Development of an enzyme immunoassay for detection of fipronil in environmental samples

An indirect competitive enzyme-linked immunosorbent assay (ELISA) was developed for the quantitative detection of fipronil in some environmental samples. Polyclonal fipronil antibodies were produced from rabbits immunized by hapten (fipronil) protein conjugate. The conjugate was prepared by activating the carboxylic groups of proteins; keyhole limpet hemocyanin (KLH) or bovine serum albumin (BSA) with 1-ethyl-3(3diaminopropyl) carbodiimide hydrochloride (EDC) followed by directly coupled to the amino group of fipronil. The influence of some factors such as concentrations of organic solvent, ionic strength and pH of buffer on the immunoassay was studied. Under optimized assay conditions, the ELISA having limit of detection (LOD) of 0.026 ng/ml and IC50 value of 0.325 μg/ml. Performance of the immunoassay was demonstrated by recovery study for spiked soil, tomato and potato samples giving recovery values in the range of 91-102 % for different concentrations in different samples. Good correlation was achieved between ELISA and conventional high pressure liquid chromatography (HPLC) analysis. The ELISA has the sensitivity to measure fipronil in soil, tomato and potato at level relevant for exposure monitoring. This study demonstrates that this proposed ELISA turned out to be a powerful tool for monitoring the residues of fipronil in environmental samples at trace levels.