times with distilled water, 50 μl of culture supernate was added per well and incubated 1 hour. The plates were washed 3 times and 50 μl per well of diluted goat anti-mouse IgG+IgM-HRPO (horseradish peroxidase) conjugate (Kirkegaard Perry Laboratories, Gaithersburg, Md.) was added to the plate for a 30 minute incubation period. The plate was washed a final time and the color development utilized O-phenylenediamine:2HCI (OPD) (Abbott Laboratories, Abbott Park, Ill.). The relative intensity of optical density readings identified hybrids #1-279 and #1-641 as 3 times that of the negative control, normal mouse serum (NMS) (Organon Teknika-Cappel, Malvern, Pa.) and the hybrids were selected as candidates for cloning and further evaluation.
Hybrid Cloning
Hybrids #1-279 and #1-641 were cloned by limiting dilutions. 1 to 10 dilutions were done starting at 1�102 up to 1�106. The cloning media used was IMDM with 10% v/v FBS and 1% v/v HT (hypoxanthine and thymidine)Supplement (Gibco, Gaithersburg, Md.). A 100 μl cell suspension was added to each of the 96 well in the TC plate. On day 7 the plates are fed with 200 μl/well of cloning media.
Clone Selection
Clones #1-279-176 and #1-641-101 were selected from the 1�106 dilution wells for further evaluation based on additional EIA screening of the clone supernate of confluent cultures. The EIA screening protocol used is described previously.
Subclone Selection
For purposes of reagent reproducibility it was necessary to ensure that a single cell line of 1-279-176 was obtained. To do so, the cell line was cloned one more time as described above. EIA screening as described above was used for subclone selection of #1-279-866.
Western Blot Evaluation
The FBP antigen, 10 μg, either reduced with 2-mercaptoethanol (Bio-Rad, Richmond, Calif.) or non-reduced was run on an 8-16%, 1.0 mm, mini-polyacrylamide gel (Novex, San Diego, Calif.) on a mini electrophoresis and transfer system (Profile�System, Schleicher & Schuell, Keene, N.H.) according to manufacturer's instructions. The protein was next transferred from the gel onto nitrocellulose. The nitrocellulose was cut into strips and antibody was incubated on the strips for several hours. The antibody binding capability to the reduced and non-reduced antigen was detected using the goat anti-mouse IgG+M-HRPO conjugate mentioned above with the color development driven by 4-chloro-napthol (Sigma, St. Louis, Mo.). Antibody from 1-279-866 was found reactive to the 32kD MW FBP in reduced and non-reduced conditions. Antibody from 1-641-101 was not found reactive to FBP antigen in the Western blot test. Based on these data, the monoclonal antibody produced by the hybrid cell lines were determined to be directed against two distinct epitope binding sites.
The isotypes of the monoclonal antibody secreted from the cell lines identified as 1-279-866 and 1-641-101 were determined on an EIA clonotyping kit (Southern Biotech, Birmingham, Ala.). The assay is performed according to the vendor recommendations and the results indicate that both were IgG1, kappa.
Isoelectric Focusing
Electrophoretic evaluation of the 1-279-866 and 1-641-101 antibodies was performed on the PhastSystem (Pharmacia-LKB, Piscataway, N.J.). Coomassie staining of the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) profile identified the typical antibody banding pattern of a single light chain band at 25 kD and a single heavy chain band at 55 kD for each antibody. The silver stained IEF profile identified a pl of 6.8�0.2 for 1-279-866 and a pl of 6.6�0.2 for 1-641-101.
Cell lines 1-279-866 and 1-641-101 have been deposited with the American Tissue Culture Collection (A.T.T.C on Jan 26, 1993) (16301 Parklawn Drive Rockville, Maryland). Cell line 1-279-866 has been designated A.T.C.C No. HB 11249 and cell line 1-641-101 has been designated A.T.C.C No. HB 11250.
The current invention teaches a method wherein a mixture of two or more anti-binding protein monoclonal or a mixture of monoclonal and polyclonal antibodies gives a better reaction rate than either monoclonal or polyclonal alone. This method of assay is applicable for many proteins and their binders including folate and vitamin B12 but is not limited to them.
The sample to be tested for the presence of analyte can be subject to various steps that denature endogenous proteins which may interfere with the assay. The present invention preferably employs a pretreatment of the sample with the DTT mixed with acetic acid, sodium chloride, and ethyldiaminetetraacetic acid (EDTA). The DTT not only denatures protein but also preserves the reduced form of 5'-mTHF. Other general denaturing agents may substitute for DTT depending on the analyte.
A second denaturing step is preferably the addition of 0.75M potassium hydroxide to the sample. This addition creates a highly basic environment that further denatures the endogenous folate binders in the sample thereby releasing the folate for measurement. Other strong bases such as NaOH, LiOH and NH4 OH can be used.
The capture technique employed utilizes a polyanion such as CMA. Coupled to the polyanion is a mixture of anti-folate binding protein antibodies complexed with folate binding protein. The antibodies act as linkers between the polyanion and the folate binding protein. The folate binding protein binds non-covalently to the antibodies associated with the polyanion. It is important that the denatured sample be neutralized by buffer before or at the time of addition of FBP. In the present invention, the neutralizing is preferably done at the time of FBP addition. The capture reagent may be diluted into a buffer of 50 mM borate, pH 8.1; 0.2% human serum albumin (HSA); 0.1% tween-20; 0.1% sodium azide; 0.003% dextran sulfate; and 1 mM EDTA. The HSA component contains no endogenous folate binding protein. Sodium azide is a preservative commonly used in laboratory reagents which can provide some antibacterial action. Dextran sulfate in the capture reagent binds to stray cations (i.e. cation dust from the matrix) which could interfere in the accuracy of the assay.
