恩诺沙星在鸡肉组织中残留的ELISA检测方法研究

分别用N-羟基琥珀酰亚胺法和氯甲酸异丁酯法把恩诺沙星(ENR)与载体蛋白牛血清白蛋白(BSA)和鸡卵清蛋白(OVA)偶联制备免疫抗原和包被抗原,免疫新西兰大白兔得到ENR的多克隆抗体,建立了ENR间接竞争ELISA方法。结果表明:抗ENR血清效价达达1∶212以上,得到标准曲线的线性回归方程为Y=-0.2341X+0.1193(R2=0.9878),中值(IC50)为36 ng/mL,最低检测限(LOD)为10 ng/mL,标准曲线的线性范围为10~1000 ng/mL。批内变异系数为3.18%~7.64%,批间变异系数为9.69%~11.94%,鸡组织中的ENR的回收率为76.5%~89.42%。本试验建立的ELISA方法能够满足恩诺沙星兽药残留检测要求。

Establishment of an indirect competitive enzyme-linked immunosorbent assay for the residues of enrofloxacin in chicken tissue

Enrofloxacin(ENR) was coupled with the carrier protein bovine serum album(BSA)to prepare the immune antigen ENR-BSA by N-hydroxysuccinimide ester(NHS) method and with chicken ovalbumin(OVA) to prepare coating detection antigen by isobutyl chloroformate method.ENR-BSA as immunizing antigen was vaccinated to New Zealand albino rabbit in order to obtain the polyclonal antibody to enrofloxacin.An enzyme-linked immunosorbent assay(ELISA) method was established.The result indicated that specificity antibody titer reached above 1∶212.The regression equation,the 50% inhibition concentration(IC50),the limits of detect(LOD) and the linear range about the standard curve of enrofloxacin were Y=-0.234 1X+0.119 3(R2=0.987 8),36ng/mL,10ng/mL and 10～1 000ng/mL,respectively.The coefficients of variation for the Ci-ELISA were 3.18%～7.64% in intra-assay and 9.69%～11.94% in inter-assay.The recoveries in chicken ranged from 76.5% to 89.42%.The established ELISA method will satisfy the determination for the residues of enrofloxacin in chicken.