Novel Hapten Design Strategy Resulting in the Improvement of Antibody Sensitivity against Phorate

The hapten design for linear organophosphorus pesticides that lacked aromatic rings, often failed to clarify the interaction between antigenic determinant and monoclonal antibodies (mAbs). To address this problem, we propose a novel hapten design strategy with a phorate as a model. Synthesis of four optimized haptens enabled, for the first time, the elucidation of the phorate recognition mechanism, identifying the alkyl chain skeleton and ethoxy groups as critical antigenic determinant. Computer-aided molecular modeling predictions and antiserum experience confirmed the strategy's feasibility, enabling the successful preparation of a high-performance anti-phorate mAb with a half-maximal inhibitory concentration of 6.27 ng/mL and a linear detection range of 2.49-15.8 ng/mL in an indirect competitive enzyme-linked immunosorbent assay. Based on this mAb, we constructed a time-resolved fluorescent immunochromatographic assay (TRFIA)detection system incorporating an independent quality control line. The good correlation between the TRFIA results and those of GC-MS/MS confirms the validity of our method.