Rational hapten design and establishment of broad-spectrum indirect competitive enzyme-linked immunosorbent assay for benzimidazoles monitoring in milk

Abstract Objectives Benzimidazoles (BZs) are commonly used for the treatment of soil-transmitted helminth infections in veterinary clinics; however, misuse and overdosing of BZs will cause residual problems and have the potential to damage human health through the food chain. Thus, the existence of BZs in foods needs more attention. This study aims to establish a broad-spectrum immunoassay for rapid detection and to simultaneously monitor BZs in milk. Materials and Methods 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 first adopted for hapten modification to obtain an ultra-sensitive and broad-spectrum antibody. An indirect competitive enzyme-linked immunosorbent assay (icELISA) was established for the detection of 18 BZs in milk sample with a single-step pretreatment. A quantitative structure–activity relationship model was constructed to interpret and predict the recognition. Results The antibody could recognize 20 BZs and the half-inhibitory concentrations 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 ranged from 76.49% to 120.40%, with a coefficient of variation <20%. Substituent R1 of BZs was considered to be the main influencing factor for recognition, and the comparative molecular field analysis model (q2=0.724, r2=0.998) was finally chosen for further prediction. Conclusions The results indicated that the established icELISA could simultaneously identify 18 BZs, with good accuracy and precision, which was suitable for rapid detection of BZs in milk.