ANTIPEPTIDE ANTIBODIES AGAINST OVERLAPPING SEQUENCES DIFFERENTIALLY INHIBIT HUMAN CYP2D6

Two peptides that correspond to sequences within the major 33-amino ac id sequence recognized by human liver-kidney microsomal-l autoantibodi es were used to elicit antibodies in rabbits (four per peptide) agains t CYP2D6. Peptide 1 (DPAQPPRDLTEAFLA) corresponded to amino acids 263- 277, and peptide 2 (LLTEHRMT-WDPAQPPRDLTE) corresponded to amino acids 254-273 of CYP2D6. The peptide-keyhole limpet hemocyanin conjugates e licited good immune responses against their respective peptides as jud ged by enzyme-linked immunosorbent assay (titers of 1/10,000 to 1/30,0 00). The antisera recognized CYP2D6 on Western blots and, to varying e xtents, inhibited recombinant CYP2D6 and liver microsomal CYP2D6 activ ity. Immunization with peptide 2 produced antisera with the greatest i nhibitory potency. Antiserum from a rabbit (#236) immunized with pepti de 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 ma rker activities. On Western blot, only a single immunoreactive protein comigrating with recombinant CYP2D6 was recognized. In liver microsom es from a CYP2D6-deficient individual, no proteins were recognized, an d the antisera did not cross-react with recombinant CYP1A2, CYP2C9, CY P2E1, or CYP3A4. There was a significant correlation between the quant ity of immunoreactive CYP2D6 as determined by immunoblotting with anti -peptide 2 antiserum and dextromethorphan O-demethylation in a panel o f 10 human liver microsomes (r = 0.95). These data identify a peptide sequence (peptide 2) that can be used to raise antisera that specifica lly recognize and inhibit CYP2D6.