A highly sensitive fluorescence immunochromatography strip for thiacloprid in fruits and vegetables using recombinant...

Thiacloprid (TCL) is a neonicotinoid insecticide. Its widespread use has led to high levels of residue in fruits and vegetables. Hence, it is important to detect TCL rapidly, accurately, and sensitively in fruits and vegetables. Recombinant antibodies (rAbs) can be synthesized rapidly with little batch-to-batch variation. In this study, recombinant single-chain variable fragment (scFv) antibody and full-length recombinant antibody against TCL were produced using three different expression systems (E. coli, yeast, and mammalian cell). The results of SDS-PAGE and non - competitive enzyme-linked immunosorbent assay (ELISA) indicated that the full-length rAb exhibited promising characteristics, and the IC₅₀ value of indirect competitive ELISA (ic-ELISA) was 2.63 μg L^-1. However, recombinant scFv antibody had little affinity for the antigen. To understand antibody recognition, the three-dimensional (3D) model of the variable fragment (Fv) was built via homologous modeling. The interaction between Fv and TCL was analyzed via molecular docking and the results of molecular docking showed that VAL-158, ALA-211, PHE-220, TRP-218, TRP-49, and ILE-100 were mainly responsible for antibody recognition. In addition, a time-resolved fluorescent microsphere-immunochromatographic test strip (TRFM-ICTS) was developed with a linear range and limit of detection of 0.01-10 ng mL^-1 and 0.003 ng mL^-1 within 15 min under optimal conditions. The IC₅₀ value was 4.268 ng mL^-1, and the recovery ranged between 79.4% and 118.6%, which was consistent with HPLC-MS. The TRFM-ICTS has great advantages in sensitivity and applicability.