传染性法氏囊病病毒VP2单克隆抗体夹心ELISA检测试剂盒的研制  

Development of a sandwich ELISA based on the monoclonal antibodies against VP2 of infectious bursal disease virus

作　　者：刘静静[1,2] 王永山[2] 欧阳伟[2] 夏兴霞[2] 潘群兴[2] 王晓丽[2] 毕振威[2] 诸玉梅[2] 朱瑞良[1] 

机构地区：[1]山东农业大学动物科技学院,山东泰安271018 [2]江苏省农业科学院兽医研究所/农业部兽用生物制品工程技术重点实验室/国家兽用生物制品工程技术研究中心,江苏南京210014

出　　处：《中国兽医学报》2014年第4期530-535,共6页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(31272537);江苏省自然科学基金资助项目(BK2010471;BK2012787)

摘　　要：为研制传染性法氏囊病病毒(IBDV)快速检测试剂盒,用重组IBDV-VP2蛋白免疫BALB/c小鼠,制备免疫脾细胞,与SP2/0骨髓瘤细胞触合,获得3株稳定分泌抗VP2蛋白单克隆抗体(mAb)的杂交瘤细胞株,分别命名为1D11、2G8和2E5,抗体亚类分别为IgG1κ、IgG2bκ和IgG1κ。间接免疫荧光试验(IFA)证明,3株单抗均与VP2发生特异性反应。相加ELISA证明3株mAb识别VP2不同的抗原表位。在病毒中和试验中,1D11和2G8腹水对IBDV的中和效价分别为104和103,而2E5无中和活性。用亲和层析方法纯化1D11和2E5,分别作为包被抗体和标记抗体,建立了IBDV夹心ELISA检测方法,优化了试验条件,测定了其主要性能指标,对IBDV的最低检出量为102 TCID50/mL。用夹心ELISA、AGP和RT-PCR 3种方法同步检测8种试验样品,夹心ELISA与RT-PCR的检测结果一致,显著高于AGP方法。组装成的试剂盒,置于37℃保存7d、4℃保存6个月和-20℃保存24个月,其检测结果没有显著差异(P>0.05)。Three hybridomas secreting monoclonal antibodies（mAb） against VP2 of infectious bur- sal disease virus （IBDV）were established by fusing SP2/0 with spleen cells from BALB/c mice im- munized with virus-like-particle recombinant VP2 protein, designated as 1Dll, 2G8 and 2E5. The subtypes of immunoglobulin were IgG2bκ,IgG2bκ and IgGlκ, respectively. Indirect immunofluo- rescence assay（IFA） proved that the three mAb could react to VP2 specifically. Additivity ELISA revealed that three mAbs recognized spatially independent epitopes of VP2. Neutralization test in- dicated that the neutralization titer of 1Dll and 2G8 was 104 and 103 respectively, while 2E5 did not have neutralizing activity. A sandwich ELISA was established using 1Dll as capture antibody and 2E5 as enzyme-labeled antibody, and the detectable minimum of IBDV was 10^2 TCID50/mL. The de- tection kit of sandwich ELISA was developed and proved to be applicable to the clinical detection of IBDV.

关 键 词：传染性法氏囊病病毒(IBDV) VP2 单克隆抗体(mAb) 夹心ELISA 

分 类 号：S852.65[农业科学—基础兽医学]