Detection of cytochrome P4501A in several species using antibody against a synthetic peptide derived from rainbow trout cytochrome P4501A1
Detection of cytochrome P4501A in several species using antibody against a synthetic peptide derived from rainbow trout cytochrome P4501A1
复制标题
使用针对虹鳟鱼细胞色素 P4501A1 衍生的合成肽的抗体检测多个物种的细胞色素 P4501A
DOI：
10.1002/etc.5620170314
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发表时间：
1998
影响因子：
4.1
通讯作者：
S. Bandiera
中科院分区：
文献类型：
--
作者：
Song Lin;P. Bullock;R. Addison;S. Bandiera
细胞色素P4501 A（CYP 1A）的诱导作用越来越多地被用作指示生物体暴露于环境污染物（如某些多环芳烃和多氯芳烃）的生物标志物。测量CYP 1A蛋白在野生动物中将有助于通过使用一种特异性抗体，识别同工酶在几个物种。在本研究中，使用对应于鳟鱼酶的氨基酸277-294的合成肽作为免疫抗原，产生针对CYP 1A 1的多克隆抗体。通过非竞争性酶联免疫吸附试验（使用几种纯化的大鼠β-内酰胺酶同工酶）和免疫印迹分析（使用不同种属的肝微粒体）评估所得抗体的特异性。在酶联免疫吸附试验中，抗体与免疫肽和纯化的大鼠细胞色素P4501 A1强烈反应，但不与大鼠CYP 1A 2（一种密切相关的同工酶）或其他6种纯化的大鼠CYP蛋白反应。在免疫印迹中，抗体识别来自测试的各种哺乳动物和鱼类物种的肝微粒体中的单一蛋白条带。在3甲基胆蒽处理鸡的肝微粒体中检测到两条蛋白条带。结果表明，抗体结合的抗原决定簇是CYP 1A独有的，并且在不同物种中是保守的。由于其特异性，这种抗肽抗体应该适合作为探针来测量野生动物中的CYP 1A蛋白水平。
Induction of cytochrome P4501A (CYP1A) is being used increasingly as a biomarker to indicate exposure of organisms to environmental contaminants such as some polycyclic and polychlorinated aromatic hydrocarbons. Measurement of CYP1A protein in wildlife would be facilitated by the use of a specific antibody that recognized the isozyme in several species. In the present study, a polyclonal antibody targeted to CYP1A1 was generated using a synthetic peptide corresponding to amino acids 277–294 of the trout enzyme as the antigen of immunization. Specificity of the resulting antibody was assessed by noncompetitive enzyme‐linked immunosorbent assay with several purified rat CYP isozymes and by immunoblot analysis with liver microsomes from diverse species. The antibody reacted strongly with the immunizing peptide and with purified rat cytochrome P4501A1 but did not react with rat CYP1A2, a closely related isozyme, or with six other purified rat CYP proteins in enzyme‐linked immunosorbent assay. On immunoblots, the antibody recognized a single protein band in hepatic microsomes from the various mammal and fish species tested. Two protein bands were detected in liver microsomes from 3‐methylcholanthrene‐treated chickens. The results suggest that the antigenic determinant to which the antibody binds is unique to CYP1A and is conserved in different species. Because of its specificity, this anti‐peptide antibody should be suitable as a probe to measure CYP1A protein levels in wildlife.