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| 双抗体夹心ELISA检测水牛γ-干扰素方法的建立及初步应用 | |
| 引用本文： | 徐贤坤,熊毅,刘棋,曾宪垠,龙剑明.双抗体夹心ELISA检测水牛γ-干扰素方法的建立及初步应用[J].畜牧与兽医,2012,44(6):10-16. |
| 作者姓名： | 徐贤坤 熊毅 刘棋 曾宪垠 龙剑明 |
| 作者单位： | 1. 广西动物疫病预防控制中心,广西南宁530001;四川农业大学生命理学院,四川雅安625014 2. 广西动物疫病预防控制中心,广西南宁,530001 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 |
| 基金项目： | 广西科技厅科技攻关项目 |
| 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。 |
| 关 键 词： | 结核病 IFN-γ ELISA ESAT-6/CFP-10 单克隆抗体 |
Establishment of double antibody sandwich ELISA for water buffalo interferon-gamma and its application in tuberculosis diagnosis |
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| XU Xian-kun , XIONG Yi , LIU Qi , ZENG Xian-yin , LONG Jian-ming.Establishment of double antibody sandwich ELISA for water buffalo interferon-gamma and its application in tuberculosis diagnosis[J].Animal Husbandry & Veterinary Medicine,2012,44(6):10-16. | |
| Authors: | XU Xian-kun XIONG Yi LIU Qi ZENG Xian-yin LONG Jian-ming |
| Institution: | 1.Guangxi Center for Animal Disease Prevention and Control,Nanning 530001,China; 2.College of Biology and Science,Sichuan Agricultural University,Ya’an 625014,China; 3.Bureau of Fishery Animal Husbandry & Veterinary of Guangxi Zhuang Autonomous Region, Nanning 530022,China; 4.College of Animal Science and Technology,Guangxi University,Nanning 530005,China) |
| Abstract: | 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. |
| Keywords: | water buffalo IFN-γ ELISA ESAT-6/CFP-10 monoclonal antibody(McAb) |
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