犬细小病毒抗体间接ELISA检测方法的建立

利用本实验室建立的昆虫细胞/杆状病毒表达系统表达犬细小病毒病毒样颗粒（CPV-VLPs）,采用硫酸铵沉淀、蔗糖密度梯度离心对表达的CPV-VLPs进行纯化,用电子显微镜、SDS-PAGE及Western-blotting方法检测纯化效果。以纯化的CPV-VLPs作为包被抗原建立CPV间接ELISA检测方法,对各反应条件进行优化并分析其特异性、敏感性、重复性。结果显示,CPV-VLPs经过纯化后纯度可达到95%以上;优化的ELISA最佳工作条件为：纯化抗原包被浓度为5.0mg/L,4℃包被过夜;1%BSA,37℃封闭2h;待检血清1∶40稀释,37℃孵育1.5h;HRP标记的酶标二抗1∶20 000稀释,37℃孵育1h;TMB室温避光显色30min;确定的血清阴性阳性临界值D450nm值为0.264。该方法可特异性检测犬细小病毒阳性血清,与犬瘟热、犬传染性肝炎、犬冠状病毒病、狂犬病阳性血清均不发生反应。该方法的敏感性为1∶640,批内重复试验变异系数小于6%,批间重复试验变异系数小于8%。42份临床血清样本的检测结果表明,与血凝抑制试验的符合率为90.48%。

Development of an indirect ELISA for detecting canine parvovirus antibody

The virus-like particles of canine parvovirus（CPV-VLPs）which were composed of VP2 protein were expressed and assembled successfully in insect baculovirus expression system.The CPV-VLPs were purified through（NH4）2SO4sedimentation and sucrose density gradient ultracentrifugation,and the purification results were analyzed by electron microscope,SDS-PAGE and Western-blotting.An indirect enzyme-linked immune sorbent assay（ELISA）was developed and optimized with the purified CPV-VLPs as coating antigen.After purification,the purity of CPVVLPs was higher than 95%.The optimal conditions of the indirect ELISA were determined as follows：the coating concentration of purified CPV-VLPs was 5.0mg/L at 4℃overnight;1%BSA as blocking agent at 37℃for 2h;the serum sample was diluted into 1∶40and incubated at 37℃for1.5h;the enzyme labeled antibody was diluted into 1∶20 000 at incubated at 37℃for 1h;the substrate TMB for ELISA being incubated for 30 min at room temperature.The threshold of positive and negative sera was 0.264 as determined by statistical analysis.The method could detect canine parvovirus antibodies specific and show no cross-reaction with the positive sera of the four dog diseases（CDV,ICHV,CCV and RABV）.The result showed that the sensitivity was 1∶640.Coefficient of variability percent（CV%）of inter-batch and extro-batch duplicative texts was less than 6%and 8%,respectively.The coincidence rate was 90.48% with HI in detecting 42 clinical serum samples.Results showed that the indirect ELISA procedures had good repeatability,high sensitivity and specificity and suited for detecting clinical samples in large-scale screening.It provides technical supports for epidemiological survey,antibody surveillance and vaccine efficacy evaluation.