Edwards R J, Adams D A, Watts P S, Davies D S, Boobis A R
Division of Medicine, Imperial College School of Medicine, Hammersmith Hospital, London, UK. r.edwards.rpms.ac.uk.
Biochem Pharmacol. 1998 Aug 1;56(3):377-87. doi: 10.1016/s0006-2952(98)00033-1.
Mono-specific antibodies against the human cytochrome P450 (P450) enzymes CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2D6, CYP2E1, CYP3A4, CYP3A5 and CYP4A11 and an antibody that binds to CYP2C8, CYP2C9 and CYP2C19 have been produced by immunising rabbits with synthetic peptides representing small regions of each of these P450 enzymes. The specificity of the antibodies was confirmed by immunoblotting using recombinant P450 enzymes and samples of human hepatic microsomal fraction. Each of the antibodies bound only to their respective target P450 enzyme(s). The relative intensity of immunoreactive bands was compared with a variety of P450 activities and correlations were found between CYP1A2 and phenacetin O-deethylase activity, CYP2A6 and coumarin 7-hydroxylase activity, CYP2C9 and tolbutamide 4-hydroxylase activity, CYP2C19 and S-mephenytoin 4-hydroxylase activity, CYP2D6 and debrisoquine 4-hydroxylase activity, CYP2E1 and chlorzoxazone 6-hydroxylase activity, CYP3A4 and midazolam 1'-hydroxylase activity, and CYP4A11 and lauric acid 12-hydroxylase activity. A proportion of the 30 liver samples examined lacked CYP2A6 (7%), CYP2C19 (10%) or CYP2D6 (13%), consistent with the polymorphic expression of these P450 enzymes in human liver. Although CYP3A5 was detected in most individuals (97%), expression was polymorphic with 20% containing substantially higher levels. CYP2B6 was expressed in 20% of the human liver samples, with one sample containing a particularly high level. No immunodetectable CYP1A1 or CYP1B1 was found, consistent with the low level of expression of these P450 enzymes in human liver. The results demonstrate the utility of the antipeptide approach for producing specific antibodies against human P450 enzymes, enabling a comprehensive panel of antibodies against human P450 enzymes to be produced.
通过用代表这些细胞色素P450（P450）酶各自小区域的合成肽免疫兔子，已产生了针对人细胞色素P450酶CYP1A1、CYP1A2、CYP1B1、CYP2A6、CYP2B6、CYP2D6、CYP2E1、CYP3A4、CYP3A5和CYP4A11的单特异性抗体，以及一种与CYP2C8、CYP2C9和CYP2C19结合的抗体。使用重组P450酶和人肝微粒体组分样品通过免疫印迹法确认了抗体的特异性。每种抗体仅与它们各自的靶P450酶结合。将免疫反应条带的相对强度与多种P450活性进行比较，发现CYP1A2与非那西丁O - 脱乙基酶活性、CYP2A6与香豆素7 - 羟化酶活性、CYP2C9与甲苯磺丁脲4 - 羟化酶活性、CYP2C19与S - 美芬妥因4 - 羟化酶活性、CYP2D6与异喹胍4 - 羟化酶活性、CYP2E1与氯唑沙宗6 - 羟化酶活性、CYP3A4与咪达唑仑1'-羟化酶活性以及CYP4A11与月桂酸12 - 羟化酶活性之间存在相关性。在所检测的30个肝脏样品中，一定比例的样品缺乏CYP2A6（7%）、CYP2C19（10%）或CYP2D6（13%），这与这些P450酶在人肝脏中的多态性表达一致。尽管在大多数个体（97%）中检测到了CYP3A5，但其表达具有多态性，20%的个体含量显著更高。CYP2B6在20%的人肝脏样品中表达，其中一个样品含量特别高。未发现免疫可检测的CYP1A1或CYP1B1，这与这些P450酶在人肝脏中的低表达水平一致。结果证明了抗肽方法在产生针对人P450酶的特异性抗体方面的实用性，从而能够产生一组全面的针对人P450酶的抗体。
