直接竞争ELISA检测氯霉素残留的方法建立及初步应用  

Development of an direct competitive ELISA for screening of chloramphenicol residue and preliminary application

作　　者：李晓云[1] 石德时[1] 王喜亮[1] 熊宁[1] 刘百红[1] 张道宏[1] 毕丁仁[1] 

机构地区：[1]华中农业大学动物医学院,湖北武汉430070

出　　处：《中国兽医学报》2007年第5期723-727,共5页Chinese Journal of Veterinary Science

基　　金："十五"国家食品安全重大科技专项基金资助项目(2001BA804A18-01);武汉市科技攻关计划资助项目(20022002062)

摘　　要：用混合酸酐法合成氯霉素的CAP抗原,用过碘酸钠氧化法合成CAP酶标记抗原,用ELISA方法对合成的CAP抗原、CAP酶标记抗原进行鉴定表明其与抗CAP单克隆抗体具有特异性反应。在此基础上建立了包被二抗的直接竞争ELISA方法。特异性试验结果表明,CAP琥珀酸酯、CAP琥珀酸钠盐的交叉反应率分别为107%、106.4%;甲砜霉素、氟甲砜霉素与CAP的交叉反应率小于0.016%;与其他结构类似物间未见交叉反应。该方法对CAP的检测限可达到0.1μg/L,IC50为11.6μg/L,线性范围0.1～100μg/L,批内变异系数小于11.5%,批间变异系数小于19.6%。对猪肉、鸡肉、蜂蜜分别添加1.0、3.0、10.0、30.0μg/L CAP标准品,回收率61.0%～102.0%,变异系数为6.6%～15.8%;对猪肉、鸡肉、蜂蜜中CAP的最低检测限分别为0.48、0.42、1.20μg/kg。A seusititive direct competitive enzyme-linked immunosobent assay（ELISA） was developed for the detection of the chloramphenicol（CAP） residues, The succinyl CAP was coupled to bovine serum albumin （BSA） with use of the mixed anhydride reaction. A horseradish peroxidase（HRP）-labeled conjugated antigen was synthesized by the sodium periodate reaction. The direct competitive ELISA was conducted by simultaneously incubating CAP and CAP-BSA-HRP with selided anti-CAP monoclonal antibody over a second antibody. The detection limit for CAP was 0.1 μg/L.The intra-and inter-assay coefficient of variation was 〈 19.6% and 〈 11.5 % in the range of 0.1 to 100μg/L, respectively. The cross reactivities with succinyl chloramphenicol and chloramphenicol sodium succinate was 107 % and 106.4%, with thiamphenicol and florphenicol was 〈 0.016 %, with L-phenylalanine and L-tyrosine was negligible. Recoveries from porcine muscle, chicken muscle, honny was in the range of 61.0%-102.0%, the coefficient of variation was in the range of 6.6%-15.8%, when 1.0,3.0, 10.0,30.0μg/L CAP was spiked, respectively. The detection limit for CAP from porcine muscle, chicken muscle, honny was 0.48,0.42,1.20 μg/kg, respectively.

关 键 词：氯霉素 药物残留 酶标抗原 直接竞争ELISA 

分 类 号：S859.796[农业科学—临床兽医学]