The addition of the capture reagent, which contains borate buffer, to the reaction well neutralizes the denaturants and allows the folate in the samples to bind to the folate binding protein. After an incubation, the reaction mixture is transferred to the matrix where the polyanion adheres to a polycation material.
There are various ways that the polycation can be added. The polycation material can be added directly to the capture reagent where it will adhere to the polyanion. Another method is to add the polycation to the reaction mixture one step prior to the reaction mixture being added to the matrix. The preferred method is to precoat the matrix with polycation and then add the reaction mixture.
The conjugate reagent contains the enzyme alkaline phosphatase conjugated to pteroic acid and diluted into 50mM Tris(hydroxymethyl)aminomethane (TRIS), pH 7.4; 0.5% HSA; 0.1M sodium chloride; 1 mM magnesium chloride; 0.1 ram zinc chloride; 0.1% dextran sulfate; and 0.1% sodium azide. The conjugate binds to the unoccupied folate binding protein sites. The conjugate reagent is not limited to the use of pteroic acid. Other folate analogs including PGA can be used in the conjugate reagent.
The standard IMx� (Abbott Laboratories, North Chicago, Ill., 60064) methylumbelliferyl phosphate substrate is used in the present invention.
As mentioned earlier, there have been problems in using 5'- mTHF as a standard or calibrator. It is unstable once exposed to light, temperature and atmosphere. Its instability negates its usefulness as a calibrator. Moreover, 5'-mTHF may necessitate using human serum in the calibration matrix. Also necessary when using 5'-mTHF is the addition of ascorbate and citrate. The addition of these increases the stability of 5'-mTHF during usage but ascorbate was found to interfere in the assay.
The present invention uses PGA as its calibration reagent. The advantages of using PGA are several. First, PGA is more stable than 5'-mTHF; second,there is better reproducibility of results with PGA calibrating the assay over 5'-mTHF; and third, ascorbate is not needed to stabilize PGA. Additionally, PGA as a calibrator allows the use of bovine serum albumin (BSA) instead of human serum as the calibrator diluent. This lessens the hazards, cost and availability problems associated with human serum.
5'-mTHF is the metabolic form of folic acid that is actually measured in patient samples. Accordingly, calibrators other than 5'mTHF must be sufficiently bound by the appropriate binding proteins to give good correlation to sample 5'-mTHF levels. Although the mechanism by which it works is unknown, the multiclonal format allows FBP to bind PGA in a similar fashion to that of 5'-mTHF. Therefore, the multiclonal format allows calibration with PGA and gives a good indication of test sample 5'-mTHF. An example of the comparison is given in FIG. 1.
Another improvement of the present invention is the finding that addition of citrate improved PGA stability of day zero values to several months. PGA day zero value stability was evaluated in the BSA diluent with and without citrate (100 mM). With citrate, PGA day zero value stability is improved over time at -20� C., 4� C., 45� C., and room temperatures. FIG. 2B shows the effects of citrate enhanced PGA day zero value stability over no citrate addition (FIG. 2A). Test points were run the IMx� instrument and MUP turnover rates were measured on the days indicated. The rates obtained were compared to the baseline runs of day zero.
The same methodology can be used to assay for vitamin B12. Vitamin B12 is preferably separated from its endogenous intrinsic 5 factor with the use of alpha-methyl thioglycerol and subsequent high alkaline enviroment. This allows the released vitamin B12 to couple to the capture reagent complex, thereby facilitating the detection process.
Patient samples were analyzed and individual folate concentrations were measured using two commonly used assays. Patient samples were analyzed by the IMx� against Bio-Rad� (Bio-Rad Chemical Div. Richmond, Calif., 94804) and Corning� (Corning Inc., Science Products Division, Corning, N.Y., 14831) assays with both the multiclonal and polyclonal format. As can be seen in Table 3, there was good agreement between the two methods. The designation "N" refers to the number of patient samples tested.
TABLE 3______________________________________ Mutliclonal Polyclonal______________________________________IMx � v. Bio-Rad �Intercept 0.84 1.14Slope 0.67 0.57R value 0.98 0.98N 47 47IMx � v. Corning �Intercept 0.87 1.40Slope 0.61 0.50R value 0.94 0.92N 46 46______________________________________
Also significant is the fact that the multiclonal reagent showed good stability after 3 days at 45� C. Two individual monoclonals, a 1:1 mix of the two monoclonals, and a polyclonal sample were tested at the different temperatures. The multiclonal reagent (1:1 mix) lost only 10% of its 4� C. activity after 3 days at 45� C. As shown in Table 4, the multiclonal format shows good ability to bind more binding protein as indicated by higher substrate turnover rates after storage at higher temperatures.
TABLE 4______________________________________[Folate] Clone A Clone B 1:1 Mix Polyclonal______________________________________Day 3-4 Degree0 ng/ml 906 1274 2245 9824 ng/ml 646 835 1184 567R4/R0 0.71 0.66 0.53 0.58Day 3-45 Degrees0 ng/ml 785 1025 2020 9034 ng/ml 565 686 1061 509R4/R0 0.72 0.67 0.53 0.56% activity 87 80 90 92remaining______________________________________
The present invention's methodology can be used to adapt it to several analytes. The antibodies used were developed to aide in eliminating reagent performance variability. The multiclonal format was initially prepared by mixing two of the CMA-antibody conjugates together in the capture reagent. This was later simplified by mixing the two antibodies together and then coupling the mixture to CMA to generate a multiclonal. The antibody mixture can be two separate monoclonal or a combination of monoclonal and polyclonal antibodies.
