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当前位置: 首页 > 文章 > 非洲猪瘟病毒p30蛋白单克隆抗体的制备及阻断ELISA检测方法的建立 中国兽医科学 2022,52 (1) 48-55
Position: Home > Articles > Preparation of monoclonal antibody against the African swine fever virus p30 protein and establishment of a blocking ELISA for detection of African swine fever virus Chinese Veterinary Science 2022,52 (1) 48-55

非洲猪瘟病毒p30蛋白单克隆抗体的制备及阻断ELISA检测方法的建立

作  者：
于浩洋;王彩霞;吴绍强;宋晓晖;刘晓飞;仇松寅;冯春燕;林祥梅
单  位：
中国检验检疫科学研究院动物检验与检疫研究所;中国动物疫病预防控制中心
关键词：
非洲猪瘟病毒;p30蛋白;真核表达;单克隆抗体;阻断ELISA
摘  要：
为建立一种能检测血清中非洲猪瘟病毒(African swine fever virus,ASFV)抗体的阻断ELISA方法,本研究以真核表达后纯化的ASFV p30重组蛋白作为包被抗原,以特异性单抗作为阻断抗体,经条件优化、特异性试验和敏感性试验,建立了一种检测血清中ASFV抗体的阻断ELISA方法。结果显示,纯化后的p30重组蛋白大小约为25 ku,建立的ASFV抗体阻断ELISA检测方法通过方阵试验优化后,最终确定了抗原最佳包被浓度为1μg/m L,待检血清最佳稀释度为1∶2,最佳封闭液为5%BSA,酶标抗体最佳稀释度为1∶20 000,阴阳性血清临界值为35.11%。本方法与PCV3灭活阳性血清、PRRSV灭活阳性血清、SVA灭活阳性血清、PRV灭活阳性血清均无交叉反应,具有较强的特异性。本方法检测ASFV阳性血清的敏感性可达1∶128。本试验建立的阻断ELISA方法具有良好的特异性和敏感性,在非洲猪瘟的临床检测中具有广泛的应用价值。
译  名：
Preparation of monoclonal antibody against the African swine fever virus p30 protein and establishment of a blocking ELISA for detection of African swine fever virus
作  者：
YU Hao-yang;WANG Cai-xia;WU Shao-qiang;SONG Xiao-hui;LIU Xiao-fei;QIU Song-yin;FENG Chun-yan;LIN Xiang-mei;Animal Inspection and Quarantine Institute,Chinese Academy of Inspection and Quarantine;China Animal Disease Control Center;
关键词：
African swine fever virus;;p30 protein;;eukaryotic expression;;monoclonal antibody;;blocking ELISA
摘  要：
To establish a blocking ELISA method for African swine fever virus(ASFV) antibody detecting in serum,the eukaryotic expressed ASFV p30 protein was used as a coating antigen,and the specific monoclonal antibody was used as blocking After optimization the antibody.condition,verification the specificity and sensitivity,an assay of blocking ELISA for detection of ASFV antibodies in serum was established.Results showed that the purified recombination protein p30 protein was 25 ku.The blocking ELISA method was optimized by square matrix test,and the optimal coating concentration of antigen was determined to be 1 μg/m L. The optimal dilution of the serum was 1 ∶ 2.The optimal blocking solution was 5% BSA,and the optimal dilution of enzyme-labeled antibodies was 1 ∶ 20 000.The prelimary cut-off value was 35.11%.The assay showed high specificity,and had no cross reaction with PCV3 positive serum,PRRSV positive serum,SVA positive serum and PRV positive serum.The sensitivity of the method for detecting ASFV positive serum was 1 ∶ 128.The established blocking ELISA method had good specificity and sensitivity,and would be widely used in detection of ASFV antibodies.

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