直接竞争ELISA法检测动物源食品中利巴韦林残留
Detection of Ribavirin Residues in Animal-Derived Food by Way of Direct Competitive ELISA
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摘要: 本研究针对动物源食品中利巴韦林残留的问题,建立了直接竞争酶联免疫分析方法,快速高效地检测利巴韦林残留。采用活泼酯法将利巴韦林(RBV)与钥孔血蓝蛋白(KLH)偶联制备利巴韦林完全抗原(KLH-RBV),通过免疫新西兰大耳白兔得到多抗血清,经Protein A-Sepharose 4B凝胶柱纯化后得到纯化抗体,在此基础上建立直接竞争ELISA检测方法。该方法的IC50为10.84 ng/mL,最低检测限达0.002 ng/mL,对鸡肉、鸡肝、猪肉、猪肝等实际样品进行检测,加标回收率为80.23%~90.07%。采用高效液相色谱法进行验证,两种方法测定结果呈现良好的线性关系(R2≥0.99)。本文建立的方法灵敏度高,简便快速,适合现场大批量快速检测动物源食品中利巴韦林残留。Abstract: Aiming at the problem of ribavirin residues in animal source foods,a direct competitive enzyme-linked immunosorbent assay(dc-ELISA)was established to detect ribavirin residues rapidly and efficiently. Ribavirin(RBV)was conjugated with keyhole limpet hemoeyanin(KLH)by active ester method to prepare the ribavirin complete antigen(KLH-RBV). The antiserum was purified with Protein A-Sepharose 4B to prepare polyclonal antibody against ribavirin. A dc-ELISA was developed to detect and quantitate ribavirin with IC50 of 10.84 ng/mL and detection limit of 0.002 ng/mL. This method was applied to determine RBV in real chicken,chicken liver,pork and pig liver samples with satisfactory result of recovery rate ranged from 80.23% to 90.07%. HPLC was used to verify the results and the results of the two methods showed a good linear relationship. The method established was sensitive and the procedure of sample pretreatment was simple and quick. It was suitable for spot rapid detection of RBV residue in animal source foods.
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