DTT reagent is added to samples at the beginning of the assay to maintain a reducing environment for preserving sample 5'-mTHF. DTT can also function as a protein denaturant by reducing disulfide bonds and making other proteins more susceptible to alkaline denaturation. Published articles have indicated that folate binding proteins are irreversibly denatured at a pH of 12 or greater. The present invention preferably uses a potassium hydroxide reagent to denature patient samples by destroying endogenous folate binding proteins. This allows the CMA-multiclonaI-FBP complex to bind with the released patient sample folate. Thus, the assay must reproducibly raise the pH for denaturation and then neutralize the base with the capture diluent. Current literature and our experience suggests that a pH of approximately 9.3 gives optimum binding of PGA in the calibrators and the 5'-mTHF in the samples. In the present invention, not only must the diluent be appropriate for the CMA-multiclonaI-FBP capture reagent to 0 function and remain stable but it must also neutralize the KOH. The capture reagent therefore buffers the reaction pH for appropriate PGA and 5'-mTHF performance. Borate is a preferable buffer with a pKA near 9.3 and is conducive for FBP binding ability. Addition of 4% sucrose increases the solubility of borate. The ability of sucrose to 5 perform this function is due to the cis-hydroxy groups which combine with the borate. Sucrose prevents the occasional precipitation of borate from capture diluent stored at 4� C.
1. Procedure
a. Load the IMx� (Abbott Laboratories, Abbott Park, Ill., 60064) carousel with the calibrators and/or controls and test samples (minimum of 100 ul each). Then 0.4 ml of dithiothreitol (DTT) is placed into the predilute well of the first reaction cell in the carousel.
b. The assay begins with each reaction well receiving 0.015 ml of the DTT from the first reaction cell's predilute well and 0.018 ml of the calibrator, control, or sample. The DTT denatures proteins and preserves the reduced form of 5'-mTHF in the samples. Each well is incubated for 8 minutes.
c. Add 0.028 ml of the 0.75M potassium hydroxide (KOH) into each reaction well and incubate 8 minutes.
d. Add 0.15 ml of capture reagent (polyanion-anti-FBP antibody complexed with FBP in borate buffer) to the reaction well. The borate buffer in the capture reagent neutralizes the denaturants (final pH near 9.3) and allows the folate in the samples to bind to the FBP. The dextran sulfate in the capture reagent binds to stray cations (i.e. cation dust from the matrix) and reduces assay variability. Incubate the wells for 12.5 minutes.
Transfer 0.22 ml of the reaction mixture to the ion capture reaction cell matrix where the polyanion (connected to the FBP through the antibodies) adheres through ionic interactions to the polycation on the reaction cell matrix.
f. Two diluent washes of the matrix remove unbound materials and then are followed by the addition of 0.06 ml of conjugate reagent. The conjugate reagent used is calf intestine alkaline phosphatase conjugated to pteroic acid. The conjugate binds to sites on the captured FBP unoccupied by folate.
g. Unbound conjugate is then washed from the surface of the matrix, 0.06 ml of methylumbelliferyl phosphate (MUP) reagent is added, and the fluorescence of the liberated MU is read. The fluorescence intensity is inversely proportional to the amount of folate in the calibrators or patient samples.
1. Procedure
a. Load the carousel with the calibrators and/or controls and test samples (minimum 100 ul each). Then 0.4 ml of alpha monothioglycerol is placed into the predilute well of the first reaction cell in the carousel.
b. The assay begins with each reaction well receiving 0.01 ml of alpha monothioglycerol and 0.06 ml of the calibrator, control, or sample. The reducing agent denatures proteins. Incubate for 8 minutes.
c. Add 0.08 ml of the KOH into each reaction well and incubate for eight minutes.
d. The addition of 0.15 ml of capture reagent (polyanion-anti-intrinsic factor antibody complexed with intrinsic factor in borate buffer) to the reaction well then neutralizes the denaturants (final pH near 9.3) and allows the vitamin B12 in the samples to bind to the intrinsic factor. The dextran sulfate in the capture reagent binds to stray cations (i.e. cation dust from the matrix) and reduces assay variability. Incubate for 12.5 minutes,
e. 0.15 ml of the reaction mixture is transferred to the ion e. capture reaction cell matrix where the polyanion (connected to the intrinsic factor through the anti-intrinsic factor antibody) adheres through ionic interactions to the polycation coated on the matrix.
f. Two diluent washes of the matrix are followed by the addition of 0.05 ml of conjugate reagent. The conjugate reagent used is calf intestine alkaline phosphatase conjugated to vitamin B12 or vitamin B12 analog. The conjugate binds to sites on the captured intrinsic factor unoccupied by vitamin B12.
g. Unbound conjugate is then washed from the surface of the matrix, 0.06 ml of methylumbelliferyl phosphate (MUP) reagent is added, and the fluorescence of the liberated MU is read. The fluorescence intensity is inversely proportional to the amount of vitamin B12 in the calibrators or patient samples.
