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 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)...
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Published in | The Journal of pharmacology and experimental therapeutics Vol. 281; no. 3; pp. 1422 - 1430 |
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Main Authors | , , , |
Format | Journal Article |
Language | English |
Published |
United States
American Society for Pharmacology and Experimental Therapeutics
01.06.1997
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Subjects | |
Online Access | Get full text |
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Summary: | 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. |
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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
ISSN: | 0022-3565 1521-0103 |