鸡肉中氟苯尼考ELISA检测方法的建立及应用  

Establishment and Application of Detecting the Residues of Florfenicol in Chicken Muscle by Indirect Competitive Enzyme-Linked Immunosorbent Assay

作　　者：孙法良[1] 刁有祥[1] 孙宁[1] 马艳芳[1] 纪巍[1] 

机构地区：[1]山东农业大学动物科技学院,山东泰安271018

出　　处：《中国农业科学》2009年第5期1813-1819,共7页Scientia Agricultura Sinica

基　　金：山东省自然科学基金项目(Y2007D32)

摘　　要：【目的】制备抗氟苯尼考(FFC)的高亲和力特异性抗体,建立检测氟苯尼考的间接竞争ELISA新方法。【方法】氟苯尼考分别与牛血清白蛋白(BSA)和人血清白蛋白(HSA)经混合酸酐法偶联,得到免疫抗原和包被抗原,其偶联比分别为14.4和14.7。用免疫原FFC-HS-BSA免疫新西兰大白兔获得了效价较高的特异性抗体。建立并优化了间接竞争ELISA检测方法。【结果】抗血清效价为1:102400。最佳包被抗原浓度为0.5μg·ml-1,最佳抗体工作浓度为1:6400,酶标二抗的工作浓度为1:20000。回归方程Y=-0.1516X+0.788(R2=0.9859),检测范围为0.18～500ng·ml-1,检出限为0.18ng·ml-1,批内和批间平均变异系数分别为4.86%和7.77%。交叉反应试验中,抗血清与FFC结构相似的氯霉素和甲砜霉素交叉反应率分别为0.094%和0.098%,与其它药物交叉反应率均小于0.01%,表明该方法具有很强的特异性。FFC以浓度0.18～500ng·g-1在鸡肉中添加,回收率为84.14%～106.1%,变异系数为2.1%～5.6%。【结论】成功获得了高效价、高特异性的抗FFC抗体,建立了鸡肉中氟苯尼考残留检测的ELISA方法。结果表明,所建立的检测方法具有灵敏、准确、简便、快速的特点。[ Objective ] The purpose of the study is to obtain special antibody and develop competitive indirect enzyme-linked immunosorbent assay （ciELISA） for florfenicol [Method] Florfenicol hapten was conjugated with the carrier proteins bovine serum albumin （BSA） as immunogen and with human serum albumin （HSA） as coating antigen, respectively, and the coupling ratio of FFC-HS-BSA was 14.4 and the coupling ratio of FFC-HS-HSA was 14.7. Polyclonal antibody against florfenicol was generated by immunizing rabbits with FFC-HS-BSA. [Result] The antiserum with high titer was 1 ： 102 400. The indirect competitive ELISA revealed that the optimal concentration of rabbit serum was 1 ： 6 400, the optimal concentration of coated antigen was 0.5 μg·ml^-1, and the optimal concentration of sheep anti-rabbit IgG was 1 ： 20 000. The regression equation was Y=- 0.1516X ＋ 0.788 （R^2=0.9859）, the lowest detection limit was 0.18 ng·ml^-1, the linear range was from 0.18 ng·ml^-1 to 500 ng·ml^-1, and the coefficients of variation of intra-assay and inter-assay were respectively 4.86% and 7.77%. The antiserum had cross reactivity of 0.094% and 0.098% with chloramphenicol and thiamphenicol, respectively, but had cross reactivity of less than 0.01% with others. The recoveries in chicken ranged from 84.14% to 106.1%. [Conclusion] The antibodies with high titer and specificity were produced successfully and a competitive indirect enzyme-linked immunosorbent assay （ciELISA） was developed for the detection of florfenicol residue in chicken. The results showed that the method is sensitive, accurate, convenient, and quick.

关 键 词：氟苯尼考 残留检测 间接竞争 ELISA 鸡肉 食品安全 

分 类 号：S859.84[农业科学—临床兽医学]