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text:
A Dozen Roses for SCALE 6x
ROSE Blog: Rikki's Open Source Exchange
On a scale from 1 to 12, I'd give SCALE 6x a dozen roses because it was one of the best conference experiences I've ever had. The SCALE staff set the tone – they were professional, friendly, helpful, and wonderful hosts. Plus, they get bonus points for the ladies t-shirt in my attendee bag. SCALE also draws a wonderful mix of attendees, vendors, and speakers.
On Friday, February 8th, I learned a bunch from the Women in Open Source conference. Allison Randall's first talk, The Art of Conference Presentations, was a hard act to follow. I took notes and plan to make a check-list for any future public speaking engagements.
In the next talk, From the Keyboard to the Boardroom and Back Again, Emma McGratten offered a list of things she wished she'd known in first grade, including:
- don't let others take credit for your work;
- negotiate, negotiate, negotiate;
- foster relationships, networks, and alliances with female coworkers across your organization and industry;
- and articulate your goals and build a plan that will take you there.
Susan Kuchinskas talked about oxytocin in her Women and Open Source: A Match Made in Hormones presentation.
Stormy Peters spoke to a full room of men and women in her talk called What Does a Community Manager Do? When Joe "Zonker" Brockmeier recently accepted a position as openSUSE's Community Manager, my first thought was, "That's cool!" My second thought was, "What's a Community Manager?" It turns out that several of us learned that we juggle many community manager tasks in our jobs already.
Google's Catherine "Cat" Allman quickly stepped in for Addison Berry, who couldn't make it to the event. (Cat, thanks for the cool pink Google coffee cups! I'm using mine already.)
Drupal's Angela Byron talked about building websites with Drupal. I didn't know that I wanted to build a website with Drupal until she started demonstrating it. Not only is Angela a fabulous public speaker, but Drupal looks like a powerful, accessible CMS.
HP's Adaora Onyia discussed masculine vs. feminine communication styles in her talk called Hello, Can Anyone Hear Me? I wish I'd seen her talk earlier in my career because I picked up a lot of her lessons the hard way.
The final talk, Danese Cooper's Why Whinging it Doesn't Work, covered accenting the positives about women in open source rather than focusing on the problems. Of course, that's part of the goal of this blog, so I give her two thumbs up for the friendly reminder. Danese then moderated our Women in Open Source panel discussion at the end of the day.
I spent Saturday and Sunday at our Linux Pro Magazine booth and had the chance to meet many of our readers, and hopefully some future authors. A big thanks for everyone who checked in to see if I needed anything, including the SCALE staff, Ohio LinuxFest's Beth Lynn Eicher, Zonker, the Shopzilla.com staff, and Jono Bacon. I didn't have much opportunity to visit the show floor, but I was able to swing by the L.A. LinuxChix booth on my way out and pick up one of their cool t-shirts.
There are many other people I need to thank for making my first time at SCALE such a wonderful experience, but I'm recovering from jetlag and can't possibly name everyone in this post, so I'll conclude with a huge THANKS! to everyone at SCALE 6x. I hope to see you again at SCALE 7x, if not before then.comments powered by Disqus
Issue 174/2015
Buy this issue as a PDF
Microsoft Frees Code for .NET Build Engine
MSBuild is now just another GitHub project as Redmond continues its path to the light.
Researchers Demonstrate Hack by Heat
Malware could pass data and commands between disconnected computers without leaving a trace on the network.
Kernel Developers Adopt Code of Conflict
New rules emphasize collegiality in coding.
Ubuntu Switches to systemd
Upstart lands in the dust bin as a new era begins for Linux.
Old Vulnerabilities Are Kept Alive Through Bad Configuration
HP's annual Cyber Risk report offers a bleak look at the state of IT.
Linus Torvalds Announces Linux 4.0
But what do the big numbers really mean?
Microsoft Frees CoreCLR
.NET Core execution engine is the basis for cross-platform .NET implementations.
New Trojan Attacks Linux Servers
The Xnote trojan hides itself on the target system and will launch a variety of attacks on command.
Cisco Releases Annual Security Report
Spammers go low-volume, and 90% of IE browsers are unpatched.
Zero Day Exploits Target Flash
Adobe scrambles to release patches for vulnerable Flash Player.

subset: commoncrawl


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Hi Jeff
Thanks a million for your rapid response. I did find the information on the N3 packaging. I got the basic handling down in a couple of...Read More ->
You know there are magic stores and then there are great magic stores. Well MJM Magic is even better than a great magic store. I use to shop over at...Read More ->


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February 2, 2014
RUMOR: Quentin Tarantino's THE HATEFUL EIGHT Back On With Sam Jackson?
It's starting to sounds like Quentin Tarantino has calmed down about the leak of his HATEFUL EIGHT script. The current rumor circling is that he's pushing forward with script rewrites and is talking with Samuel L. Jackson about taking a lead role. I've been hearing this rumor over the week from different sources, but Jeff over at TheWrap is posting it on his Twitter account, being clear it's only a rumor at this point. I'd love for it to be true, but unless it comes out of the mouth of Quentin, we never really know if it's actually going to happen.
I really hope we end up hearing new names such as Michael K. Williams (in the running for Django) and Kurt Russell (dropped out of Django), being in the mix for the film. No matter what happens, we should expect Christoph Waltz will also have a role.
5 comments:
- AnonymousFeb 5, 2014, 12:19:00 PM
add joe don baker and michael j pollard to the cast.ReplyDelete
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VIEWS: 3 PAGES: 1 CATEGORY: Business POSTED ON: 7/29/2012
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A no-nonsense boot without any of the unnecessary bells or whistles that's willing to go anywhere that you can get to.
