双抗体夹心ELISA检测ASFV抗原方法的建立与评价  

Establishment and evaluation of a method for detection of ASFV antigen by double-antibody sandwich ELISA

作　　者：李其炫 岳慧贤 蒋依倩 张艳艳[1] 陈腾 王述超 张守峰 扈荣良 LI Qixuan;YUE Huixian;JIANG Yiqian;ZHANG Yanyan;CHEN Teng;WANG Shuchao;ZHANG Shoufeng;HU Rongliang(Changchun Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Changchun 130122,China)

机构地区：[1]中国农业科学院长春兽医研究所,吉林长春130122

出　　处：《中国兽医学报》2024年第8期1579-1584,1592,共7页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(U19A2039)。

摘　　要：非洲猪瘟(African swine fever,ASF)是一种猪的急性、热性、高度接触传染性出血性传染病,具有高度致死性,给养猪业造成巨大经济损失。ASF的实验室确诊目前多采用特异性核酸扩增技术,但该方法受多种因素影响,尤其是环境中核酸扩增产物的污染给扩增结果判定带来诸多困扰。为了对ASF临床样品进行高通量诊断检测,本研究以ASF阳性猪血清IgG为捕获抗体,HRP标记的p72单克隆抗体为检测抗体,以细胞培养的非洲猪瘟病毒(African swine fever virus,ASFV)绘制标准曲线,建立了双抗体夹心ELISA抗原检测方法,并评价了其敏感性、特异性和重复性,进一步比较了不同因素对抗原检测的影响。结果显示,本研究建立的夹心ELISA检测方法对重组蛋白和ASFV的最低检测限分别为0.1 mg/L和10^(3.7)TCID_(50)/mL,与5种常见致病性猪源性病毒均不发生交叉反应,试验批间变异系数小于10%;与实时荧光定量PCR对临床血液病毒检测的总符合率为92%(23/25)。在对灭活ASFV抗原的检测中证明,BEI灭活会显著降低ASFV抗原检测的灵敏度,铝胶佐剂和纳米佐剂会干扰抗原的检出。综上,本研究建立的双抗体夹心ELISA抗原检测方法具有良好的特异性、灵敏度和重复性,与qPCR法有良好的一致性,为ASFV临床诊断以及灭活ASFV抗原评价提供了一种高通量检测方法。African swine fever(ASF)is an acute and highly pathogenic hemorrhagic disease of pigs,causing huge economic losses to pig industry.In order to quantitatively detect clinical samples of ASF and inactivated ASFV antigens,the IgG of ASF positive serum was used as capture antibody and the HRP-labeled p72 monoclonal antibody was used as detecting antibody.The standard curve was drawn with the cell-cultured ASFV,and a sandwich ELISA detection of antigen was established.The specificity,sensitivity and stability of the method were evaluated.The effects of different inactivation methods and adjuvant addition on antigen detection were further evaluated.The results showed that the minimum detection limits of the recombinant protein and the ASFV were 0.1 mg/L and 10^(3.7)TCID_(50)/mL,respectively.There was no cross-reaction with five common porcine pathogenic viruses,and the coefficient variations between batches was less than 10%.The total coincidence rate with real-time fluorescence quantitative PCR was 92%(23/25).The sensitivity of antigen detection was significantly reduced when antigen was treated by BEI inactivation,and the detection results were severely interfered by aluminum adjuvant and nano-adjuvant.In summary,the sandwich ELISA antigen detection method established is specific,sensitive,and repeatable,with a good consistency to the qPCR method,which provides an effective clinical diagnostic method for ASFV antigen.

关 键 词：非洲猪瘟病毒 高免血清 单克隆抗体 夹心ELISA 

分 类 号：S858.28[农业科学—临床兽医学] S852.65[农业科学—兽医学]