摘要
为建立检测非洲猪瘟病毒(ASFV)血清抗体的阻断ELISA方法,本研究对ASFV p72蛋白进行原核表达并纯化,将其免疫BALB/c小鼠并通过间接ELISA方法筛选到1株稳定分泌p72蛋白的杂交瘤细胞株3F8。以纯化的p72蛋白作为包被抗原,辣根过氧化物酶(HRP)标记的单克隆抗体(MAb)作为检测抗体,采用方正滴定法对反应条件优化后初步建立了检测ASFV的阻断ELISA方法。利用该方法检测133份ASFV阳性血清和73份ASFV阴性血清,通过ROC曲线分析确定临界值。结果显示,该方法的临界值为25.62%时,ROC的曲线面积最大,为0.9989,诊断敏感性和特异性分别为98.63%和98.5%。利用该方法检测ASFV、O型口蹄疫病毒(FMDV)、A型FMDV、猪繁殖与呼吸综合征病毒、猪流行性腹泻病毒、猪伪狂犬病毒、猪瘟病毒阳性血清,结果显示,除ASFV阳性血清外,其他阳性血清均为阴性,表明特异性强;利用该方法和商品化试剂盒对2倍倍比稀释(1∶4~1∶2048)的两种ASFV灭活阳性血清(P1、P2)检测,结果显示该方法检测结果与商品化试剂盒基本一致,敏感性高。利用该方法对3份灭活的ASFV阳性血清进行批内和批间的重复性检测,结果显示批内和批间重复性试验的变异系数均小于10%,重复性好。利用该检测方法和商品化试剂盒同时检测216份临床血清样品,两种方法检测结果符合率为99.07%。本研究所建立的阻断ELISA方法特异性强,敏感性高,重复性好,可用于ASFV抗体的检测,为非洲猪瘟流行病学调查及弱毒株疫苗抗体评价提供技术支撑。
The present study developed a p72 monoclonal antibody-based blocking ELISA for detecting African swine fever virus(ASFV)serum antibodies.P72 protein was expressed in prokaryotic cells,purified and was used to immunize BALB/c mice to generate p72 monoclonal antibody producing hybridoma cell lines.A hybridoma cell line designated as 3F8 that secreted p72protein monoclonal antibody(MAb)was produced and characterized.The purified p72 protein was used as the coating antigen and the HRP conjugated monoclonal antibody was used as the detection antibody.Based on the p72-3F8 MAb,a blocking ELISA was established for ASFV antibody detection.The newly assay was optimized through a series of reaction conditions and was evaluated for sensitivity,specificity,reproducibility and compliance.The ROC curve analysis revealed that the method's cut-off value was25.62%after testing 133 ASFV positive sera and 73 ASFV negative sera.When the cut-off value was 25.62%,the ROC curve had the largest area under curve accounting of 0.9989,and the sensitivity and specificity were 98.63%and 98.5%,respectively.Moreover,the ELISA did not show any cross-reaction with positive sera of O-type FMDV,A-type FMDV,porcine reproductive and respiratory syndrome virus,porcine epidemic diarrhoea virus,porcine pseudorabies virus and swine fever virus,and the sensitivity was equivalent to the commercial kit.The intra-batch and inter-batch coefficients of variation were less than 10%,indicating the assay is reproducible.Also,a high compliance rate of 99.07%was achieved when 216 serum samples were tested simultaneously using our assay and a commercial kit.Thus,we have successfully developed p72 monoclonal antibody-based blocking ELISA method for the detection of ASFV antibodies which is highly specific,sensitive,and reproducible.The newly established assay would provide a technical support for the epidemiological investigation of ASF and the evaluation of attenuated vaccine antibody.
作者
石正发
马源
袁洪
王由森
朱晓霞
车亮
李甲
羊倩倩
孙晓林
SHI Zheng-fa;MA Yuan;YUAN Hong;WANG You-sen;ZHU Xiao-xia;CHE Liang;LI Jia;YANG Qian-qian;SUN Xiao-lin(College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070,China;Lanzhou Biological Pharmaceutical Factory,Lanzhou 730100,China)
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2023年第5期488-493,547,共7页
Chinese Journal of Preventive Veterinary Medicine
基金
国家自然科学基金项目(32160843)。
作者简介
石正发(1986-),男,甘肃白银人,硕士,主要从事动物传染病病原学与流行病学研究;通信作者:孙晓林,E-mail:sunxl@gsau.edu.cn。
