The Hapten Design, Monoclonal Antibody Preparation, and Immunoassay Development for Rapid Detection of Isofenphos-Methyl

Isofenphos-methyl (IFP), a highly toxic and persistent organophosphate pesticide (OP), is widely used for soil pest control in crops but poses severe risks to ecological safety and human health because of its environmental accumulation and bioaccumulation. Herein, a sensitive and specific indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was developed for rapid IFP detection in vegetables. A novel IFP hapten was rationally designed and synthesized via computer-aided molecular simulation, and its structure was validated by liquid chromatography–tandem mass spectrometry (LC–MS/MS) and nuclear magnetic resonance (NMR). High-specificity anti-IFP monoclonal antibodies (mAbs) with strong anti-matrix interference were prepared for the first time using a matrix effect-enhanced screening strategy. The optimized ic-ELISA showed high sensitivity, with an IC50 of 6.087 ng/mL and a detection range of 1.165–30.490 ng/mL, and no cross-reactivity with other common OPs. Spiked recovery experiments in celery and chili pepper matrices yielded recoveries of 81.87–97.95% (RSD < 5.44%), with highly consistent LC–MS/MS results. The method exhibited a weak positive matrix effect in vegetable matrices, eliminating complex pretreatment and enabling rapid onsite detection.