- Classically styled mountina boot
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Newest Products
White's Boots News
- 01/01/2015 Contest Winner 4th Quarter 2014
- 12/12/2014 Spokane Sales Floor will be closed on December 30, 31, and January 1st.
- 11/29/2014 Santa Pictures at the White's Boots Showroom!
Spokane Information


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Conference Agenda
Please note that the agenda is subject to change.
Session Five
5-1: Are There Choices in Fertility Management after Breast Cancer? / Elizabeth McGee, MD / What are the options for fertility and family building after one has been treated for breast cancer? How can you know if you are still fertile? Can infertility be treated? Is it safe? Could it be an option for me? This session will answer these and other important questions about fertility management after breast cancer.
5-2: PHYSICIAN TRACK: The Many Benefits of Palliative Care as a Part of a Comprehensive Cancer Team / Jaina Clough, MD / Palliative care is an essential piece of treatment that many providers need to know more about. This session will discuss palliative treatments from a provider's standpoint and give a comprehensive discussion on how every provider can benefit from knowing more about this field. This session is specifically geared toward primary care providers and other health care professionals. Please note that in order to fill the palliative care portion of the new CME requirements, this session will run until 3:15 pm.
5-3: Everyday Chemical Exposures and Breast Cancer: Why are We Concerned? What can We Do? / Janet Gray, PhD / Many common products we use in our everyday life contain chemicals, including endocrine disrupting compounds, which have been linked to increased risk for developing breast cancer (and many other diseases). We will look at some of the scientific evidence that raises concerns about these chemicals, and then talk about ways that we as individuals can decrease exposures. And we will talk very briefly about pending federal policy and market-based initiatives that, if implemented, would make our homes and environment safer.
5-4: Management of the Axilla in 2014: Can the Past Guide the Future? / Kari Rosenkranz, MD / The role of axillary surgery in the management of breast cancer has been a constantly evolving thing over the years. This session will explore the history of this procedure and discuss what the past can tell us about the future of treatment.
5-5: Clinical Trials and You / Chris Holmes, MD, PhD / Clinical trials are among the greatest weapons we have in fighting cancer. This panel discussion will explore the world of clinical trials, how they help find new treatments for cancer, and how you can become a part of this important process.
5-6: Aromatase Inhibitors: Managing Pain, Hot Flashes, and Bone and Sexual Health / Joanna Schwartz, PharmD / This session will focus on emerging data and treatments for aromatase inhibitor-induced muscle and joint pains, vaginal dryness and pain with intercourse, and bone health. Dr Schwartz, who is a pharmacist, will also provide information on access to the treatments in the studies, such as if they are over-the-counter, prescription only, or compounding pharmacy only.
5-7: What to do When you Find a Breast Lump / Ted James, MD / This session is geared for anyone who wants to understand the nature of breast lumps and what to do when you find one. The talk will review the various causes of breast lumps and walk participants through the diagnostic process for evaluating a new breast lump. Recommendations to ensure timely, safe and high-quality care will be discussed.
5-8: What's New in Breast Cancer Reconstruction? / Robert Nesbit, MD / In this session, we will focus on newer techniques for reconstruction after breast cancer surgery. Some of the techniques discussed may include oncoplastic surgery after lumpectomy, nipple sparing mastectomy, and new technologies that may improve reconstructive outcomes.
5-9: Komen for the Cure Grant Workshop / Terry Farkas / We will discuss the various breast cancer programs currently being funded by the Vermont/New Hampshire affiliate of Susan G. Komen for the Cure and the unique nature of Komen Community Grants. Additionally, we will offer a presentation on how your breast health program can become involved in our granting process.
5-10: Grow Your Wellness and Joy After Cancer : Survivorship NOW VT / Linda Dyer and Susie McNamara / Come learn about Dragonheart's Survivorship NOW free weekly cancer wellness programs. Up your fitness and strength. Tap into your creative side. Stir up some delicious healthy meals. Broaden your mind and your connections. Find out how you can be a part of these empowering programs as a participant or presenter. It's all about empowering all cancer survivors in our community to live life well.
5-11 Coffee Chats / Bring your coffee and enjoy a casual session in the Emerald III ballroom. Coffee chats available in this time slot will include:
- You are What you Breathe / Martin Wolf
- Light, Darkness, Color, and Sound in Healing / Martha Loving
- Performing a Self Breast Exam / UVM Nursing Students
- Fight Cancer Deliciously! / Chef Curtiss Hemm
- Coping with the Physical and Emotional Pain of Cancer Treatment / Suzanne Hall, RN, BSW
- Laughter-cize and You / Traci Pena
5-12 Culinary Demonstration / Join local chefs for a culinary demonstration outside G's Restaurant. This demo will give you a chance to learn some nutritional recipes and techniques that you can apply to create fast, healthy, and delicious meals anywhere and anytime.
Back to Conference Agenda
Last modified September 11 2014 01:05 PM


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Traffic infraction in Snohomish County South Division, managed to get my fine more than doubled. Rarely heard from him, had to call the court the day...
Posted by: Charlie, 10 months ago.
12 endorsements
A real excellent lawyer. If you need a ticket dismissed or help with other litigation I would not hesitate one moment to hire him.
I endorse Yan. He is a highly skilled attorney that is well respected in the legal community.
14 years since Yan Siprin was first licensed to practice law.
We have not found any instances of professional misconduct for this lawyer.


