Phage display-derived single-chain variable fragment antibody for highly sensitive and specific detection of thiamethoxam residues in food matrices.

Thiamethoxam (TMX), as a widely used pesticide, poses potential risks to food safety due to residual contamination, underscoring the need for highly sensitive recognition elements for its detection. This study constructed a phage-displayed immune antibody library via mouse immunization with a titer of 1.6 × 1010 PFU/mL. Following four rounds of biopanning, a soluble single-chain variable fragment (scFv) antibody was isolated, which can specifically recognize TMX. Based on this antibody, an indirect competitive ELISA (ic-ELISA) and colloidal gold immunochromatographic assay (CG-ICA) were established. Under the optimal conditions for ic-ELISA, the IC50 value was 25.95 ng/mL, with a linear response range of 8.13 ng/mL - 137.97 ng/mL. Additionally, the scFv showed acceptable specificity against other structurally related neonicotinoids. In spiked real samples, average recoveries ranged from 71.14% to 110.49% with coefficients of variation of 2.22%-7.92%, demonstrating good accuracy and precision. Furthermore, the visual limit of detection (vLOD) for the optimized CG-ICA was 0.06 mg/kg - 0.10 mg/kg. This work offers theoretical insight and technical assistance for developing scFv antibodies against TMX residues in various samples.