Monoclonal antibodies to rat liver microsomal cytochrome b5

Abstract

Hybridomas obtained by the fusion of spleen cells from rat cytochrome b 5-immunized mice with mouse myeloma cells produced five groups of monoclonal antibodies (MAbs) with three mouse immunoglobulin subtypes: IgG1, IgG2b and IgM. All of the MAbs bound strongly to rat cytochrome b 5 as measured by radioimmunoassay (RIA). Four clones of MAbs were also strongly immunoreactive with cytochrome b 5 when tested by Western blotting, but only one of the MAbs (1-39-2) weakly immunoprecipitated cytochrome b 5 in an Ouchterlony double-immunodiffusion test. Two of the MAbs partially inhibited cytochrome b 5-mediated NADH cytochrome c reduction catalyzed by liver microsomes (24–36%). Expression of immunodetectable cytochrome b 5 was highest in the liver, next highest in the kidney, and quite low in the other tissues examined with MAb 1-17-1 by Western blotting. This MAb recognized homologous cytochrome b 5 of human liver microsomes and in homogenates of TK − cells infected with recombinant vaccinia virus encoding human cytochrome b 5. These MAbs to cytochrome b 5 will be useful for the identification, quantification, and purification of cytochrome b 5 from animal and human tissues, and for understanding its role in cytochrome P450 catalyzed drug metabolism and carcinogen activation with respect to tissue, organ and individual differences.