牛奶中替米考星间接竞争酶联免疫吸附检测法的建立

本研究通过棋盘法优化替米考星抗体、包被原浓度,并考察了最佳包被条件,最佳反应温度、时间和酶标二抗最佳反应时间等,建立了替米考星残留的间接竞争酶联免疫吸附试验(ELISA)并应用到牛奶样本的检测。建立的间接竞争ELISA方法半数抑制浓度(IC₅₀)为2.1 ng/mL,牛奶中替米考星的最低检测限(LOD)为2 μg/L。在5、10和20 μg/L添加浓度下,回收率为79.2%~90.1%,变异系数不高于9.0%。与其他常见的大环内酯类药物无交叉反应。本研究建立的替米考星间接竞争ELISA方法灵敏度达到了残留限量标准要求,为开发可用于牛奶中替米考星的快速检测试剂盒奠定基础。

Establishment of Indirect Competitive Enzyme-linked Immunosorbent Assay for Tilmicosin in Milk

An indirect competitive enzyme-linked immunosorbent assay (ELISA) for detecting tilmicosin residue in milk was developed and applied to milk samples after the determination of dilution of coating antigen and antibody to timicosin by using checkerboard method,and the optimization of coating condition,reaction time and temperature,and reaction time of enzyme-labled secondary antibody.The results showed that the 50% inhibition concentration (IC₅₀) of the indirect competitive ELISA was 2.1 ng/mL and the limit of detection (LOD) of tilmicosin in milk was 2 μg/L.The recoveries of tilmicosin added in milk at 5,10 and 20 μg/L ranged from 79.2% to 90.1% with the coefficients of variation (CV) not higher than 9.0%.The indirect competitive ELISA would not react with other common macrolide drugs.The sensitivity of indirect competitive ELISA developed in the study complied to the maximum residue limit of tilmicosin set by China,and laid the foundation for the test kit of the rapid detection of tilmicosin in milk.