Development of a high-specificity nanobody-based enzyme-linked immunosorbent assay for the rapid detection of mebendazole residues

Mebendazole (MBZ) residues in animal-derived foods pose significant public health risks, necessitating robust monitoring tools. In this study, a high-specificity nanobody (Nb) against MBZ was isolated from an alpaca-derived phage display library. The selected Nb exhibited superior thermal stability, retaining over 90% of its binding activity even after 40 min of incubation, and maintaining more than 60% activity after 90 min, whereas conventional rabbit IgG and chicken IgY against MBZ lost all activity under identical conditions. A competitive ELISA developed using this Nb achieved an IC₅₀ of 4.42 ng/mL and a limit of detection of 0.13 ng/mL. Cross-reactivity with structurally related benzimidazoles (albendazole, fenbendazole, oxfendazole, flubendazole, and oxibendazole) was negligible (<1%), while the assay recognized the major metabolite amino-MBZ with 90.9% cross-reactivity. Validation in chicken and mutton matrices showed recoveries between 75 and 114% with strong correlation to HPLC-MS/MS analysis (0.995). These results confirm the Nb-based ELISA is a robust, rapid screening tool for MBZ residues in foods.