摘要
利用大肠杆菌表达的牛病毒性腹泻病毒(BVDV)重组E2蛋白作为抗原,建立了检测BVDV血清抗体的间接Dot-ELISA。最佳工作条件为:E2蛋白抗原的最适包被质量浓度2.0 mg/L(2.0 ng/点),酶标抗体的工作浓度为1∶500,血清稀释度为1∶100,3%明胶-TBS作为封闭液,封闭45 min效果最佳。通过重复性试验、交叉试验、特异性试验和稳定性试验等证明,该方法重复性好、特异性强、灵敏度高;与用BVDV全病毒为抗原的IDEXX ELISA试剂盒相比,特异性96.67%,灵敏度90%,符合率95%。应用建立的检测方法对河北省4个奶牛场采集的178份腹泻奶牛血清样本进行检测,结果BVDV血清抗体阳性率40.45%,与IDEXX ELISA试剂盒的检出率无明显差异。
An indirect Dot-ELISA for detection of antibodies against BVDV was established by the recombinant E2 protein antigen expressed in E. coli prokaryotic expression system. The optimal coating concentration of E2 recombinant protein was 2.0 mg/L. The dilution of serum sample was 1 ： 100 and optimal concentration of HRP labeled antibody IgG was 1：500. The optimal blocking reagent was 3G glutin-TBS and blocking 45 min. Compared with the routine IDEXX ELISA test kit with the whole virus,its specificity and sensitivity of the ELISA were 96. 67% and 90%, respectively. In addition, 178 serum samples collected from 4 cow farms in Hebei province were detected by the developed assay and the IDEXX BVDV Antibody Test Kit,it was showed that the BVDV antibody positive rate was 40.45 %. The result demonstrated that the established method was specificity, sensitivity, reiterativity, whereas it ＇ s results were easily interpreted without an ELISA reader. The method was practical for detection of BVDV and antibody surveillance.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2009年第9期1093-1096,共4页
Chinese Journal of Veterinary Science
基金
国家"十一五"科技支撑计划资助项目(2006BAD04A10-4)
河北省重大技术创新专项计划资助项目(07227146Z-4)
河北农业大学科研基金资助项目
