An Indirect Enzyme-Linked Immunosorbent Assay for Determination of New Herbicide H-9201 in Water

A sensitive indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) was developed for the detection of new herbicide H-9201, and the performance of ELISA for real sample of environmental water test was evaluated. Starting with phosphorus thiochloride and methanol, an analog with similar structure of hapten was synthesized, and carboxyl group was introduced by reacting with succinic anhydride, then the derivative was conjugated with bovine serum albumin or ovalbumin by means of active ester as immunogen or coating antigen, respectively. IC-ELISA method for H-9201 was developed based on the polyclonal antibody generated in rabbits by immunizing with synthesized hapten, with a linear range of 0.05–20 mg L−1. For standard, 50% reduction of the maximum ELISA signal (IC50) was 1.331 mg L−1, whereas the detection limit (IC10) was 0.017 mg L−1. The concentrations of coating solution and primary antibody were optimized to be 1/512 and 1/18000, respectively. No significant cross-reactivity for related compounds, such as DMNT, p-nitrophenol, Methamidophos, Phorate, Chlorpyrifos, and Parathion, was observed. The antibody showed negligible cross-reactivity with S-Carboxylethyl-O,O-Dimethyl-phosphorodithioate, dimethoate, and malathion, but exhibited high cross-reactivity with analogs of hapten. Recovery of H-9201 in environmental water was (80.5% ± 4.5%)-(95.2% ± 3.2%) based on the developed IC-ELISA method. This is the first report for demonstration of new herbicide H-9201 in water by IC-ELISA method.