摘要
为建立检测牛病毒性腹泻病毒(Bovine viral diarrhea virus,BVDV)抗体的间接ELISA方法,利用昆虫细胞真核表达系统成功表达E2蛋白,将纯化后的E2蛋白作为包被抗原,用方阵滴定方法对影响ELISA的各个因素进行优化,并进行特异性、敏感性和重复性试验。结果表明,在昆虫细胞中表达了BVDV E2蛋白,Western blot证实目的蛋白可与BVDV阳性血清发生特异性反应。ELISA优化结果显示,E2蛋白最佳包被浓度为0.5μg/mL,最佳封闭液为1%明胶,最佳血清稀释度为1∶400,最佳血清作用方式为37℃作用30 min,酶标抗体的最佳作用方式为1∶2000稀释、37℃作用30 min,最佳底物作用时间为室温20 min,阳性临界值为OD 450≥0.423。与血清中和试验法进行比较,总符合率为97.8%,板内和板间重复性试验的变异系数均小于10%。该方法与牛常见病毒阳性血清均无交叉反应。说明建立的间接ELISA抗体检测方法特异性、敏感性和重复性良好,可用于大批量样本的临床检测和流行病学研究。
In order to establish an indirect ELISA method for the detection of bovine viral diarrhea virus antibodies,E2 protein was successfully expressed in eukaryotic insect expression system.The purified protein was used as the coating antigen,and the factors of ELISA test were optimized by square matrix method.Specificity,sensitivity and repeatability tests were performed.The optimal conditions of ELISA were as follows:0.5μg/mL E2 antigen coated at 4℃for overnight,1%gelatin sealed at 37℃for 1 h,1∶400 dilution serum incubation at 37℃for 30 min,1∶2000 dilution enzyme labelled antibody incubation at 37℃for 30 min critical value was OD 450≥0.423.Compared with serum neutralization test,the total coincidence rate was 97.8%,and the coefficient of variation of intra-plate and inter-plate repeatability tests was less than 10%.This method had no cross reaction with common bovine virus positive serum.The results indicated that the established indirect ELISA antibody detection method has good specificity,sensitivity and repeatability,and can be used for antibody detection of large quantities of samples and epidemiological studies.
作者
刘丹
黄小洁
吴华伟
孙淼
陈延飞
秦义娴
侯力丹
薛麒
LIU Dan;HUANG Xiao-jie;WU Hua-wei;SUN Miao;CHEN Yan-fei;QIN Yi-xian;HOU Li-dan;XUE Qi(China Institute of Veterinary Drug Control,Beijing 100081,China)
出处
《动物医学进展》
北大核心
2024年第4期51-56,共6页
Progress In Veterinary Medicine
基金
中国兽医药品监察所公益性专项(GY202103)。
