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HOMOGENEOUS FLUOROIMMUNOASSAY OF A PYRETHROID METABOLITE IN URINE. (R825433)
Citation:
Matveeva, E. G., G. Shan, I. M. Kennedy, S. J. Gee, D. W. Stoutamire, AND B. D. Hammock. HOMOGENEOUS FLUOROIMMUNOASSAY OF A PYRETHROID METABOLITE IN URINE. (R825433). ANALYTICA CHIMICA ACTA 444(1):103-117, (2001).
Description:
Pyrethroids are widely used in agriculture as insecticides. In this study, we describe a simple one-step homogeneous fluoroimmunoassay for the glycine conjugate of phenoxybenzoic acid (PBAG), a putative pyrethroid metabolite that may be used as a biomarker of exposure to pyrethroids. Quenching fluoroimmunoassay (QFIA) is based on the competition of labeled and non-labeled pesticide for binding with antibodies and the resulting calibration curve is based on the relationship between analyte concentration and fluorescence quenching of labeled pesticide by specific antibodies. We developed a QFIA for PBAG in aqueous solution using fluorescein-labeled PBAG and polyclonal antibodies. The estimated IC50 (analyte concentration giving 50% inhibition of quenching) for PBAG was 4.5 nM. The detection limit (DL) was 0.9 nM. The dynamic range of the calibration curve was 2–50 nM. The average analytical recovery obtained by applying the method to urine samples (400- or 1000-fold urine dilution) was 85–111%. This demonstrates the QFIA to be a very simple and rapid detection method for PBAG; no washing steps and no enzyme conjugates were required.
Author Keywords: Homogeneous fluoroimmunoassay; Fluorescence quenching; Pyrethroid metabolite; Polyclonal antibodies; Urine
Abbreviations: Ab, polyclonal antibody (antibodies) against PBAG; DL, detection limit; DMF, dimethylformamide; ELISA, enzyme-linked immunosorbent assay; F-PBAG, PBAG labeled with fluorescein; IC50, analyte concentration giving 50% inhibition (quenching); PBAG, glycine conjugate of phenoxybenzoic acid, N-(3-phenoxybenzoyl)glycine; QFIA, quenching fluoroimmunoassay; r.u., relative units; Tris, tris(hydroxymethyl)aminomethane