Organophosphorus pesticides pose significant risks to the ecosystem and human health due to their environmental persistence and potential accumulation in medicinal herbs. This study aimed to develop rapid immunoassays for detecting four phosphorothioate organophosphorus pesticides isofenphos-methyl (Fen), isocarbophos (Car), isofenphos (Phe), and isofenphos-oxon (Oxon) in complex Codonopsis pilosula and Angelica sinensis matrices. Five novel haptens (H1-H5) were synthesized. Among them, a broad-spectrum hapten (H1) was rationally designed through computer-aided modeling, which optimized its spatial conformation, electrostatic potential, and electronic structure for maximum similarity to those of the target pesticides. This hapten yielded a high-affinity monoclonal antibody (mAb 2B1) with excellent sensitivity (IC50 values of 0.78, 1.53, 6.89, and 8.36 ng/mL for Fen, Car, Phe, and Oxon, respectively) and specificity. Homology modeling and molecular docking revealed the structural basis for the mAb's differential affinity; amino acid GLY-99(A), TYR-36(B), and TRP-106(A) play a central role in and could be a key target for subsequent antibody affinity maturation to broaden its cross-reactivity profile against structural analogs. GLY-99(A) and TYR-36(B) are responsible for polar complementarity and precise positioning, while TRP-106(A) contributes to hydrophobic stabilization and energy gain. Based on mAb 2B1, a high-throughput ic-ELISA and a rapid dual-test-line immunochromatographic strip (AuNP-LFIA) were developed; it enabled on-site, visual detection within 15 min, with detection limits compliant with regulatory standards. The liquid chromatography-mass spectrometry (LC-MS/MS) analysis of the positive samples was consistent with the results of the AuNP-LFIA method, further validating the reliability, accuracy, and practical applicability of the developed assay in this study for monitoring phosphorothioate organophosphorus pesticide residues in environmental and agricultural products.
Wu et al. (2026) studied this question.