Highly broad-specific and sensitive direct competitive enzyme-linked immunosorbent assay for screening multi-antibacterial synergists: assay optimization and application to animal-derived food

Abstract

The optimum conditions of a direct competitive enzyme-linked immunosorbent assay (dcELISA) in regard to different monoclonal antibodies (MAbs), coating conditions, blocking conditions and physicochemical factors of the assay buffer were investigated to develop a broad-specific and sensitive immunoassay for detection of antibacterial synergists (ASGs) in milk, eggs, chicken and pork. Under optimized conditions, a highly broad-specific and sensitive dcELISA for ASGs was obtained based on 5C4 and hapten-C-HRP, demonstrating a 50% specific binding (IC50) for five ASGs of 0.208–9.24 ng mL−1 with cross-reactivity (CR) of 138.2–7.5%. The optimized dcELISA was used to quantify the five ASGs in milk, egg, chicken muscle and beef with the average recoveries of 79.5–105.4% and coefficients of variation (CV) less than 13.4%. The established method with broad specificity and uniform affinity, offered a simple, sensitive, and high-throughput screening tool for the detection of multi- ASGs in animal-derived food.

Keywords

• antibacterial synergists
• direct competitive enzyme-linked immunosorbent assay
• optimization
• animal-derived food