Identification of the epitope of an anti-peptide antibody which binds to CYP1A2 in many species including man

An anti-peptide antibody was raised against the sequence Thr-Gly-Ala-Leu-Phe-Lys-His-Ser-Glu-Asn-Tyr-Lys which occurs at positions 283–294 in the rat cytochrome P450 enzyme CYP1A2. Compared with its binding to the peptide used for immunization, the antibody bound with only slightly reduced affinity to the truncated peptides Thr-Gly-Ala-Leu-Phe-Lys-His-Ser and Leu-Phe-Lys-His-Ser. However, binding to the peptide Ser-Glu-Asn-Tyr-Lys-Asp-Asn, which overlaps with the C-terminal region of the immunizing peptide, was very low. Thus, a major epitope for the anti-peptide antibody is Leu-Phe-Lys-His-Ser, which corresponds to a region of CYP1A2 that is conserved in many species. The antibody was tested by immunoblotting for its ability to bind to hepatic microsomal fractions from a number of species. Where possible animals were treated with compounds which induce CYP1A2 and the results compared with those with untreated animals. It was found that the antibody bound to rat, mouse, rabbit, hamster, guinea pig, pig, marmoset monkey and human CYP1A2. No evidence was found for binding to dog CYP1A2. The region corresponding to the major epitope at residues 286–290 of rat CYP1A2 was identical in mouse, hamster, rabbit and human CYP1A2. The sequence of marmoset and guinea pig CYP1A2 are not known but are predicted to be very similar to the sequence in the rat. The lack of binding of the antibody to dog CYP1A2 may be explained by two differences in this region compared with rat CYP1A2. Maximum inhibition of CYP1A2 activity by this antibody, as measured by high-affinity phenacetin O-deethylase activity, was 20%. This is in contrast to a previously described anti-peptide antibody directed to an adjacent region which caused 65% inhibition of this activity. Thus, the edge of an inhibitory region on the surface of cytochrome P450 has been identified. The ability of the antibody to bind to CYP1A2 from a number of animals should make this antibody of use for studying the levels of CYP1A2 apoprotein in many species.