Cribb A, Nuss C, Wang R
Department of Drug Metabolism, Merck Research Laboratories, West Point, PA 19486, USA.
Drug Metab Dispos. 1995 Jul;23(7):671-5.
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.
两种与人类肝肾微粒体-1自身抗体识别的主要33个氨基酸序列中的序列相对应的肽，被用于在兔子体内（每种肽4只兔子）诱导产生针对CYP2D6的抗体。肽1（DPAQPPRDLTEAFLA）对应于CYP2D6的263-277位氨基酸，肽2（LLTEHRMTWDPAQPPRDLTE）对应于CYP2D6的254-273位氨基酸。通过酶联免疫吸附测定判断，肽-钥孔戚血蓝蛋白缀合物对其各自的肽引发了良好的免疫反应（效价为1/10,000至1/30,000）。抗血清在蛋白质免疫印迹法中识别CYP2D6，并在不同程度上抑制重组CYP2D6和肝微粒体CYP2D6的活性。用肽2免疫产生的抗血清具有最大的抑制效力。用肽2免疫的一只兔子（#236）的抗血清在测试的最高浓度（体积比40%）下，可抑制人肝微粒体中高达95%的右美沙芬O-脱甲基酶活性，但对CYP1A2、CYP2C9、CYP2E1或CYP3A4标记活性没有显著抑制作用。在蛋白质免疫印迹法中，仅识别到一种与重组CYP2D6共迁移且具有免疫反应性的蛋白质。在来自一名CYP2D6缺陷个体的肝微粒体中，未识别到蛋白质，且抗血清与重组CYP1A2、CYP2C9、CYP2E1或CYP3A4无交叉反应。在用抗肽2抗血清进行免疫印迹测定所确定的免疫反应性CYP2D6的量与一组10个人肝微粒体中的右美沙芬O-脱甲基化之间，存在显著相关性（r = 0.95）。这些数据确定了一个肽序列（肽2），该序列可用于产生能特异性识别和抑制CYP2D6的抗血清。
