PT  - JOURNAL ARTICLE
AU  - Roberts, D W
AU  - Pumford, N R
AU  - Potter, D W
AU  - Benson, R W
AU  - Hinson, J A
TI  - A sensitive immunochemical assay for acetaminophen-protein adducts.
DP  - 1987 May 01
TA  - Journal of Pharmacology and Experimental Therapeutics
PG  - 527--533
VI  - 241
IP  - 2
4099  - http://jpet.aspetjournals.org/content/241/2/527.short
4100  - http://jpet.aspetjournals.org/content/241/2/527.full
SO  - J Pharmacol Exp Ther1987 May 01; 241
AB  - The hepatotoxicity of acetaminophen may be mediated by the reactive metabolite N-acetyl-p-benzoquinone imine which binds covalently to protein primarily as 3-(cystein-S-yl)acetaminophen. We have developed an avidin biotin-amplified competitive enzyme-linked immunosorbent assay to detect protein-bound acetaminophen. This assay utilizes antisera from rabbits immunized with 3-(N-acetyl-L-cystein-S-yl)acetaminophen coupled via the carboxyl group to primary amino groups on the protein keyhole-limpet hemocyanin. The competitive enzyme-linked immunosorbent assay utilizes metallothionein derivatized with N-acetyl-p-benzoquinone imine (acetaminophen-bound metallothionein) and quantitation was obtained by competition of acetaminophen-derivatives for a limited amount of antibody in the presence of excess solid phase acetaminophen-bound metallothionein. Synthetic 3-(N-acetyl-L-cystein-S-yl)acetaminophen, acetaminophen bound to mouse 9,000 X g supernatant, 100,000 X g supernatant, microsomes, as well as acetaminophen-bound metallothionein were inhibitory. The 50% inhibition for 3-(N-acetyl-L-cystein-S-yl)acetaminophen was 110 fmol/well. In contrast, free acetaminophen was 6200 times less efficient as an inhibitor. The mean 50% inhibition for protein-bound acetaminophen was 2.89 pmol/well. The utility of the method to detect acetaminophen-protein adducts in biological samples was confirmed by detection of NADPH-dependent binding of acetaminophen to microsomal proteins.