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

Norbert Staimer, Shirley J. Gee, Bruce D. Hammock

Research output: Contribution to journalArticle

11 Scopus citations

Abstract

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: 1/60000). 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.

Original languageEnglish (US)
Pages (from-to)27-36
Number of pages10
JournalAnalytica Chimica Acta
Volume444
Issue number1
DOIs
StatePublished - Oct 12 2001

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Keywords

  • ELISA
  • Glucuronides
  • Immunoassay
  • Integrated HPLC-ELISA
  • Phenolic compounds

ASJC Scopus subject areas

  • Biochemistry
  • Analytical Chemistry
  • Spectroscopy
  • Environmental Chemistry

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