Development of a monoclonal antibody-based enzyme-linked immunosorbent assay for detection of the furaltadone metabolite, AMOZ, in fortified shrimp samples

摘要

3-Amino-5-morpholinomethyl-2-oxazolidone (AMOZ) is a tissue bound metabolite of furaltadone, a synthetic nitrofuran antibiotic widely used in veterinary practices both to treat infectious diseases and as a growth promoter. Because AMOZ is carcinogenic and poses a potential health hazard for human consumers, its parent compounds, the nitrofurans, are banned from being used in animals destined for human food consumption in various countries. To enforce this, foods are routinely monitored for nitrofuran metabolite residues including AMOZ. Thus, reliable high throughput analytical methods to detect AMOZ have been developed but these are mostly based on chemical analysis. In contrast, sensitive and specific detection methods based on immunoassays for AMOZ using monoclonal antibodies have yet to be established. In this study, we report the generation of two monoclonal antibodies with high specificity for AMOZ and the development of a monoclonal antibody-based ELISA to detect AMOZ in shrimp samples using one of these clones (clone 2E5.1). Clone 2E5.1 yielded the highest sensitivity and specificity and cross reacted solely to furaltadone and AMOZ and not to other antibiotics. Competitive ELISA with 2E5.1 gave IC_(50) values for AMOZ, CPAMOZ and furaltadone of 5.33, 0.023 and 1.330 ng/ml, respectively. When applied in ELISA to detect AMOZ in fortified shrimp samples, the detection capability and limit of detection were 0.3 and 0.16 μg/kg, respectively. Taken together, a sensitive and specific monoclonal antibody-based ELISA for AMOZ detection has been developed that could facilitate the economic and reliable high throughput monitoring of AMOZ in shrimps and potentially other food.