SPECIFICITY AND CROSS-REACTIVITY OF MONOCLONAL AND POLYCLONAL ANTIBODIES AGAINST CYTOCHROME-P-450E OF THE MARINE FISH SCUP
SPECIFICITY AND CROSS-REACTIVITY OF MONOCLONAL AND POLYCLONAL ANTIBODIES AGAINST CYTOCHROME-P-450E OF THE MARINE FISH SCUP
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DOI：
10.1016/0003-9861(87)90660-6
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发表时间：
1987-02-15
影响因子：
3.9
通讯作者：
STEGEMAN, JJ
中科院分区：
文献类型：
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作者：
KLOEPPERSAMS, PJ;PARK, SS;STEGEMAN, JJ
抗肝细胞色素P-450 E（P-450 E）的单克隆抗体（MAb）1-12-3在免疫印迹分析中仅与P-450 E反应。这一个和其它六个抗P-450 E的MAb识别纯化的P-450 E，以及β-CD中的单个条带。萘啶酮（BNF）诱导的scup微粒体与真实的P-450 E共迁移。与MAb 1-12-3一样，多克隆抗P-450 E与P-450 E反应，但不与其他scup P-450组分反应，并与BNF处理的scup微粒体中与P-450 E一致的条带强烈反应。然而，多克隆抗体（PAb）也微弱地识别其他微粒体蛋白。单克隆抗体1-12-3识别由3，3“，4，4”，5，5“-六氯联苯和多氯联苯混合物诱导的扇贝微粒体中的P-450 E，并识别由BNF或3-甲基胆蒽（MC）诱导的其他硬骨鱼（包括两种鳟鱼、鳉鱼和比目鱼）微粒体中的单一条带。这些鱼中的P-450 E对应物的含量以及未处理的scup中的含量与诱导的乙氧基试卤灵O-脱乙基酶（EROD）活性一致。MAb 1-12-3强烈抑制了鲑鱼和鳟鱼中诱导的EROD活性，进一步证明了鳟鱼中P-450 E和诱导的P-450之间的关系。单克隆抗体1-12-3，另外两个单克隆抗体，和抗P-450 E PAb识别一个带与P-450 c在BNF诱导的大鼠微粒体共迁移。MAb 1-12-3也识别纯化的大鼠P-450 c。MAb 1-12-3和抗P-450 E PAb识别BNF大鼠微粒体中分子量低于P-450 c的第二条带，其可能对应于P-450 d，MC和异黄樟素诱导的大鼠同工酶。这些结果坚定地确立了scup P-450 E的同一性，其他硬骨鱼中BNF诱导的P-450与P-450 E的关系，以及P-450 E与大鼠P-450 c的免疫化学关系。此外，未经处理的鱼的结果表明，环境化学品的影响，可以通过免疫印迹与单克隆抗P-450 E检测。
Monoclonal antibody (MAb) 1-12-3 generated against liver cytochrome P-450E (P-450E), an aryl hydrocarbon hydroxylase of the marine fish Stenotomus chrysops (scup), reacted only with P-450E when tested in immunoblot anaysis with five P-450 fractions from scup liver. This and six other MAbs against P-450E recognized purified P-450E, as well as a single band in .beta.-naphthoflavone (BNF)-induced scup microsomes that comigrated with authentic P-450E. Like MAb 1-12-3, polyclonal anti-P-450E reacted with P-450E but not with other scup P-450 fractions and reacted strongly with a band coincident to P-450E in BNF-treated scup microsomes. However, the polyclonal antibody (PAb) also faintly recognized additional microsomal proteins. MAb 1-12-3 recognized P-450E induced by 3,3'',4,4'',5,5''-hexachlorobiphenyl and by polychlorinated biphenyl mixtures in scup, and a single band induced by BNF or 3-methylcholanthrene (MC) in microsomes of other teleosts, including two trout species, killifish and winter flounder. The content of the P-450E counterpart in these fish and also in untreated scup coincided with induced ethoxyresorufin O-deethylase (EROD) activity. Induced EROD activity in scup and trout was strongly inhibited by MAb 1-12-3, further demonstrating the relationship between P-450E and induced P-450 in trout. MAb 1-12-3, two other MAbs, and anti-P-450E PAb recognized a band comigrating with P-450c in BNF-induced rat microsomes. MAb 1-12-3 also recognized purified rat P-450c. MAb 1-12-3 and anti-P-450E PAb recognized a second band of lower molecular weight than P-450c in BNF rat microsomes which may correspond to P-450d, the MC- and isosafrole-inducible rat isozyme. The results firmly establish the identity of scup P-450E, the relationship of BNF-induced P-450 in other teleosts with P-450E, and the immunochemical relationship of P-450E with rat P-450c. Furthermore, results with untreated fish suggest that effects of environmental chemicals may be detected by immunoblotting with monoclonal anti-P-450E.