鸡传染性法氏囊病病毒(IBDV)抗体间接ELISA检测方法的建立

Development of an Indirect ELISA to detect Antibodies Against Infectious Bursal Disease Virus

关键词:  鸡传染性法氏囊病病毒  抗体  VP2  间接ELISA

Key words:  Infectious Bursal Disease virus  Antibody  VP2  Indrect ELISA

摘要:

为建立鸡传染性法氏囊病病毒(IBDV)抗体的间接ELISA检测方法,应用原核表达的IBDV VP2蛋白作为包被抗原,经方阵试验确定间接ELISA试验的最佳反应条件：包被抗原的浓度为8 μg/mL,标准阴、阳性血清的稀释度为1:400,封闭液选用10 %马血清,酶标抗体最佳稀释倍数为1:15000,最佳抗原稀释液为0.01 M PBS(PH 7.2)；抗原最佳包被条件为4 ℃过夜,待检血清和酶标二抗反应条件为37 ℃ 60 min,底物作用时间为15 min。待检血清的OD450nm≥0.288判为阳性,反之判为阴性。特异性试验、敏感性试验和重复性试验结果显示,该方法的特异性好、敏感高、重复性好。用建立的间接ELISA方法与商品化IDEXX-ELISA试剂盒对临床血清样品进行检测,符合率为93.5 %。该方法的建立为检测IBDV抗体提供了一种安全、特异、敏感、方便经济的检测方法,为鸡传染性法氏囊病免疫监测和预防控制提供了科学的技术手段。

Abstract:

To develop an indirect enzyme-linked immunosorbent assay(ELISA) to detect antibodies against Infectious Bursal Disease virus(IBDV),the recombinant VP2 protein of IBDV were used as the coated antigen. The best ELISA reaction system was determined by array titrimetric test. The final concentration of the coated antigen was 8 μg /mL and the dilution of the normal positive and negative serum was 1:400. The confining liquid was 10 % horse serum, and the working concentration of conjugate antibody was 1:15000. The best diluents was 0.01 mole phosphate buffer solution . The best packing condition was 4℃overnight. The reaction condition for serum samples and conjugate antibody was 37℃60 min. And 15 min were needed for chromato-geic substrate to display. OD450nm of 0.288 was set as the positive cutoff. The result of specificity test, sensitivity test and duplicability test showed that the method was specific, sensitive and repeatable. The serum samples were detected by the established ELISA method and the IDEXX-ELISA kit. The coincidence was 93.5 % . The results showed that the indirect ELISA was rapid, simple, specific, sensitive, suggesting it is a valuable method for immunity surveillance, prevention and control of infectious bursal disease.