Donald W. Stoutamire

Development of a class-specific immunoassay for the type I pyrethroid insecticides

A general enzyme-linked immunosorbent assay was developed for the type I pyrethroid insecticides, such as permethrin, phenothrin, resmethrin and bioresmethrin. Polyclonal antibodies were generated by immunizing with a permethrin derivative, 2,2-dimethyl-3-(5 � -carboxy-pent-1 � -en-yl)cyclopropanecarboxylic acid-(3-phenoxybenzyl)ester conjugated with thyroglobulin, bovine serum albumin or ovalbumin. Antisera were screened against eight different coating antigens. The antibody–antigen combination with the lowest background, and highest sensitivity for permethrin was further optimized and tested for solvent and detergent tolerance. The I50’s of the optimized immunoassay were 30g/l for permethrin and 20g/l for phenothrin, respectively. No cross-reactivities were measured to the type II pyrethroids, such as esfenvalerate, cyfluthrin, cypermethrin, deltamethrin, fenvalerate and fluvalinate. This assay can be used in monitoring studies to distinguish between types I and II pyrethroids.

Rapid assays for environmental and biological monitoring.

Rapid, inexpensive, sensitive, and selective enzyme-linked immunosorbent assays (ELISAs) now are utilized in environmental science. In this laboratory, many ELISAs have been developed for pesticides and other toxic substances and also for their metabolites. Compounds for which ELISAs have recently been devised include insecticides (organophosphates, carbaryl, pyrethroids, and fenoxycarb), herbicides (s-triazines, arylureas, triclopyr, and bromacil), fungicides (myclobutanil), TCDD, and metabolites of naphthalene and toluene. New rapid assays have been developed for mercury.

Homogeneous fluoroimmunoassay of a pyrethroid metabolite in urine

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 (400or 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.

Immunoassays for pesticide monitoring

This study compares two formats of rapid assays for the detection of pesticides (bromacil and pyrethroid based metabolites): enzyme linked immunosorbent assay (ELISA) and immunoassay with near-infrared (NIR) fluorescence detection. NIR dye immunoassay (NIRDIA) measurements were carried out by using two different instruments, both having a silicon photodiode as the detector and a laser diode for excitation. ELISA and NIRDIA were performed in a tracer format, where the specific antibody is bound to the surface of a microtiter plate well and the tracer with enzyme or fluorescent dye label competes with the analyte for the antibody binding site. It was demonstrated that the NIRDIA is at least as sensitive as the ELISA. Both assays detect pesticides in the (mu) g/L (ppb) range. Hapten- macromolecule-NIR dye-conjugates have been synthesized with various biopolymers (e.g., proteins) as carriers. The use of carrier macromolecules enables convenient purification of the cyanine dye derivatives. The mild conjugation method of the dye is based on isothiocyanate chemistry.