Development of a class-selective enzyme-linked immunosorbent assay for mercapturic acids in human urine

Epidemiological and toxicological studies often require the analysis of large numbers of samples for biological markers of exposure. The goal of this work was to develop a class-selective ELISA to detect groups of structurally closely related mercapturic acids with small nonpolar S-substituents. An assay was developed with strong recognition for mercapturates including S-benzylmercapturic acid (IC50 = 0.018 micromol/L), S-n-hexylmercapturic acid (IC50 = 0.021 micromol/L), S-phenylmercapturic acid (IC50 = 0.024 micromol/L), and S-cyclohexylmethylmercapturic acid (IC50 = 0.042 micromol/L). The same assay also showed weaker recognition for S-(1-hydroxynaphthal-2-yl)mercapturic acid and S-allylmercapturic acid (IC50 = 1.1 and 1.7 micromol/L, respectively). Subtle modifications to the hapten linker structure of the coating antigen proved to have a strong impact on the selectivity and the specificity of the assay. A slightly modified assay showed high recognition for S-benzylmercapturic acid (IC50 = 0.018 micromol/L) and weaker recognition for seven other mercapturic acids (IC50 = 0.021-10 micromol/L). Strong positive assay responses were detected in 12 urine samples obtained from persons with no known occupational exposure to exogenous electrophilic xenobiotics. Solid phase extraction and cross-reactivity indicated that the presumptive immunoreactive materials were similar in size and polarity to S-benzylmercapturic acid. The assay was more selective to mercapturic acids than the spectrophotometric thioether assay.