新霉素的直接竞争酶联免疫分析法

Direct Competitive Enzyme-linked Immunosorbent Assay (ELISA) for Neomycin

首先用碳二亚胺(EDC)法将新霉素(NEO)偶联于载体蛋白-卵清白蛋白(ovalbumin,OVA),合成包被原OVA-NEO,SDS-PAGE进行鉴定;用高碘酸钠法连接辣根过氧化物酶(horseradish peroxidase,HRP),制备酶标抗原NEO-HRP并建立直接ELISA检测方法。通过一系列参数的优化,包括包被溶液、封闭溶液、竞争时间、抗体稀释液、pH值、反应温度、显色时间等,最终得到其IC20(抑制率为20%时的标准溶液质量浓度)<1ng/mL、半数抑制量(IC50)为7.6ng/mL,线性方程为y=-0.2798x+0.7456,R2=0.991。直接ELISA总耗时只需大约1h。

The neomycin（NEO） was coupled with ovalbumin（OVA） to form a coating antigen NEO-OVA by EDC method and determined by SDS-PAGE.The NEO was coupled with horseradish peroxidase（HRP） by NaIO4 method to establish enzymatic tracer NEO-HRP and direct competitive ELISA.Then,the parameters coating liquid,pH,incubation time and incubation temperature of direct ELISA were optimized.The IC20 value（20% inhibitory concentration） was less than 1 ng/mL and IC50 value（50% inhibitory concentration） was 7.6 ng/mL.The linear equation was y =-0.2798x ＋0.7456,R2 = 0.991.The total consumption time of direct competitive ELISA was 1 h.