Development of anti-thiamethoxam full-length recombinant antibody and investigation of the specific recognition mechanism

In this study, the recombinant antibody against thiamethoxam was prepared and the specific molecular recognition mechanism between the antibody and thiamethoxam was studied via computer-assisted homology modeling and molecular docking. Firstly, surface plasma resonance (SPR) technique was used to evaluate the recognition features of monoclonal antibody (mAb) against thiamethoxam. Secondly, the specific variable regions of heavy and light chains (VH and VL) in anti-thiamethoxam mAb were amplified from a hybridoma cell line, and full-length recombinant antibody (rAb) was successfully in vitro expressed by mammalian cell HEK 293 (F). At last, the molecular recognition mechanism of the antibody’s high specificity and sensitivity to thiamethoxam was investigated by homology modeling and molecular docking, based on the correct sequences of VH and VL. The results showed that the mAb could specifically recognize thiamethoxam and had a high binding affinity with dissociation equilibrium constant K_D of 7.995 × 10⁻¹¹ mol/L. As evaluated by indirect competitive enzyme-linked immunosorbent assay (ic-ELISA), the performance of full-length rAb was similar to that of the parental mAb, exhibiting high specificity and high sensitivity to thiamethoxam, with the IC₅₀ value of 0.41 μg/L to thiamethoxam. No cross reactivity to other neonicotinoid pesticides was observed (< 0.04%). The results demonstrated that Asn39 (L-CDR1) and the other 8 amino acid residues in the hydrophobic binding pocket interacted with the target mainly through hydrogen bond and van der Waals, which appeared to be the predominant contributor to the selectivity (specificity) of the antibody. His35 (H-CDR1) and Trp108 (H-CDR3) residues located in the VH affected on the binding affinity of the antibody against thiamethoxam. In conclusion, the full-length rAb prepared in this study can replace traditional monoclonal antibody as the core reagent to establish a variety of immunoassay methods for the detection of thiamethoxam residues in environmental samples and agricultural products. The study of the recognition mechanism can provide theoretical guidance for the further improvement of antibody affinity.