Development of a Sensitive Enzyme Immunoassay Using Phage-Displayed Antigen-Binding Fragments for Zearalenone Detection in Cereal Samples

Zearalenone (ZEN), a non-steroidal estrogenic mycotoxin, contaminates animal feed and grain crops, thereby entering the food chain and posing a significant threat to human health. Consequently, there is an urgent need for a sensitive and rapid method for detecting trace levels of ZEN. In this study, we developed a phage-displayed antigen-binding fragment (Fab-phage) and established a Fab-phage-based enzyme-linked immunosorbent assay (Fab-pELISA) for ZEN detection. Under optimal conditions, this method exhibits a half-maximal inhibitory concentration of 0.36 ng/mL, with a linear range from 0.07 to 3.89 ng/mL and a detection limit of 0.03 ng/mL. The method demonstrates high selectivity towards ZEN and good recovery rates of 97.35-122.66% with relative standard deviations not exceeding 3.5%. Furthermore, the detection results obtained using Fab-pELISA on real cereal samples are consistent with those from high-performance liquid chromatography, meeting practical application requirements. Therefore, the Fab-phage serves as a valuable biochemical reagent, and the established Fab-pELISA represents a promising analytical strategy for detecting ZEN and other trace toxic contaminants in cereals.