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

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IC_(50)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(R^2≥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。

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(KLHRBV).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.