传染性法氏囊病病毒VP2结构蛋白抗体ELISA检测方法的建立  

Establishment of an ELISA for the detection of IBDV antibody by using the viral specific VP2 structural proteins

作　　者：王晓丽[1] 王永山[1] 欧阳伟[1] 夏兴霞[1] 潘群兴[1] 毕振威[1] 诸玉梅[1] WANG Xiao-li;WANG Yong-shan;OUYANG Wei;XIA Xing-xia;PAN Qun-xing;BI Zhen-wei;ZHU Yu-mei(Institute of Veterinary Medicine/Key Laboratory of Veterinary Biological Engineer-ing and Technology,Ministry of Agriculture/National Center for Engineering Research of Veterinary Bio-products,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China)

机构地区：[1]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

出　　处：《中国兽医学报》2018年第10期1864-1871,共8页Chinese Journal of Veterinary Science

基　　金：国家重点研发计划资助项目(2016YFD0500800);国家自然科学基金资助项目(31572504);江苏省自然科学基金资助项目(BK20151366);江苏省农业科技自主创新资金资助项目(EX[16]1052)

摘　　要：为建立敏感、特异、稳定的传染性法氏囊病病毒（IBDV）VP2结构蛋白抗体ELISA检测方法,本试验以杆状病毒表达系统表达的重组IBDV-VP2结构蛋白为抗原,通过包被VP2单克隆抗体捕捉VP2结构蛋白的形式,建立鸡血清VP2结构蛋白抗体ELISA检测方法（VP2-ELISA）。条件优化结果显示：单克隆抗体最适包被质量浓度为3mg/L,VP2最佳稀释度为1∶20,夹心ELISA效价1∶12.8,被捡血清的稀释度为1∶400。该方法与禽流感、鸡新城疫、鸡马立克氏病、鸡传染性喉气管炎病毒及杆状病毒阳性血清无交叉反应,对琼扩效价为1∶32的IBDV阳性血清的最低检出量为1∶12 800,ROC曲线确定的D450nm临界值为0.236,组内、组间重复性试验变异系数均小于10%。用本试验建立的VP2-ELISA方法与进口IBDV-ELISA抗体试剂盒平行检测100份背景已知的临床鸡血清样品,总符合率VP2-ELISA（97%）高于进口ELISA试剂盒（96%）。本试验为进一步研制鸡传染性法氏囊病病毒VP2结构蛋白抗体ELISA检测试剂盒奠定基础。To establish an effective ELISA method for the detection of the IBDV VP2 antibody,the baculovirus expressed recombinant IBDV-VP2 proteins were used as an capture antigen and an EI.ISA assay was developed for the detection of IBDV infection in chicken. The working conditions of this assay were optimized according to the following protocol, the coating concentration of cap ture antibody was 3 mg/L,the optimized dilution for VP2 antigen was 1 ： 20 （1 ： 12.8 for sand wich ELISA）,and the optimized dilution for the sample serum was approximately 1 ： 400. There were no visible cross reaction to the antibodies against AIV, NDV, MDV, AI.TV and BacV. The detection efficiency of this assay was verified to react to the IBDV positive serum （AGP titer was 1 ：32） ,which was 1 ： 12 800. The cut-off D450 nm value was 0. 236 according to the ROC curve. The intra- and inter-assay coefficients of variability between experiments were all less than 10%. Compared the detection efficiency of this method with the imported commercialized detection Kit by measuring 100 clinical serum samples, our results showed that the coincidence rates from VP2- ELISA Kit was 97%, compared with the 96% that of imported commercialized Kit. This study provided a solid experimental evidence showed that to using the VP2 ELISA Kit for detection of antibody seems to be a good approach detection of IBDV infection.

关 键 词：传染性法氏囊病病毒 VP2 单克隆抗体 ELISA 

分 类 号：S852.65[农业科学—基础兽医学]