猪链球菌抗体GDH重组蛋白PPA-ELISA检测方法的建立  

Development of PPA-ELISA for the detection of antibodies against Streptococcus suis using recombinant GDH protein as antigen

作　　者：夏小静[1,2] 李书光[1] 王金良[1] 陈金龙 谢金文[1] 姜世金[2] 沈志强[1,3] 

机构地区：[1]山东省滨州畜牧兽医研究院,山东滨州256600 [2]山东农业大学动物科技学院,山东泰安271018 [3]山东绿都生物科技有限公司,山东滨州256600

出　　处：《中国兽医科学》2012年第9期932-937,共6页Chinese Veterinary Science

基　　金：山东省滨州畜牧兽医研究院自主创新基金项目(201002);山东绿都生物科技有限公司技术创新项目(201003)

摘　　要：以纯化的猪链球菌重组谷氨酸脱氢酶(GDH)蛋白作为包被抗原,酶标葡萄球菌A(PPA)蛋白为二抗,建立了检测猪链球菌抗体的间接PPA-ELISA诊断方法。经方阵滴定确定了抗原的最适包被浓度为12.5mg/L,血清样品的最佳稀释倍数为1∶80,PPA-ELISA阳性反应的临界值为D450nm≥0.378,交叉反应试验结果表明,该重组抗原与其他常见的6种猪病的阳性血清不发生交叉反应,批内和批间重复试验的变异系数均小于10%。经与武汉科前动物生物制品有限责任公司的商品化猪链球菌抗体检测试剂盒比较,二者的符合率为96.7%;对320份血清进行猪链球菌抗体检测,阳性检出率为73.1%。试验证明,所建立的PPA-ELISA检测法重复性好、特异性强、灵敏度高,为大规模地进行猪链球菌病的流行病学调查和血清学诊断提供了有效的技术手段。An indirect enzyme-linked immunosorbent assay was developed for the detection of specific antibody against Streptococcus suis by using the recombinant expressed glutamate dehydrogenase（rGDH） protein as a coating antigen,with horseradish peroxidase labeled SPA（PPA） as the second antibody.Result of chessboard titration test showed that the optimal concentration of coating antigen was 12.5 mg/L.The optimal dilution of serum sample was 1∶80 in the cross assay.The cut-off was chosen as an D450 nm≥0.378 for positive response.The specificity test indicated that rGDH was highly specific and had no cross-reaction with the positive sera of other swine diseases.The variation coefficient of intra-batch and the inter-batch in the repeatability tests was less than 10%.Compared with commercial S.suis antibody test kit,the PPA-ELISA approach showed 96.7% coincidence with the commercial kit based on 120 clinical sera detection results.A total of 320 sera were detected and 73.1% of them were positive.The experiments then confirmed that the PPA-ELISA was a convenient,rapid,more sensitive and specific diagnostic method.The present study provided a new tool for the large-scale epidemiological surveys and serological diagnosis of streptococcal infection.

关 键 词：猪链球菌 酶标葡萄球菌A蛋白-酶联免疫吸附试验 谷氨酸脱氢酶 

分 类 号：S852.611[农业科学—基础兽医学]