Antipeptide antibodies against overlapping sequences differentially inhibit human CYP2D6.

Two peptides that correspond to sequences within the major 33-amino acid sequence recognized by human liver-kidney microsomal-1 autoantibodies were used to elicit antibodies in rabbits (four per peptide) against CYP2D6. Peptide 1(DPAQPPRDLTEAFLA) corresponded to amino acids 263-277, and peptide 2 (LLTEHRMTWDPAQPPRDLTE) corresponded to amino acids 254-273 of CYP2D6. The peptide-keyhole limpet hemocyanin conjugates elicited good immune responses against their respective peptides as judged by enzyme-linked immunosorbent assay (titers of 1/10,000 to 1/30,000). The antisera recognized CYP2D6 on Western blots and, to varying extents, inhibited recombinant CYP2D6 and liver microsomal CYP2D6 activity. Immunization with peptide 2 produced antisera with the greatest inhibitory potency. Antiserum from a rabbit (#236) immunized with peptide 2 inhibited up to 95% of dextromethorphan O-demethylase activity in human liver microsomes at the highest concentration tested (40% v/v) but did not significantly inhibit CYP1A2, CYP2C9, CYP2E1, or CYP3A4 marker activities. On Western blot, only a single immunoreactive protein comigrating with recombinant CYP2D6 was recognized. In liver microsomes from a CYP2D6-deficient individual, no proteins were recognized, and the antisera did not cross-react with recombinant CYP1A2, CYP2C9, CYP2E1, or CYP3A4. There was a significant correlation between the quantity of immunoreactive CYP2D6 as determined by immunoblotting with anti-peptide 2 antiserum and dextromethorphan O-demethylation in a panel of 10 human liver microsomes (r = 0.95). These data identify a peptide sequence (peptide 2) that can be used to raise antisera that specifically recognize and inhibit CYP2D6.