A sensitive monoclonal antibody-based ELISA integrated with immunoaffinity column extraction for the detection of zearalenone in food and feed samples

Abstract

Zearalenone (ZEN) is one of the most toxic mycotoxins widely found in agricultural products. In this study, a sensitive enzyme-linked immunosorbent assay (ELISA) integrated with immunoaffinity column extraction for the detection of ZEN in food and feed samples was developed. A ZEN derivative containing a carboxylic group was first synthesized and then linked to bovine serum albumin (BSA). The formed ZEN–BSA conjugate was used as the immunogen for the production of the monoclonal antibody (mAb) against ZEN. The hybridoma clones (1G5) capable of secreting antibodies against ZEN were successfully selected. Based on this mAb, the IC₅₀ and LOD of the ELISA for ZEN were 0.37 ng mL⁻¹ and 0.04 ng mL⁻¹, respectively, which were 1.6–308.1 times lower than those in the published ELISAs, indicating the high sensitivity of our assay. There was no cross-reactivity of the mAb with other four mycotoxins (patulin, AFB₁, DON, and OTA). Due to the high similarity in molecular structures among ZEN and its homologs (α-zearalanol, β-zearalanol, zearalanone, α-zearalenol, β-zearalenol), the CR values of the mAb with the homologs were within 3.59%–105.71%. Taking advantage of plenty of mAb, the immunoaffinity column was prepared by immobilizing the mAb on Sepharose-4B gel and filling it into an SPE column. ZEN spiked samples (corn, wheat, feed) were extracted using an immunoaffinity column and measured by ELISA and HPLC-FLD simultaneously. The recoveries of the ELISA for ZEN in the spiked samples were 92.46–105.48% with RSDs of 4.87–10.11%. A good correlation between ELISA (x) and HPLC-FLD (y) with the linear regression equation y = 1.0589x + 1.43815 (R² = 0.998, n = 6) was obtained. To verify the applicability, the proposed ELISA was also applied to some real samples randomly collected from a local market. It was proven that the newly produced mAb-based ELISA was a feasible and sensitive method for the detection of ZEN in food and feed samples.