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King of Prussia Mall
Robbers Sought In Armed Smash-And-Grab At King Of Prussia Mall
A $40,000 reward is being offered in connection to an armed smash-and-grab robbery at a high-end watch store in King of Prussia.
Stigall Show Log 1.5.12
Chris discusses Rick Santorum's Presidential campaign and Michele Bachmann's decision to drop out, a grenade found at the King of Prussia Mall, a dispute over a tribute to Michael Landon in Collingswood, and talks to Daily News Editor Larry Platt about the city's DROP program.
Grenade Replica Prompts Scare At King Of Prussia Mall
A portion of the King of Prussia Mall was evacuated after the discovery of a suspicious device that was later determined to be a replica of a grenade.
Hi Ho, Hi Ho, It's Back To Work (In Retail) Men Go
According to the Bureau of Labor Statistics, more men are back at work, but in different positions. Instead of construction, you'll find more of the guys in retail or other positions traditionally held by women.
Early Birds Snag Black Friday Deals
Who doesn't like a good sale? They're everywhere here at the Cherry Hill Mall – 50% off or more at many stores.
King of Prussia Mall Opens Electric Car Charging Stations — And They're Free
"Charge it" now has another meaning at the Montgomery County shopping mecca.
Fundraiser Set For 'New Leash On Life,' Program That Pairs Inmates With Dogs
A fashion show fundraiser tomorrow night at the King of Prussia Mall launches a new program that pairs rescue dogs and prison inmates in an effort to make them both ready for society.
Mother's Day Gifts For Mom
Want to give your mom a personal gift this Mother's Day? Assistant Marketing Manager at the King of Prussia Mall, Ashley Pompei, stopped by the morning show on Wednesday to share some Mother's Day gift […]
Celebrity Chef Lidia Bastianich Signs Books At King Of Prussia Mall
She's a restaurant owner, a TV presenter, and an author. But all this chef really wants to do is to bring a taste of Italy to your dinner table.
Sink Hole Opens Up On Route 202
A 75-foot wide sink hole suddenly opened up along route 202 north near mall boulevard in Upper Merion Township's "King-of Prussia" section Friday morning not far from the King of Prussia Mall.
Six Shopping Days Left To Christmas
As expected, holiday shoppers are flooding retail stores this weekend.
King Of Prussia Mall: There's An App For That
There's a free iPhone app that could make its 400 stores easier to find.
Putting The 'Jean Machine' To The Test
Jeans are the basis of many women's wardrobes, but searching for the perfect pair can be frustrating for a lot of women. Janine Watkins is one of them.
Stores At KoP Mall Still Closed Because Of Flood Damage
KING OF PRUSSIA, Pa. (CBS) – It's been more than a month, but the King of Prussia Mall continues to deal with the effects of water damage from a major rainstorm, as the height of holiday […]
Top Shopping Malls
The Philadelphia area lays claim to one of the biggest shopping centers in the country, with little time-wasting filler. Go on – get your shop on, and leave the games to the kiddies out in Minneapolis.


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by Patrick Rall
There have been seemingly endless rumors about the future of the SRT Dart and whether or not it will see production or – if it does arrive in dealerships – what will be found under the hood. It has long been expected that the SRT Dart would follow in the footsteps of the uber popular …
Photo of the Week: merc225hp's 2nd gen Ram 150011October
For this week's Photo of the Week, I have selected the shot above of merc225hp's 1996 Dodge Ram 1500 SLT Laramie. This truck features a big suspension lift to make room for the beefy offroad tires and custom rims while the matching black wheel opening flares extend the body for better fitment of this big …
Black Fridays: 2g Cummins Ram Rolls Coal on the Dyno11October
There is just something special about watching a truck roll coal in a confined space and for this week's Black Friday feature – we check out a 2nd gen Dodge Ram 3500 filling a room in the dyno shop with thick, black diesel soot. We can see exhaust removal piping hanging from the ceiling but …
by Patrick Rall
If you've ever had a vehicle with a non-functioning fuel gauge it can prove to be a tremendous headache – especially if your Dodge Dakota happens to run out of gas because you forgot to keep track of how many miles you had driven since your last fill-up. Fortunately, there are a few common problems …
by Patrick Rall
One of my favorite kinds of threads to feature are model specific member's rides pictorial threads because it gives us a look at all of the ways the different members can make their car or truck more distinct and this week we look at the 1st generation Dodge Dakota pictorial show off thread. With the …


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Cullera de Boix Boqueria
Hospital, 5
The content on this page is provided by a Time Out partner.
Posted: Thu Jan 8 2015
Cullera de boix restaurant is located at Hospital street, in Raval neighborhood, a very magical and vital area, where you can sense history at every step. This restaurant offers you Mediterranean cuisine and it's specialized in delicious rices, one of the most appreciated dishes of Catalan cuisine. At Cullera de Boix you'll be able to enjoy a very wide selection of dishes, elaborated with fresh, local and high quality products. Besides, you'll have the option of choosing dishes 100% vegetarian, for those of you who don't want to eat meat. Broken eggs with foie and ham; Tomatoes dressed with olive oil, young garlic and tuna; Veal burger with 3 cheeses, bacon and barbeque sauce with species bread or Rice with chicken and vegetables are just a little taste of the exquisite dishes you'll find here. This restaurant is the ideal place for those of you who want to enjoy Catalan cuisine at its best, as well as for those of you who want to recharge batteries while doing some tourism since Cullera de Boix is located in the very centre of the city, close to a lot of interesting places to visit such as Ramblas, Gothic neighborhood, Catalunya Square and the well-known Liceu theatre. You can also enjoy in its partenaire Cullera de Boix located at Ronda Sant Pere.
