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8μg/m L,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 mol/L PBS(p H7.2),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OD_(450nm)≥0.288判为阳性,反之判为阴性。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95.7%。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。
To develop an indirect enzyme-linked immunosorbent assay(ELISA)to detect antibodies againstinfectious bursal disease virus(IBDV),the recombinant VP2 protein of IBDV was used as the coated antigen.The best ELISA reaction system was determined by array titrimetric test.The final concentration of the coatedantigen was 8μg/m L and the dilution of the normal positive and negative serum was 1∶400.The confining liquidwas 10%horse serum,and the working concentration of conjugate antibody was 1∶15000.The best diluents was0.01 mol/L phosphate buffer solution(pH7.2).The best packing condition was 4℃overnight.The reactioncondition for serum samples and conjugate antibody was 37℃60 min.The substrate action time was 15 minOD_(450nm) of 0.288 was set as the positive cutoff.The result of specificity test,sensitivity test and duplicability testshowed that the method was specific,sensitive and repeatable.The serum samples were detected by theestablished ELISA method and the IDEXX-ELISA kit.The coincidence was 95.7%.The results showed that theindirect ELISA was rapid,simple,specific,sensitive,suggesting it was a valuable method for immunitysurveillance,prevention and control of infectious bursal disease.
作者
黄小洁
吴华伟
张兵
杨承槐
孔冬妮
侯力丹
杨飞
薛麒
刘丹
HUANG Xiao-jie;WU Hua-wei;ZHANG Bing;YANG Chen-huai;KONG Dong-ni;HOU Li-dan;YANG Fei;XUE Qi;LIU Dan(China Institute of Veterinary Drug Control,Beijing 100081,China;Key Laboratory of VeterinaryBioproduction and Chemical Medicine of the Ministry of Agriculture,Engineering and Technology Research Center for BeijingVeterinary Peptide Vaccine Design and Preparation,Zhongmu Institutes of China Animal Husbandry Industry Co.Ltd,Beijing 100095,China)
出处
《中国兽药杂志》
2022年第9期14-21,共8页
Chinese Journal of Veterinary Drug
