Monoclonal antibody-directed determination of cytochrome P-450 types expressed in a human lymphoblastoid cell line.
Fred K. FriedmanRobert RobinsonByeong Jun SongS S ParkCharles L. CrespiMichael A. MarlettaHarry V. Gelboin
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Abstract:
Cytochrome P-450-dependent aryl hydrocarbon hydroxylase (AHH) and 7-ethoxycoumarin O-deethylase activities of a cloned line of human lymphoblastoid AHH-1 cells are inhibited by a monoclonal antibody (MAb 1-7-1) prepared to a 3-methylcholanthrene-induced rat liver cytochrome P-450. The monoclonal antibody inhibition determined that a single MAb 1-7-1-sensitive type of cytochrome P-450 is responsible for all of AHH expression in both the basal and benz[a]anthracene-induced cells. Partial inhibition by the MAb 1-7-1, however, indicates that at least two forms of cytochrome P-450 catalyze 7-ethoxycoumarin O-deethylase in both the basal and the induced cells, one form of which is identical to the MAb-sensitive cytochrome P-450 responsible for all of the AHH. Thus, a single cloned cell line is capable of expressing two classes of cytochromes P-450, and the observed multiplicity of cytochrome P-450 in animal tissues does not necessarily depend on cell heterogeneity. A sensitive MAb 1-7-1-based radioimmunoassay also directly demonstrates the presence in these cells of a MAb 1-7-1-specific type of cytochrome P-450 as well as its elevation in the induced cells. These MAb-based methods thus can determine the contribution of specific MAb-defined types of cytochromes P-450 to the cellular metabolism of specific xenobiotics.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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Hydroxylation
Cytochrome b5
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Monoclonal antibodies (MAb) are antibodies that are identical because they are produced by one type of immune cell; all are clones of a single parent cell. Given (almost) any substance, it is possible to create monoclonal antibodies that specifically bind to that substance; they can then serve to detect or purify that substance. Monoclonal antibodies (MAbs) are an integral part of targeted therapy approach for various diseases which result in decrease in adverse effects and increase in efficacy. They target various receptors or various growth factors on the cell surface and modulate their vital functions and cause cell death by various mechanisms. They are produced by conventional hybridoma technique. They have been modified to humanized forms to decrease adverse effects. Since then many MAbs have been produced which have changed the clinical course of many diseases. In this article, briefly the technique of production of these monoclonal antibodies, their mechanism of action in therapy and clinically important monoclonal antibodies will be discussed.
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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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An Indian isolate of P. falciparum was propagated in vitro and monoclonal antibodies were raised against this parasite. Of 11 positive hybrids obtained, 3 were cloned and characterized. When tested in IFA with synchronized cultures dominated by mature schizonts, all three monoclonal antibodies showed distinct fluorescent patterns. These three monoclonals reacted with seven of the eight P. falciparum isolates tested. One of these monoclonal antibodies, which showed a fluorescence pattern similar to the merozoite dot fluorescence reported by previous investigators, also inhibited parasite growth in vitro. The antigens that reacted with the monoclonal antibodies have been isolated by using MAb-affinity columns.
Direct fluorescent antibody
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