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Cullera de Boix Boqueria Hospital, 5
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Cullera de Boix Boqueria
- Hospital, 5
- www.restalo.es/en/cullera-de-boix-boqueria-restaurant
- 41.381165,2.172891
- Hospital, 5
- Cullera de Boix Boqueria


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October 18, 2012 Sterne Agee came out with positive comments Thursday on Crocs (CROX), the Niwot, Colo., maker of shoes with holes in them, that half a decade ago had a cult following before catering. The firm believes you should get long the stock, and it's happy affirming a previously issued buy rating and a $30 target. That's well above the $23.86 consensus price shown on factsheet, and it's 85% above where the stock closed in the prior session.
Sentiment: Strong Buy
Then they trash it 3 weeks later. They are crooks in plain English.


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Artist-DJ-Producer, Minimal, Deep House, Techno, Experimental, Berlin.
Steve Freedom was born in Cape Town, South Africa and trained in classical and jazz music with violin, guitars, keys, and percussion from the age of seven. Today he lives in Berlin, Germany, where he produces Acoustic/Electronic music in multiple Genres. Steve is also a Performing Electronic Music Improviser and Singer and plays two (2) Native Instruments Maschine's, synced via Ableton Live to spontaneously create House Music, Minimal-techno, Detroit-techno, Deep-House, Nu-disco, Progressive experimental sets and funky soulful dance music.
At six years old (in 1972) Steve's mom surprised him by taking him to see the Cape Town Symphony Orchestra! He was so excited when he met Pierre Amoyal, the famous violinist, backstage, that he demanded from his mom to learn the violin. That night was the beginning of his life-long love of music and within weeks he received a good quality violin from an aunt in the UK and began his sixteen (16) year-long training in classical and jazz music. At thirteen (13) years old he won a local Lego competition and received his first ever vinyl record, by Michael Jackson, called 'Off The Wall'. Throughout his teens Steve was influenced by Kwela and Reggae, as well as Blue-Note Jazz, The Classicists, Soul, Funk, Disco and Rock and he played in cover bands with tunes like "Who Can It Be Now", by the Police, "Night Fever", the Bee Gees, and "Ladies Night" by Cool and the Gang. He also played in school and local classical orchestras as well as jazz bands.
Throughout school life Steve studied violin, piano and electronic keys, rhythm and lead guitars, bass guitar and drums, djembe and bongos, continuing on with Jazz Harmony and Arrangement and African Rhythm under tutelage by Michael Campbell at the University of Cape Town, while simultaneously studying with world renowned Vibraphone virtuoso Merton Barrow, at the Jazz Workshop, at Die Waterkant.
Steve has collaborated with Australian Artists like Faith Anderson, Ghislaine Saucedo, Trent Chapman, Oshry Masty and DJ Richie. Then from July 2010 he began performing live in Berlin and Paris alongside Dj's like Jake The Rapper (Bar 25) and Hippie Stress (Bachstelzen) and performed at venues like Bar 25, Renata and ZMF. He also collaborates with top Berlin Artist's like Rafael Gimelstein, Ricardo Ferreira, Andrey Wakko, Mama-Simone, Yannick Robins, Sander Markey and Marcela Donato.
Lately Steve performed in Goa, Mamalapuram and New Delhi, India, where he played Djembe as a guest with Tiago Oudman (The Bastards, Portugal), Andre Gardeja (Freizeitglauben, Berlin) and Hippie Stress (Bachstelzen, Berlin), at TLR 'The Living Room". Steve runs workshops on NI Maschine and Ableton Live and does session-work in Berlin today.
Steve Freedom is one of the founding members of "Playground Int.", that was established in 2010, an International Artist Collective located in Berlin, Germany. It is dedicated to Collaboration, Creation and Presentation of all forms of art, using a variety of media. The Group creates extravagant experiences in mutant spaces and addresses themes like installation, recycling, DJ-Culture and Synthesthesia, as well as providing visibility for emerging International Artists.


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Most storage vendors are happy to just add flash to their existing product lines, often using it as cache, or as a storage tier handled transparently within the array. Few vendors take the opportunity to rethink the way storage works, though, from the basics of performance to how it meshes with the idea of public & private clouds. Coho Data, coming out of stealth mode with their first product, the DataStream, does just that.
Storage Networking
Caching is taking over the world of virtualization in an attempt to use flash, SSD, and RAM to decouple storage performance from storage capacity.
Cisco announced today their intent to acquire Whippany, NJ based WHIPTAIL, a manufacturer of Solid-State Disk (SSD) storage.
In order to sell the Software Defined Data Center, VMware needs to prove that it delivers a hard dollar ROI to the customers of the Software Defined Data Center. Commoditizing the expensive networking hardware business and commoditizing the expensive storage hardware business are two excellent ways for VMware to deliver that hard dollar ROI, and for VMware to be able to justify its price premium over competitive offerings.
Keeping in mind that the best server and storage IO is the one that you do not have to do, then second best is that which has the least impact combined with best benefit to an application. This is where SSD, including DRAM- and NAND-flash-based solutions, comes into the conversation for storage performance optimization.
