抗恩诺沙星抗血清的制备及其ELISA检测

Preparation of enrofloxacin antiserum and its determination by enzyme-linked immunosorbent assay(ELISA)

目的:建立快速、灵敏的恩诺沙星残留酶联免疫检测方法(ELISA)。方法:采用混合酸酐法,将恩诺沙星与牛血清白蛋白和卵清蛋白分别合成免疫抗原(Enrofloxac in-BSA)和包被抗原(Enrofloxac in-OVA)。通过背部多点免疫法免疫日本大白耳兔,制备抗恩诺沙星的特异性抗血清。结果:利用所获得的特异性抗血清,建立的恩诺沙星ELISA检测方法,其线性范围为5 ng.mL-1～2.5μg.mL-1(R2=0.9935),且与HPLC方法具有良好的相关性(R2=0.9892,n=9)。牛奶、大鼠血浆和尿液中的加样回收率分别为98.4%～105.8%,91.7%～101.2%,97.0%～110.3%。运用所建立的方法,对大鼠体内血浆及药品中恩诺沙星的含量进行了测定。结论:本研究所建立的检测恩诺沙星的ELISA方法具有样品前处理简单,灵敏度高及分析速度快等优点,适合用于恩诺沙星的体内分析及动物性食品中恩诺沙星的残留分析。

Objective：To develop a rapid and sensitive enzyme-linked immunosorbent assay（ELISA） for determination of enrofloxacin（ENR）.Method：Immunogen enrofloxacin-bovine serum albumin（Enrofloxacin-BSA） and coating antigen enrofloxcin-ovum albumin（Enrofloxacin-OVA） were prepared by a succinic anhydride method.By subcutaneous injection,the polyclonal antiserum was produced from big ear rabbits immunized with conjugates ENR-BSA.Result：After the assay procedure was optimized,the standard curve of ENR was established and the practical measuring range of the ELISA extended from 5 ng·mL-1to 2.5 μg·mL-1（R2=0.9935）.In a comparison of the assay results obtained by ELISA and HPLC,there is a good correlation between these two methods（R2=0.9892,n=9）.The proposed method has been satisfactorily applied for the determination of ENR in rat plasma and pharmaceuticals with recoveries in the range of 98.4% to 105.8% for milk sample,91.7% to 101.2% for rat plasma and 97.0% to 110.3% for rat urine.Conclusion：The experimental data indicated that in some extent the ELISA method was more suitable for high throughput and real-time ENR analysis in biological samples and animal food with lower detection limit,low background and no requirement of sample pre-treatment.