| 牛布鲁氏菌间接ELISA抗体检测方法的建立 |
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| 引用本文： | 王 芳,冯 宇,张 阁,蒋 卉,朱良全,丁家波.牛布鲁氏菌间接ELISA抗体检测方法的建立[J].中国农业科学,2016,49(9):1818-1825. |
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| 作者姓名： | 王 芳 冯 宇 张 阁 蒋 卉 朱良全 丁家波 |
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| 基金项目： | 国家高技术研究发展计划（“863”计划）（2012AA101302）、北京市科技新星计划（XX2013099） |
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| 摘 要： | 【目的】建立高通量牛布鲁氏菌间接ELISA诊断方法,为科学高效诊断牛布鲁氏菌病提供技术手段。【方法】以提纯的猪布鲁氏菌S2株脂多糖(LPS)作为包被抗原,采用棋盘滴定法确定了抗原包被浓度、包被液、抗原包被时间、封闭液、样品稀释液、血清稀释倍数、兔抗牛酶标抗体稀释液、兔抗牛酶标抗体稀释浓度、最佳TMB底物反应时间等参数,初步建立了牛布鲁氏菌间接ELSIA方法。并用上述条件初步建立的方法对176份牛布鲁氏菌病阳性血清和132份牛布鲁氏菌病阴性血清进行检测。检测结果经SPSS17.0软件分析,构建受试者工作特征曲线(ROC)及曲线下面积,确定敏感性和特异性;通过Youden指数确定阴阳性判定的临界点。使用质控阴、阳性血清评价试剂盒的批内和批间重复性。用本研究建立的ELISA试剂盒及商品化试剂盒同时对临床上1 200份牛血清样本进行比对检测,比较其符合率。【结果】通过棋盘法确定的试剂盒中各组分的最佳条件为:抗原包被浓度为10μg·m L-1,最适包被液为碳酸盐缓冲液,最佳包被时间为2—8℃、16 h,最适血清稀释度为1﹕50,最佳封闭液为含有3%明胶的PBST,最佳样品稀释液为含有0.5%蔗糖的PBST,最佳兔抗牛酶标抗体稀释液为含有5%马血清的PBST,最佳兔抗牛酶标抗体工作浓度为1﹕20 000,最佳TMB底物反应时间为15 min。按上述条件建立的间接ELISA方法检测176份牛布鲁氏菌病阳性血清和132份牛布鲁氏菌阴性血清,并统计结果后使用SPSS17.0软件分析该方法受试者工作特征曲线(ROC)线下面积为0.98。最佳判定临界点为样本OD值/阳性OD值(S/P)×100%=20%。在此临界值上时,敏感性为97.7%,特异性为95.5%。对1 200份临床样本的比对检测显示,本研究建立的间接ELISA方法与进口商品化试剂盒的总符合率为96.25%。【结论】建立了特异性和敏感性良好的牛布鲁氏菌间接ELISA抗体检测方法。
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| 关 键 词： | 布鲁氏菌 间接ELISA 特异性 敏感性 |
| 收稿时间： | 2015-08-18 |
Development of Indirect ELISA for Antibody of Brucella abortus |
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| WANG Fang,FENG Yu,ZHANG Ge,JIANG Hui,ZHU Liang-quan,DING Jia-bo.Development of Indirect ELISA for Antibody of Brucella abortus[J].Scientia Agricultura Sinica,2016,49(9):1818-1825. |
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| Authors: | WANG Fang FENG Yu ZHANG Ge JIANG Hui ZHU Liang-quan DING Jia-bo |
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| Affiliation: | 1.Department of Inspection Technology Research, China Institute of Veterinary Drug Control, Beijing 100081;2.College of Animal Science and Veterinary Medicine, Shandong Agriculture University, Taian 271018, Shandong |
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| Abstract: | 【Objective】 To develop a high-throughput indirect ELISA diagnostic method for detecting antibodies against bovine brucella. 【Method】 The indirect ELISA method was developed by using the purified LPS from brucella S2 strains as the coating antigen, and confirming coating concentration of antigen, coating buffer, coating time, blocking buffer, sample diluent buffer, serum dilution, rabbit anti-bovine antibody diluent buffer, rabbit anti-bovine antibody diluting concentration, and the reaction time of substrate by chessboard titration. We detected 176 brucella positive samples and 132 negative samples using the developed method. The detecting result was analyzed by SPSS17.0. At the same time, ROC curve and area under the curve, sensitivity and specificity values, the critical value by Youden index was also constructed. The into-batch and inter-batch repeatability were also evaluated by using the positive and negative control serum. We detected the 1 200 clinical samples to compare the coincidence rate between bovine brucellosis indirect ELISA antigen kits and the commercial imported kits. 【Result】 The optimal conditions for each of the components of the indirect ELISA kit was described below. The coating concentration of antigen is 10 μg·mL-1, the coating buffer is carbonate buffer solution, the coating time is 16 hour in 2-8℃, the serum degree of dilution is 1﹕50, the blocking buffer is PBS containing 3% gelatin, and the sample dilution buffer is PBS containing 0.5%sucrose, the rabbit anti-bovine antibody diluent buffer is PBST containing 5% horse serum, the rabbit anti-bovine antibody diluting concentration is 1﹕20 000 and the reaction time of substrate is 15 minutes. The critical value is P%=20% (P%=OD450 sample/OD450 positive control×100%). In this critical value, the sensitivity and specificity values of the developed method is 97.7% and 95.5% respectively. By detecting the 1 200 clinical samples, it is showed that the coincidence rate was 96.25% between our indirect ELISA antigen kit and the commercial imported kit. 【Conclusion】 We established the indirect ELISA method for detecting bovine brucella antibody with good specificity and sensitivity. |
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| Keywords: | brucella indirect ELISA specificity sensitivity |
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