VMworld 2013 is upon us and one of our tasks is to figure out which vendor's booths to go see. With over 230 booths to choose from this is a daunting task. If you are interested in finding creative new solutions to your management, monitoring, deployment, security, data protection, and desktop management problems, this list will help you.
The benefits of virtualizing networking and storage will be very different than were the benefits of virtualizing CPU and memory. VMware's success to date has not come at the expense of server vendors. However VMware's success with virtualized networking will come at the expense of Cisco. The success with virtualizing storage will come at the expense of enterprise storage vendors. By commoditizing networking and storage VMware will deliver substantial benefits to its customers, and create strained relationships with vendors who used to be partners.
Performance Management ...
The New Management Platforms for the SDDC Management Stack and the Cloud
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Legacy management frameworks are going to get replaced by SDDC Management Platforms that combine big data back ends, analytics and ecosystem friendly data collection and integration strategies to give customers the best of both worlds. Customers will be able to choose from among best of breed solutions, and then integrate them at the data level via a big data back end data store. This will revolutionize the management software industry, give rise to a new set of leaders in this industry, and completely destroy the legacy management frameworks.
The Cloud Management layer of the Software Defined Data Center is where the flexibility of the SDDC is translated into tangible benefits for the business constituents of the virtualized data center, the private clouds, the hybrid clouds and the public clouds. Without a robust Cloud Management layer, the IT Operations flexibility of the SDDC cannot translate into the relevant level of Business Agility for the business.


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Low Heel Zip Shoes
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Add a dash of glamour to an everyday look with these charming black booties. In soft faux-suede, this sassy pair boasts glittery trimmings, dainty bow accents, and ultra-cushioned footbeds - because youll shine if you want to!
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The Old Soundman: Guitar Cab Mics & Urban Myths"X" marks the magic spot... Or does it?
Wedge Management: Combating "More Of Me"…
In The Studio: Recording Acoustic Guitars
RE/P Files: Construction Of A Live Echo Chamber
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Now let's switch to the application side. When adding effects to instruments and vocals, keep in mind that in most situations, it all should be done in a complimentary manner. (Except in very rare cases.)
For example, if mixing a slow ballad with a long reverb applied on vocals, then chances are that a short-gated reverb on the drums will sound awkward.
I've also heard different delay times and reverb settings on lead and background vocals. Again, for the most part this is not a good idea. If all vocals are intended to be part of a unified pallet, which is almost always the case, then they should be presented as unified.
In other words, adding a random setting to each vocal - without any specific artistic purpose - will result in vocals that are a jumbled-up mess.
I once found this out the hard way. Experimenting with a new, original song, I set up different delay times for the background vocals.
Everything sounded great - until all four vocalists started singing a certain passage together. Then it became three badly matched background voices topped by a lead voice that also didn't remotely fit.
It suddenly sounded like a different song had started! (By the way, if you ever find yourself in this position, slap the side of your rack and act like the gear is causing the problem, not you.)
A very common question with effects: how much is enough? I think we've all come across mixes that were compromised or even ruined by the overuse of effects. (On the other hand, I've also heard some so-so bands that were saved by a good mix and heavy doses of effects!)
Particularly in a smaller environment like a club, certain elements of a mix tend to stand out: guitar lead, keyboard lead, thundering kick drum, and so on, while most of the time, the effects are not intended to stand out.


text: Word of the Day – Ruck By Bradley James June 29, 2012 5:28 AM Share on Facebook Share on Twitter Tim Boyle, Getty Images Today's Word of the Day: ruck Dictionary.com defines ruck as: A large number or quantity; mass. We're gonna be playing a ruck of Good-Time Oldies this morning – hope you're ready! Category: Bob & Marianne in the Morning, Word of the Day Share on Facebook Share on Twitter More Leave a comment Send to a friend! Print this page Pin it! Reddit This! Share on Tumblr! Best of WOBM AM 1160 & 1310 Recommended For You Around the Web


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Microsoft developer builds robot to keep his dog company during the day
Jeff Horwich
Feb 10, 2012 Rover, meet R2
Consumer sentiment slips
Joel Patterson
Feb 10, 2012 Macroeconomic signs point to a brighter future, but average Americans are still struggling with the basics.
Could that new toy robot be hacked?
John Moe
Dec 26, 2011 How about the pacemaker?
New Facebook feature lets you exist at all moments in your life simultaneously
John Moe
Dec 16, 2011 At first glance, Facebook's Timeline feature just seems to be another in a long series of redesign elements. But upon examination, it reveals itself to be a fundamental shift in how you present yourself to the world.
ROBOT ROUNDUP: Videos of the bots in action
John Moe
Dec 15, 2011 Now I don't have to describe them
Robot prison guard
Marc Sanchez
Nov 28, 2011 The headline pretty much says it all.
VIDEO: Robots doing crazy things in our Robot Roundup
John Moe
Nov 23, 2011 Inflatable ant roaches? Swimming bots? Really fast micebots? We got 'em!
Can't stop snoring?
Marc Sanchez
Nov 17, 2011 There's a bear for that.
Robot resembles your friend Todd from high school who was way into Judas Priest and drove an old Camaro
John Moe
Nov 9, 2011 The latest version of Honda's Asimo robot is lighter, smaller, and more versatile than previous editions. By way of explaining the headline above,...
Now you can live through a Star Wars Episode 1 robot through TELEXISTENCE
John Moe
Nov 8, 2011 As discussed on our Wednesday show, now you can see and hear and feel everything that the robot you control does. Wha? Yeah! Huh? Whoa!