Preparation and Characterization of Monoclonal Antibodies Recognizing Unique Epitopes on Sexually Differentiated Rat Liver Cytochrome P-450 Isozymes

Cytochrome P-450 isozymes P-450_16a, and are immunochemically related, as indicated by mutual cross-reactivity with polyclonal antibody preparations. We have isolated five monoclonal antibodies to and one antibody to that show selectivity for the respective antigens. High frequencies of cross-reactivity were observed, indicating a high degree of homology among and All of the antibodies bound to the same epitope, or closely grouped epitopes, supporting this conclusion. The specificity of each monoclonal antibody was characterized by enzyme-linked immunosorbent assay, Western immunoblotting, and antibody-Sepharose immunoad-sorption of solubilized rat liver microsomes. Antibodies F22 and F23, which were apparently identical, were specific for by these criteria. However, the apparent specificities of antibodies F3 and F20 for and of M16 for P-450_16a, were highly dependent on the analytical technique used. The five antibodies all inhibited the catalytic activity of microsomal by a maximum of 70%. However, they also produced a similar inhibition of microsomal activity, indicating that even F22 and F23 have a low-affinity interaction with an epitope on The antibody M16 was not inhibitory. The results indicate that the apparent specificity of a monoclonal antibody for an antigen determined by, e.g., Western blotting does not allow the conclusive identification of a protein in another system, e.g., immunoprecipitation of in vitro translation reaction products. The specificities of such antibodies should be characterized under the precise conditions in which they are to be used.