A monoclonal antibody recognizing the activation domain of protein C in its calcium‐free conformation
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Abstract:
A monoclonal antibody (mAb) binding to protein C (PC) heavy chain but not to activated PC was found to inhibit PC activation by free thrombin, suggesting that epitope involved the activation site. Using a set of overlapping synthetic peptides, we confirmed that this mAb recognizes the sequence encompassing the thrombin cleavage site (165QVDPRLI(171)). Surprisingly, epitope was only accessible in the absence of calcium, half-maximal inhibition of mAb binding occurring at 100 microM Ca2+. Thus, our antibody provides direct evidence that conformation and/or accessibility of the activation site differ between the apo and Ca2+-stabilized conformers of PC.Oxalic Acid
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In this report,five mouse hybridoma cell lines secreting monoclonal antibodies to recombi-nant human interferon—α2a(rHu IFN—α2a)were raised.All the monoclonal antibodies with IgGl subclass react with rHu IFN—α2a detected by indirect ELISA with the titers ranging from 1:320,000 to 1:10,240,000 but have no cross reactivity with rHu IFNαl,γ and proteins from recombinant E.coli.Two of monoclonal antibodies,designated 3F1 and 5G10,have the ability to neutralize antiviral activity of rHu IFN—α2a.By a competitive ELISA the five monoclonal anti-bodies can be divided into three categories based on their epitope specificities.A sandwich ELISA established by employing 3F1 and 5G10 monoclonal antibodies could detect rHuIFN-α2a or rHuIFN-α2b as few as 60pg/ml·Moreover,3F1 monoclonal antibody displayed a satisfactory
property in affinity chromatography for rHuIFNα2a,which is able to match a monoclonal anti-
body NK2 from CellTech Corporation.
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Epitope mapping
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Vitamin D-binding protein
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Human anti-murine antibodies (HAMA) can be found in serum of many patients who have received murine monoclonal antibodies for diagnosis or therapy. These antibodies are known to give false positive results in sandwich-type assays (e.g. ELISA or RIA). This interference problem will increase in the future as more patients are treated with murine monoclonal antibodies in vivo. HAMA can also be found in sera from patients that has not been treated with monoclonal antibodies. In this work we have studied the interference of HAMA in sandwich ELISAs containing antibodies from different species. HAMA, present in the sample, may react both with the capture antibody and the detection antibody in these assays to give a false positive reaction. HAMA did not react with chicken IgG, and if one of (or both) the capture and detection antibody was of avian origin, the interference of HAMA in sandwich assays could be avoided.
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Idiotopes
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