Characterization of nine monoclonal antibodies against rat hepatic cytochrome P-450c. Delineation of at least five spatially distinct epitopes.
Spleen cells from a BALB/cByJ mouse previously immunized with purified rat liver microsomal cytochrome P-450c were fused with myeloma cells (P3X63Ag8.653) and 10 hybridoma clones secreting antibody against cytochrome P-450c were selected for characterization. The monoclonal antibodies (C1-C10) were purified from mouse ascites fluid and nine were determined to be distinct immunoglobulins. C6 was an IgG2b, whereas the rest were of the IgG1 subclass. A competitive enzyme-linked immunoassay was used to show that the antibodies were directed against at least five spatially distinct epitopes on cytochrome P-450c. Additional evidence for the recognition of distinct epitopes was provided by Ouchterlony immunoprecipitation of cytochrome P-450c with mixtures of appropriate monoclonal antibodies. Differences in antibody reactivity provided evidence for a sixth overlapping epitope that was recognized by two antibodies (C4 and C6). Three monoclonal antibodies to the same epitope on cytochrome P-450c, (CD2, CD3, and CD5) cross-reacted strongly with cytochrome P-450d, another isozyme induced by 3-methylcholanthrene treatment of rats. The antibodies that did not cross-react with cytochrome P-450d contained kappa light chains, whereas the three cross-reacting antibodies contained lambda light chains. None of the monoclonal antibodies cross-reacted with purified cytochromes P-450a, P-450b, P-450e, P-450f, P-450g, or P-450h or any other cytochrome P-450 in "Western blots" of liver microsomes from untreated or 3-methylcholanthrene-treated rats. C8 was a potent inhibitor of metabolism catalyzed by cytochrome P-450c in a reconstituted system as well as microsomes from 3-methylcholanthrene-treated rats. This antibody effected maximal inhibition of catalytic activity at an approximately 0.5:1 molar ratio of IgG to cytochrome P-450c, i.e. one antibody-binding site per epitope on cytochrome P-450c.
D000818:Animals; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis* / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D008214:Lymphocytes / Q000276:immunology; D051379:Mice; D008815:Mice, Inbred Strains; D008862:Microsomes, Liver / Q000378:metabolism*; D008970:Molecular Weight; D010954:Plasmacytoma / Q000276:immunology; D051381:Rats
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D003577:Cytochrome P-450 Enzyme System