Development of a class-selective enzyme immunoassay for urinary phenolic glucuronides Original Research Article

Class-selective immunoassays for the measurement of glucuronides in human urine can aid evaluation of human exposure to complex mixtures of xenobiotics. Therefore, an enzyme immunoassay (EIA) for the group-selective detection of phenolic β-d-glucuronides was developed. The immunoconjugate was formed by coupling p-aminophenyl-β-d-glucuronide to the carrier protein thyroglobulin leaving an exposed glucuronic acid. Rabbits were injected with the immunogen in order to raise polyclonal antibodies. The resulting antibody #1339 (serum dilution: 1/5000) has been used in combination with a p-aminophenyl-1-thio-β-d-glucuronide-peroxidase-conjugate as an enzyme tracer (tracer dilution: 1/60 000). The resulting competitive assay is sensitive for phenyl-β-d-glucuronide (IC50=401 ng/ml), p-nitrophenyl-β-d-glucuronide (IC50=244 ng/ml), p-acetamidophenyl-β-d-glucuronide (IC50=239 ng/ml) and p-aminophenyl-β-d-glucuronide (IC50=434 ng/ml). Little or no cross-reactivities were observed for potential urinary cross-reactants. A combination of high-performance liquid chromatography (HPLC) and immunoassay was developed in an approach to apply the developed EIA for the determination of four different phenolic glucuronides in complex matrices such as human urine. The hyphenated technique HPLC–EIA may be used to monitor human exposure to a combination of related toxic agents including benzene, phenol and phenylamines which are all known to be excreted in urine as their phenolic glucuronic acid conjugates.