Inhibitory anti-peptide antibody against human CYP3A4

An inhibitory anti-peptide antibody was raised against a 21-amino acid peptide (VKRMKESRLEDTQKHRVDFLQ) corresponding to residues 253-273 of human cytochrome P450 3A4. High titer antibodies were produced by rabbits immunized with this peptide coupled to keyhole limpet hemocyanin, as judged by ELISA. Anti-peptide antibody recognized a single protein band in microsomes prepared from cells expressing recombinant human CYP3A4 in immunoblotting analysis. No immunodetectable proteins were found in microsomes containing other cytochrome P450 isoforms. In addition, the antibody did not recognize CYP3A5, a closely related isoform in the CYP3A family. In human liver microsomes, only one protein band which comigrated with human CYP3A4 was recognized by this antibody and the relative blotting intensity of this protein band correlated significantly with human CYP3A4-catalyzed testosterone 6 beta-hydroxylase activities (r = 0.96). More importantly, this antibody exhibited greater than 90-95% inhibition of testosterone 6 beta-hydroxylation, while other cytochrome P450-mediated reactions in human liver microsomes were not inhibited. Because of its specificity and inhibitory potency, this anti-peptide antibody should be a valuable tool in evaluating the role of CYP3A in mediating in vitro metabolism of therapeutic agents.