Hapten Synthesis and Monoclonal Antibody Preparation for Simultaneous Detection of Albendazole and Its Metabolites in Animal-Origin Food

Abstract

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%.

Development of a Lateral Flow Immunoassay Based on a Highly Specific Monoclonal Antibody To Detect 4-Methylaminoantipyrine

“…CG particles ∼25 nm in size were prepared and used to label mAb (CG-mAb) as previously described. − The detailed procedure is included in the Supporting Information. The LFA strip consisted of four sections: an absorbent pad, a sample pad, an NC membrane, and a PVC backing sheet.…”

“…The performance of mAb was evaluated by indirect ELISA and icELISA, for which protocols were almost the same as those previously described. , The sensitivity of mAb was assessed using the IC 50 value, defined as the value of the half-maximal inhibitory concentration of the analyte. The specificity of mAb was evaluated using cross-reactivity (CR) values calculated using the following formula: CR 0.25em ( % ) = ( IC 50 of MAA / IC 50 of analyte false[ ng / mL false] ) × 100 …”

Immunosensor of Nitrofuran Antibiotics and Their Metabolites in Animal-Derived Foods: A Review

“…Therefore, the basis and key to developing highly sensitive immunoassays is to obtain antigens and antibodies with high affinity and high specificity ( Dengl et al, 2016 ; Guillermo et al, 2019 ). The key to develop antibodies with desired affinity and specificity is hapten design ( Shao et al, 2021 ). Since the molecular mass of nitrofuran metabolites is less than 1,000 Da, they are small molecule compounds that are not immunogenic and cannot easily bound to carrier protein ( Cooper et al, 2017b ).…”

“…Obtain relevant specific antibodies by immunizing animals with antigens, its performance was characterized by enzyme-linked immunoassay (ELISA) in liquid phase system. The binding ability of antibodies to compounds can also be studied through computational chemistry analysis, which provides 3D conformational and quantitative information such as conformation, electrostatic potential, and molecular hydrophobicity ( Shao et al, 2021 ). In 2007, Cooper et al successfully prepared the first polyclonal antibodies against SEM and AOZ ( Cooper et al, 2004 ; Cooper et al, 2007 ).…”

Application of Antibody and Immunoassay for Food Safety

“…An immunoassay based on a broadly specific antibody is an emerging trend in the sensitive and simple detection of a group of similar compounds in a single assay. Shao et al [ 9 ] designed and synthesized an unreported hapten, 5-(propylthio)-1H-benzo[d]imidazol-2-amine, which maximally exposed the characteristic sulfanyl group of albendazole (ABZ) to the animal immune system to induce the expected antibody. One mAb that can simultaneously detect the sum of ABZs (ABZ and its metabolites, i.e., ABZSO 2 , ABZSO, and ABZNH 2 SO 2 ) was obtained.…”