INHIBITORY ANTIPEPTIDE 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 rabb its immunized with this peptide coupled to keyhole limpet hemocyanin, as judged by ELISA, Antipeptide antibody recognized a single protein b and in microsomes prepared from cells expressing recombinant human CYP 3A4 in immunoblotting analysis, No immunodetectable proteins were foun d 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 whi ch comigrated with human CYP3A4 was recognized by this antibody and th e relative blotting intensity of this protein band correlated signific antly with human CYP3A4-catalyzed testosterone 6 beta-hydroxylase acti vities (r = 0.96), More importantly, this antibody exhibited greater t han 90-95% inhibition of testosterone 6 beta-hydroxylation, while othe r cytochrome P450-mediated reactions in human liver microsomes were no t inhibited, Because of its specificity and inhibitory potency, this a nti-peptide antibody should be a valuable tool in evaluating the role of CYP3A in mediating in vitro metabolism of therapeutic agents.