Development of a sensitive enzyme immunoassay for the detection of phenyl-beta-D-thioglucuronide in human urine

Immunoassays for the measurement of glucuronides in human urine can be a he lpful tool for the assessment of human exposure to toxic chemicals. Therefo re an enzyme immunoassay (EIA) for the specific detection of phenyl-beta -D -thioglucuronide was developed. The immunoconjugate was formed by coupling p-amino-phenyl-beta -D-thioglucuronide to the carrier protein thyroglobulin leaving an exposed glucuronic acid. The hapten-protein conjugate was adsor bed to gold colloids in order to enhance the immunogenic effect. Rabbits we re injected with the immunogold conjugates to raise polyclonal antibodies. The resulting competitive assay showed an inhibition by phenyl-beta -D-thio glucuronide at sample concentrations of 23.0 +/- 1.3 ng/mL (50% B/B-o) and a high cross-reactivity to p-aminophenyl-beta -D-thioglucuronide (120%). Li ttle cross-reactivities (< 2%) were observed for potential urinary cross re actants. In addition human urine samples were incubated with <beta>-glucuro nidase in order to investigate the EIA for specific matrix effects. An inte gration of high-performance liquid chromatography (HPLC) and EIA was develo ped in an attempt to decrease the matrix effects and increase the sensitivi ty of the overall method. The hyphenated technique HPLC-EIA map be used to monitor human exposure to toxic thiophenol which is excreted by mammals as urinary phenyl thioglucuronide.