双抗体夹心ELISA检测水牛γ-干扰素方法的建立及初步应用  

Establishment of double antibody sandwich ELISA for water buffalo interferon-gamma and its application in tuberculosis diagnosis

作　　者：徐贤坤[1,2] 熊毅[1] 刘棋 曾宪垠[2] 龙剑明[4] 

机构地区：[1]广西动物疫病预防控制中心,广西南宁530001 [2]四川农业大学生命理学院,四川雅安625014 [3]广西水产畜牧兽医局,广西南宁530022 [4]广西大学动物科学技术学院,广西南宁530005

出　　处：《畜牧与兽医》2012年第6期10-16,共7页Animal Husbandry & Veterinary Medicine

基　　金：广西科技厅科技攻关项目(桂科攻0719004-3F)

摘　　要：纯化2株针对水牛γ-干扰素的单克隆抗体,其中1株进行HRP标记作为检测抗体,另1株单抗作为捕获抗体,建立检测水牛γ-干扰素的双抗体夹心ELISA(DAS-ELISA)。通过对封闭液、抗体稀释液等条件进行优化,经过方阵滴定试验确定捕获抗体的最佳浓度为625 ng/mL,检测抗体的工作效价为1∶100;所建立的DAS-ELISA方法检测灵敏度25 ng/mL,批内变异系数为0.52%～6.86%,批间变异系数为5.6%～7.07%。采集60头单次皮内注射结核菌素的水牛肝素钠抗凝全血,利用牛结核杆菌特异性抗原rHIS-CFP10/ESAT-6融合蛋白体外刺激外周血淋巴细胞释放IFN-γ,用所建立的夹心ELISA检测所有样本,与皮内变态反应试验比较,结果显示,自制夹心ELISA方法检测水牛结核病的敏感性为62%,特异性为87%,符合率为75%,表明可应用于水牛结核病的检测。Double antibody sandwich ELISA(DAS-ELISA) for IFN-γ in water buffalo was developed.In this method,two mice ascites McAbs against water buffalo interferon-γ were purified by ammonium sulfate precipitation(AS),and McAbs 1E4 was used as a capture antibody and HRP-labeled McAbs 4A11 as a detection antibody.The optimal reaction conditions for the assay were determined.The result indicated that the optimal concentration of capture antibody was 625ng/mL,the working concentration of HRP-labeled 4A11 was 1∶100,and the detection limit of the sandwich ELISA for IFN-γ was 25ng/mL.Compared with tubculin skin test(TST),the diagnosis specificity,sensitivity and coincidence test of DAS-ELISA were 62%,87% and 75%,respectively.Therefore,this DAS-ELISA assay will be a useful tool in the bovine tuberculosis diagnosis in water buffalo.

关 键 词：结核病 IFNγ- ELISA ESAT-6/CFP-10 单克隆抗体 

分 类 号：S855.2[农业科学—临床兽医学]