Development of an Immunoassay for the Detection of the Phenylpyrazole Insecticide Fipronil

Abstract

Phenylpyrazole insecticides such as fipronil have been used as replacements for organophosphates. The wide application of fipronil raises concern about environmental contamination and risk for fish, birds, other non-targeted beings and human health. A sensitive, competitive indirect heterologous enzyme-linked immunosorbent assay (ELISA) was developed. Antibodies with different specificities to fipronil and its metabolites were produced. Two ELISAs having IC50 values of 0.58 ± 0.06 and 2.6 ± 0.4 ng/mL were developed. Design of different haptens and coating antigens resulted in two assays with distinct cross-reactivity patterns for structurally related compounds: 96%, 38% and 101% vs 39%, 1.4% and 25% for fipronil-sulfide, fipronil-detrifluoromethylsulfonyl and fipronil-desulfinyl, respectively. Performance of the immunoassays was demonstrated by a recovery study from spiked water, human serum and urine matrices, giving recovery values in the range of 85–111% for different concentrations. The assays demonstrated good correlation in fipronil recovery with conventional LC-MS/MS analysis. The generic assay 2265 has the sensitivity to measure fipronil and its analogs in serum at levels relevant for exposure monitoring. The assays were used to analyze human urine samples obtained from exposure studies and serum samples from rats treated with fipronil-containing diet.

Hydroxy-fipronil is a new urinary biomarker of exposure to fipronil

“…We previously developed 2 immunoassays using different sera for the detection of fipronil (Vasylieva et al 2015b). Cross-reactivity (CR) to hydroxy-fipronil was tested with these polyclonal antibodies 2265 and 2268 (pAbs).…”

“…S5). The assay 2268 is more robust to changing sample conditions like pH or ionic strength (Vasylieva et al 2015a). We therefore chose this assay to assess the applicability of the immunoassay for screening of urine samples for the presence of biomarkers of exposure to fipronil.…”

“…Substrate solution contained 0.2 mL of 0.6% TMB (in dimethyl sulfoxide, DMSO w/v), 0.05 mL of 1% H 2 O 2 in 12.5 mL of substrate buffer. Stop solution was 2 MH 2 SO 4 .The immunoassay was performed as previously described (Vasylieva et al 2015a). Briefly, plates were coated with 1 μg/mL antigen BDH-297-7-CON diluted in coating buffer (100 μL/well).…”

Sensitive immunoassay for simultaneous determination of tetrabromobisphenol A bis(2-hydroxyethyl) ether and tetrabromobisphenol A mono(hydroxyethyl) ether: An effective and reliable strategy to estimate the typical tetrabromobisphenol A derivative and byproduct in aquatic environments

“…As shown in Table 1, the pAB could simultaneously recognize TBBPA DHEE and TBBPA MHEE, manifesting low cross-reactivity to the other selected compounds. Meanwhile, although a linker arm of 3e5 carbon atoms was generally considered an optimal distance between hapten and the carrier protein (Vasylieva et al, 2015), our hapten design showed that linker arms of less than 3 also met the requirements for generating the antibodies.…”

“…Since TBBPA-DHEE belongs to a nonpolar chemical, methanol is useful to keep the hydrophobic targets in immunoassay solution and prevent nonspecific binding on the container walls (Vasylieva et al, 2015). In order to improve the performance of the ELISA method, methanol was added to the assay system.…”