氯霉素竞争ELISA检测试剂盒的研制

目的应用抗氯霉素多克隆抗体,建立竞争ELISA分析方法,研制氯霉素残留快速检测试剂盒,并对其性能进行测定。方法选用抗氯霉素高特异性抗体,直接包被抗体后,将酶标记氯霉素和氯霉素标准品或样品加入预先包被了特异性抗体的孔内,建立直接竞争ELISA检测试剂盒。结果所选用的抗体对氯霉素亲和力高,与链霉素、四环素、土霉素、青霉素4种抗生素及克伦特罗的交叉反应率均小于0.01%;抗体的特异性强;检测质量浓度为50、10.5、0.5mg/ml样本时,平均变异系数为11.4%;对氯霉素残留检测的标准曲线为y=-14.843logx+56.692,R2=0.9965,线性范围为0.02～64.0ng/ml,检测限IC10为0.451ng/ml,灵敏度IC50为2.82ng/ml,样品的加标回收率为72%～113%。结论试剂盒检测的准确性和重现性较好,可用于批量虾肉、鸡肉、牛奶等样品中氯霉素残留的检测。

DEVELOPMENT OF A CHLORAMPHENICOL COMPETITIVE ELISA TEST KIT

Objective To develope a direct competitive enzyme-linked immunosorbent assay （ELISA） test kit with coated polyclonal antibodies （PAbs） for the determination of chloramphenicol （CAP） residue in food of animal origin,and to evaluate its performance. Methods Enzyme labled CAP （enzyme conjugate） and CAP standard or sample were added to the precoated wells which were coated by the specific antibody （rabbit anti-CAP）. Results PAbs had high affinity and specificity to CAP.The cross reaction rates to streptomycin,terramycin,cyclomycin,penicillin,and clenbuterol were less than 0.01%.The average variation coefficient was 11.4% when the sample concentration was 50,10.5,0.5 mg/ml. A standard curve was obtained and the regression equation was y =-14.843logx＋56.692,R^2=0.9965. The linear range of the CAP immunoassay was from 0.02 to 64.0 ng/ml, with the detection limit （IC10） of 0.451 ng/ml, and sensitivity （IC50） 2.82 ng/ml. The recoveries of CAP ranged from 72% to 113%. Conclusion The ELISA test kit showed good repeatability and accuracy, could be used for the screening analysis of residual CAP in a large number of samples such as shrimp,chicken, and milk, etc.