Forkert P G, Collins K S, Dowsley T F, Ross G M
Department of Anatomy and Cell Biology, Queen's University, Kingston, Ontario, Canada.
J Pharmacol Exp Ther. 1997 Jun;281(3):1422-30.
Cytotoxicities induced by 1,1-dichloroethylene (DCE) are ascribed to cytochrome P450-dependent metabolism to an epoxide. Conjugation of the DCE-epoxide with glutathione (GSH) results in the formation of the conjugates 2-S-glutathionyl acetate (GTA) and 2-(S-glutathionyl) acetyl glutathione (GAG); GAG undergoes hydrolysis to form GTA, and thus GTA is a major metabolite of DCE metabolism. Our objective is to develop an antiserum against the chemically synthesized GTA, and for immunization, we have used a hapten that consists of GTA conjugated to bovine serum albumin (BSA) as the carrier protein and glutaraldehyde (GLUT) as a chemical cross-linker. The antisera were raised in rabbits and were characterized by using the following synthesized structural analogs: GTA, glycine-GLUT-BSA (GLY-GLUT-BSA), GTA-GLUT-ovalbumin (GTA-GLUT-OVB), GTA-1-ethyl-3-(3-dimethylaminopropyl) carbodiimide-BSA (GTA-EDC-BSA), TRIS-GLUT-BSA, glutathione-GLUT-BSA (GSH-GLUT-BSA). The enzyme-linked immunosorbent assay (ELISA) and slot immunoblotting were used to characterize the specificity of the antisera. Noncompetitive ELISA experiments showed that the reaction of the antiserum with the antigen was concentration-dependent. In the competitive ELISA, GTA-GLUT-BSA inhibited binding efficiently; in contrast, the unconjugated GTA did not inhibit binding to the antigen. Competitive studies with the other analogs indicated low or minimal reactivities with the antibodies, which were blocked by incubation with GLY-GLUT-BSA. However, there was residual reactivity with the antigen that was not competitively inhibited by either the GTA-EDC-BSA or the GSH-GLUT-BSA conjugates. Slot-blotting experiments confirmed the findings of the ELISA studies and revealed high specificity of the antiserum to detect the hapten. These results demonstrated the successful development of polyclonal antibodies to detect GTA and hence DCE-epoxide.
1,1-二氯乙烯（DCE）诱导的细胞毒性归因于细胞色素P450依赖性代谢生成环氧化物。DCE-环氧化物与谷胱甘肽（GSH）结合会形成结合物2-S-谷胱甘肽基乙酸酯（GTA）和2-（S-谷胱甘肽基）乙酰谷胱甘肽（GAG）；GAG会水解形成GTA，因此GTA是DCE代谢的主要代谢产物。我们的目标是开发一种针对化学合成的GTA的抗血清，在免疫过程中，我们使用了一种半抗原，它由与牛血清白蛋白（BSA）结合的GTA作为载体蛋白，以及戊二醛（GLUT）作为化学交联剂组成。在兔子体内产生抗血清，并使用以下合成的结构类似物进行表征：GTA、甘氨酸-GLUT-BSA（GLY-GLUT-BSA）、GTA-GLUT-卵清蛋白（GTA-GLUT-OVB）、GTA-1-乙基-3-（3-二甲基氨基丙基）碳二亚胺-BSA（GTA-EDC-BSA）、TRIS-GLUT-BSA、谷胱甘肽-GLUT-BSA（GSH-GLUT-BSA）。采用酶联免疫吸附测定（ELISA）和狭缝免疫印迹法来表征抗血清的特异性。非竞争性ELISA实验表明，抗血清与抗原的反应呈浓度依赖性。在竞争性ELISA中，GTA-GLUT-BSA能有效抑制结合；相比之下，未结合的GTA不能抑制与抗原的结合。与其他类似物的竞争性研究表明，它们与抗体的反应性较低或极低，这些反应会被与GLY-GLUT-BSA孵育所阻断。然而，与抗原仍有残余反应性，且未被GTA-EDC-BSA或GSH-GLUT-BSA结合物竞争性抑制。狭缝印迹实验证实了ELISA研究的结果，并揭示了抗血清检测半抗原的高特异性。这些结果表明成功开发出了用于检测GTA以及DCE-环氧化物的多克隆抗体。
