Albendazole (ABZ) is one of the benzimidazole anthelmintics, and the overuse of ABZ in breeding industry can lead to drug resistance and a variety of toxic effects in humans. Since the residue markers of ABZ are the sum of ABZ and three metabolites (collectively referred to as ABZs), albendazole-sulfone (ABZSO2), albendazole-sulfoxide (ABZSO), and albendazole-2-amino-sulfone (ABZNH2SO2), an antibody able to simultaneously recognize ABZs with high affinity is in urgent need to develop immunoassay for screening purpose. In this work, an unreported hapten, 5-(propylthio)-1H-benzo[d]imidazol-2-amine, was designed and synthesized, which maximally exposed the characteristic sulfanyl group of ABZ to the animal immune system to induce expected antibody. One monoclonal antibody (Mab) that can simultaneously detect ABZs was obtained with IC50 values of 0.20, 0.26, 0.77, and 10.5 μg/L for ABZ, ABZSO2, ABZSO, and ABZNH2SO2 in ic-ELISA under optimized conditions respectively, which has been never achieved in previous reports. For insight into the recognition profiles of the Mab, we used computational chemistry method to parameterize cross-reactive molecules in aspects of conformation, electrostatic fields, and hydrophobicity, revealing that the hydrophobicity and conformation of characteristic group of molecules might be the key factors that together influence antibody recognition with analytes. Furthermore, the practicability of the developed ic-ELISA was verified by detecting ABZs in spiked milk, beef, and liver samples with recoveries of 60% to 108.8% and coefficient of variation (CV) of 1.0% to 15.9%.

Ampicillin (AMP) was coupled with mcKLH by the method of EDC intermediate and injected into Balb/c mice as immunogen. The hybridomas were obtained by fusing mouse myeloma cells SP2/0 with splenocytes from the mice immunized with AMP-KLH. The chromosomal numbers were 97-104, the results indicated that the subclass of the McAb was IgG2a, the titer of ascitic fluid was 2×10 6 , the affinity constant was 3×10 -10 mol/L, the molecular weight was 154KD. The cross-reactivities of McAb to other antibiotics were below 0.01%. The linear detecting range of calibration curves to detect AMP was 0.5 ng/mL-100 ng/mL. The formular was Y = 0.0569X+0.1308, R 2 = 0.9948. The limit of detection was 0.3 ng/mL.