Rational Hapten Design and Establishment of Broad-spectrum Indirect Competitive Enzyme-linked Immunosorbent Assay for Benzimidazoles Monitoring in Milk

Benzimidazoles were commonly used for the treatment of soil-transmitted helminth infections in veterinary clinics, however, misuse and overdose of BZs will cause residual problems and have potential damage to human health through the food chain. Thus, the existence of BZs in foods need more attention. This study aimes to establish broad-spectrum immunoassay for rapid and simultaneously monitor benzimidazoles in milk. Based on structure analysis, a "zero epitope loss" strategy which introduced a spacer arm into the imino group of the imidazole ring of albendazole was firstly adopted for hapten modification to obtain ultra sensitive and broad-spectrum antibody. An indirect competitive ELISA was established for the detection of eighteen BZs in milk sample with a single-step pretreatment. And quantitative structure-activity relationship model was constructed to interpret and predict the recognition. The antibody could recognize twenty benzimidazoles and the half-inhibitory concentrations were ranged from 0.054 to 417.58 ng/mL, the limits of detection of icELISA ranged from 0.4 to 89.4 ng/mL, and the mean recovery rates were ranged from 76.49% to 120.40%, coefficient of variation < 20%. Substituent R1 of benzimidazoles was considered to be main influencing factor for recognition, and CoMFA model (q 2 =0.724, r 2 =0.998) was finally chosen for further prediction. The results indicated that the established icELISA could simultaneously identify 18 benzimidazoles, with good accuracy and precision, which was suitable for rapid detection of benzimidazoles in milk.