Time-resolved Fluorescence Immunoassay and Molecular Recognition Mechanism of the Single Chain Variable Fragment against the Cry2Aa Toxin
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Abstract:
Cry2Aa toxin is a new type of biopesticide, and analysis of the interactions between this toxin and a specific single chain variable fragment (scFv) to enable the establishment of a rapid method for toxin-residue detection is important for maintenance of food and ecological security. Here, a homologous protein was used as a template for modeling the three-dimensional structure of anti-Cry2Aa scFv in order to allow simulations of molecular docking between Cry2Aa and scFv and determination of key binding sites. The results showed that svFv was suitable for use as an antibody to establish an indirect competitive time-resolved fluorescence immunoassay (IC-TRFIA), which was applied for the detection of Cry2Aa toxin in rice samples. The three-dimensional structure of scFv was modeled and refined using bioinformatics tools. The molecular-docking results showed that the amino acid residues of the heavy chain variable regions 81NY82 (VH-CDR2) and 121SGNY124 (VH-CDR3) and the light chain variable region 245YSSN248 (VL-CDR3) played important roles in the recognition of the Cry2Aa domain, providing a basis for the establishment of a highly efficient detection method. The limit of the detection was 0.08 ng/mL, the 50% inhibition rate was 7.99 ng/mL, and the linear range (IC20~IC80) was from 0.24 ng/mL to 263.77 ng/mL. Furthermore, IC-TRFIA results and those of conventional double-antibody sandwich ELISA showed good linear correlation.
