Epitope characterization of acetaminophen bound to protein and nonprotein sulfhydryl groups by an enzyme-linked immunosorbent assay.

The oxidation of acetaminophen to N-acetyl-p-benzoquinone imine and its subsequent binding to protein sulfhydryl groups may be important in the observed toxicity of acetaminophen. An avidin biotin-amplified competitive enzyme-linked immunosorbent assay has been developed which utilizes solid phase acetaminophen bound to metallothionein and antiserum obtained from rabbits that were immunized with 3-(N-acetyl-L-cystein-S-yl)acetaminophen covalently bound to keyhole-limpet hemocyanin. Over 25 synthetic analogs of acetaminophen conjugates and structurally similar compounds have been ranked relative to their ability to compete in the avidin biotin-amplified enzyme-linked immunosorbent assay. The most effective inhibitor was 3-(N-acetyl-L-cystein-S-yl)acetaminophen, which had an observed 50% inhibitory concentration of 120 fmol/well. Approximately 6200-fold higher concentrations of unbound acetaminophen and 5.2 x 10(6)-fold higher concentrations of N-acetyl-L-cysteine were required for comparable inhibition. It was demonstrated with acetaminophen analogs that the hydroxyl group and the N-acetyl moiety of acetaminophen were important in epitope recognition. A 5000-fold decrease in detection was observed when the analog did not contain the hydroxyl group or when the N-acetyl moiety was replaced with a hydroxyl substituent. Recognition by antibody was also dependent upon the stereochemistry of the analogs. The 50% inhibitory concentration for 3-(L-cystein-S-yl)acetaminophen was 2300 fmol/well, whereas a 25-fold higher concentration of 3-(D-cystein-S-yl)acetaminophen was required for 50% inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)