Development of an Ultrasensitive Monoclonal Antibody-Based Time-Resolved Fluorescence Immunochromatographic Assay for Mebendazole Detection

Mebendazole (MBZ), a broad-spectrum benzimidazole anthelmintic widely applied in veterinary medicine and aquaculture, has raised increasing concerns about its potential residues in foods of animal origin. In this work, a high-affinity and specific monoclonal antibody (mAb) was generated using a newly designed hapten containing a carboxylic spacer at the N1 site of the benzimidazole ring. The obtained mAb exhibited an IC₅₀ of 0.13 ng/mL with negligible cross-reactivity against related compounds. By combining this mAb with time-resolved fluorescent microspheres, a time-resolved fluorescence immunochromatographic assay (TRFIA) was developed for rapid and quantitative MBZ detection. The TRFIA achieved a detection limit of 1.02 μg/kg in fortified mutton and carp samples, with recovery rates ranging from 83.5% to 114.5% and coefficients of variation below 10.8%. Results correlated strongly with LC–MS/MS (R² > 0.97). This TRFIA provides a highly sensitive, reliable, and cost-effective platform for on-site screening of MBZ residues in animal-derived foods.