Optimized Electrofusion for Efficient Generation of Monoclonal Antibodies against Chlorpyrifos and Its Application in...

Immunoassays are reliable methods for pesticide residue detection, where antibody quality determines assay performance. In this study, we synthesized four haptens and generated 17 monoclonal antibodies (mAbs) against chlorpyrifos (CHP) using an optimized electrofusion. Through negative sorting of B lymphocytes and treatment with the optimal inducer (10 μg/mL interleukin-4 and 2.5 ng/mL lipopolysaccharide), the electrofusion rate was increased to 0.206%, which is 10-fold higher than that of conventional electrofusion. An AuNP-based lateral flow immunoassay (AuNP-LFIA) developed with mAb 7A2F8E5 achieved a visual detection limit of 1.25 ng/mL for CHP in buffer and 0.005 to 0.02 mg/kg in various crops, all below the maximum residue limits of the U.S., Japan, and China. The accuracy of the AuNP-LFIA was further validated through UPLC-MS/MS. These findings demonstrate a novel and efficient electrofusion strategy for generating high-affinity mAbs, and establish a rapid, sensitive, and reliable AuNP-LFIA for practical monitoring of chlorpyrifos residues in agricultural products.