Development of a phage-displayed antigen-binding fragment-based enzyme immunoassay for sensitive detection of...

Deoxynivalenol (DON) is a widely prevalent mycotoxin in food crops and poses a potential threat to human and animal health due to its multiple toxic effects. Therefore, sensitive detection tools are urgently required for monitoring DON in food. In this study, we developed a phage enzyme-linked immunosorbent assay (P-ELISA) using a phage-displayed antigen-binding fragment (Fab) as the detection probe of DON in corn samples. By constructing a pDong-V_L-V_H plasmid, Fab fragments were successfully displayed on the surface of the phage (Fab-phage) through phage rescue. The proposed method for DON was achieved by utilizing the principle of DON competing with the coating antigen bovine serum albumin-conjugated DON (DON-BSA) to bind to the Fab-phage. After optimization, the P-ELISA demonstrated a limit of detection of 0.33 ng mL^-1, a half inhibitory concentration (IC₅₀) of 3.83 ng mL^-1, and a linear range (IC₂₀-IC₈₀) of 0.82-29.85 ng mL^-1. No cross-reactions were observed between the Fab-phage and four common mycotoxins. The spiked recovery experiments demonstrated good accuracy and precision. In actual corn sample testing, the results obtained by HPLC-UVD showed a good correlation to those of the P-ELISA (R² = 0.997). Thus, the Fab-based P-ELISA was successfully developed and demonstrated to be accurate, reliable, and suitable for detecting DON in corn samples.