非洲猪瘟病毒抗体竞争ELISA检测方法的建立  

Establishment of a Competitive ELISA Method for Detection of African Swine Fever Virus Antibodies

作　　者：李海 宋煜 吴汉 李桂珍 陈纯 李海涛 邵敏 王晓虎 扈荣良 周鹤峰 LI Hai;SONG Yu;WU Han;LI Guizhen;CHEN Chun;LI Haitao;SHAO Min;WANG Xiaohu;HU Rongliang;ZHOU Hefeng(School of Biological Engineering,Zhuhai Campus,Zunyi Medical University,Zhuhai 519000,China;Institute of Animal Health,Guangdong Academy of Agricultural Sciences,Guangzhou 510000,China;Institute of Military Veterinary Medicine,Academy of Military Medical Sciences,Changchun 130062,China)

机构地区：[1]遵义医科大学珠海校区生物工程学院,珠海519000 [2]广东省农业科学院动物卫生研究所,广州510000 [3]军事科学院军事医学研究院军事兽医研究所,长春130062

出　　处：《病毒学报》2024年第4期802-810,共9页Chinese Journal of Virology

基　　金：贵州省科技计划项目(项目号:黔科合支撑[2023]一般259),题目:新型冠状病毒(2019⁃nCoV)抗原快速检测试剂盒的研制。

摘　　要：利用非洲猪瘟病毒(African swine fever virus,ASFV)p30、p72和MGF360⁃12L重组蛋白及其酶标单克隆抗体(Monoclonal antibodies,mAbs),建立一种快速、准确检测ASFV抗体的竞争ELISA法。分别利用纯化的ASFV p30、p72和MGF360⁃12L重组蛋白免疫BALB/c小鼠,通过聚乙二醇(PEG)诱导融合获得阳性杂交瘤细胞,腹水经辛酸⁃硫酸铵法纯化得到相应的mAbs,并使用HRP标记,建立竞争ELISA法并优化反应条件,对该法的临界值、敏感性、特异性和符合率进行评估。制备的p30、p72和MGF360⁃12L重组蛋白纯度良好,融合后的细胞株经Western blot鉴定具有良好的反应性和特异性,筛选的杂交瘤细胞株均为IgG类抗体,纯化后的mAbs符合抗体IgG分子量大小,经HRP标记后活性较高。竞争ELISA优化结果显示,血清与酶标mAbs最佳稀释倍数分别为1∶8和1∶3200,临界值判定PI≤14.71%为阴性,PI≥18.97%为阳性,在14.71%≤PI≤18.97%期间为可疑,试剂盒敏感性为1∶128,经检测临床样品表明符合率极高。成功建立了ASFV抗体的快速检测方法,为ASFV的预防和大规模血清样品检测提供有效监测工具,并为ASFV疫苗免疫效果评估提供有效技术手段。To establish a competitive ELISA method for rapid and accurate detection of AFSV antibody by using recombinant proteins of African swine fever virus p30,p72 and MGF360⁃12L,and their enzyme⁃labeled monoclonal antibodies.BALB/c mice were immunized with purified recombinant proteins of ASFV p30,p72 and MGF360⁃12L,respectively,and positive hybridoma cells were obtained by conventional fusion method.The corresponding monoclonal antibodies were purified by caprylic acid⁃ammonium sulfate method,and HRP labeled monoclonal antibodies were prepared.A competitive ELISA method was established and the reaction conditions were optimized.The critical value,sensitivity,specificity and coincidence rate of this method were evaluated.The purified recombinant proteins p30,p72 and MGF360⁃12L were of good purity,and the fused cell lines were identified by Western blot with good reactivity and specificity.All the hybridoma cell lines screened were IgG antibodies,and the purified monoclonal antibodies met the molecular weight of antibody IgG.The monoclonal antibodies were highly active after HRP labeling.The optimized results of competitive ELISA showed that the dilution of serum and enzyme⁃labeled monoclonal antibody were 1∶8 and 1∶3200,respectively.The critical value of PI≤14.71%was negative,PI≥18.97%was positive,and it was suspicious when 14.71%≤PI≤18.97%.The sensitivity of the kit was 1∶128,and the coincidence rate was very high.The rapid detection method of ASFV antibody has been successfully established,which provides an effective monitoring tool for the prevention of ASFV and the detection of large⁃scale serum samples,and provides an effective technical means for the evaluation of the immune effect of ASFV vaccine.

关 键 词：非洲猪瘟病毒 单克隆抗体 竞争ELISA P30 p72 MGF360 

分 类 号：Q819[生物学—生物工程]