IMMUNOCHEMICAL ASSAY FOR RECOGNITION OF 2-S-GLUTATHIONYL ACETATE, A GLUTATHIONE CONJUGATE DERIVED FROM 1,1-DICHLOROETHYLENE-EPOXIDE

Cytotoxicities induced by 1,1-dichloroethylene (DCE) are ascribed to c ytochrome P450-dependent metabolism to an epoxide. Conjugation of the DCE-epoxide with glutathione (GSH) results in the formation of the con jugates 2-S-glutathionyl acetate (GTA) and 2-(S-glutathionyl) acetyl g lutathione (GAG); GAG undergoes hydrolysis to form GTA, and thus GTA i s a major metabolite of DCE metabolism. Our objective is to develop an antiserum against the chemically synthesized GTA, and for immunizatio n, we have used a hapten that consists of GTA conjugated to bovine ser um albumin (BSA) as the carrier protein and glutaraldehyde (GLUT) as a chemical cross-linker. The antisera were raised in rabbits and were c haracterized by using the following synthesized structural analogs: GT A, 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 char acterize the specificity of the antisera. Noncompetitive ELISA experim ents showed that the reaction of the antiserum with the antigen was co ncentration-dependent. in the competitive ELISA, GTA-GLUT-BSA inhibite d binding efficiently; in contrast, the unconjugated GTA did not inhib it 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 r eactivity with the antigen that was not competitively inhibited by eit her the GTA-EDC-BSA or the GSH-GLUT-BSA conjugates, Slot-blotting expe riments confirmed the findings of the ELISA studies and revealed high specificity of the antiserum to detect the hapten. These results demon strated the successful development of polyclonal antibodies to detect GTA and hence DCE-epoxide.