TARGETED ANTIPEPTIDE ANTIBODIES TO CYTOCHROME-P450 2C18 BASED ON EPITOPE MAPPING OF AN INHIBITORY MONOCLONAL-ANTIBODY TO P450 2C5

The epitope recognized by the inhibitory monoclonal antibody designate d 2F5, which was raised against P450 2C5, was mapped to amino acids 23 7-260 by immunoblotting using a combination of recombinant antigens an d chimeric and partial fusion proteins constructed from rabbit P450s 2 C2, 2C4, 2C5, and 2C16, which are recognized by 2F5, and from 2C1 and 2C3, which are not. When the sequence of the epitope for 2F5 (amino ac ids 237-260) was compared with those of other rabbit 2C P450s, a singl e lysine residue at position 253 appeared to be a likely determinant o f 2F5 immunoreactivity. Substitution of lysine for glutamic acid 253 i n P450 2C3 (2C3E253K) conferred immunoreactivity and the ability of 2F 5 to inhibit progesterone metabolism catalyzed by P450 2C3E253K. Seque nce alignment revealed that this epitope lies in close proximity to th e epitope identified for LKM-1 autoantibodies to P450 2D6. Based on th ese results, an antipeptide antibody was raised to the corresponding r egion (amino acids 252-263) of human P450 2C18. The resulting antipept ide antiserum recognizes P450 2C18 but not P450 2C8, 2C9, or 2C19. How ever, the antipeptide 2C18 antiserum did not inhibit SC18-catalyzed di azepam N-demethylation. Human 2C P450s were also quantitated by immuno blot analysis in a panel of six human liver microsomes using Escherich ia coli expressed P450s as standards. Analysis of immunoblots indicate d that, if present, P450 2C18 was expressed at very low levels (<2.5 p mol/mg), whereas P450s 2C8, 2C9, and 2C19 were easily detected. (C) 19 97 Academic Press.