A Tailored Monoclonal Antibody for Visual Detection of an Emerging Insecticide Chlorfenapyr: Rational Hapten Design and Molecular Insights

Chlorfenapyr (CFP), a novel pyrrole-class insecticide, in the environment and agricultural products pose a threat to human health and ecological balance. This study designed five CFP-hapten conjugates. By computer simulations predict highly feasible hapten structures, leading to the development of a CFP monoclonal antibody resistant to matrix interference (IC₅₀: 1.07 ng/mL). Docking simulations reveal that TRP 90, THR 50, PRO 95, PRO 93, and TYR 59 in the antibody binding pocket are key residues. These residues interact with the core recognition domain of CFP (chlorophenyl ring, ether bond, and pyrrole ring), with the ether bond emerging as a crucial exposed group in the recognition process. The binding pose analysis indicates that the bromine atom and cyano group are oriented outward within the recognition pocket, providing an ideal site for protein conjugation and validating the reliability of the hapten design strategy. Colloidal gold immunosorbent assay utilizing CFP-mAb enables rapid, accurate, and highly sensitive detection of CFP residues in fruits, vegetables, and environmental matrices. Its visual LOD reaches as low as 5 μg/kg, while the calculated LOD achieves 0.31 μg/kg. This method has demonstrated significant advantages in terms of cost-effectiveness, regulatory applicability, high-throughput detection, and portability. This study not only develops a sensitive CGIA for CFP detection but also, for the first time, provides molecular-level insights into the antibody-hapten recognition mechanism, offering a new paradigm for rational design of immunoassays for small molecule pesticides.