Development of an enzyme-linked immunosorbent assay for atrazine mercapturic acid in human urine

Improved assessments of human exposure to electrophilic chemicals require rapid and inexpensive analytical techniques that can detect specific urinary metabolites at low levels as needed for epidemiological screenings of large populations. The first aim of this study has been to apply rational hapten design strategies to develop a more sensitive and selective enzyme-linked immunosorbent assay for atrazine mercapturic acid. Polyclonal sheep antiserum was generated against an improved hapten, numerous coating antigen chemistries were evaluated, and assay conditions were optimized. An assay was developed with an IC50 of 0.08 +/- 0.02 micrograms/L (K approximately with 10(-)10 M) for atrazine mercapturic acid. The assay exhibited greatest recognition of atrazine mercapturic acid relative to other known urinary metabolites of atrazine as well as other triazine herbicides. The assay was surprisingly selective to atrazine mercapturic acid over the structurally similar simazine mercapturic acid. Urine samples presented matrix effects due in part to the nonspecific effects of urinary salts, but 4-fold dilution of urine achieved an overall method limit of quantitation of 0.3 micrograms/L. Solid-phase extraction strategies were also developed in an attempt to increase the sensitivity of the overall method. However, a weak positive assay response was present in the solid-phase extracts of unspiked urines, resulting in accurate recovery of atrazine mercapturic acid at microgram/L.