DHAV-33D蛋白单克隆抗体制备及其DAS-ELISA检测方法的建立  

Preparation of monoclonal antibodies against DHAV-33Dprotein and establishment of DAS-ELISA detection method

作　　者：冯旭东 伍琳楠 陈天泽 赵梦茹 刘言言 周学慧 杨晓伟 郁蕾 张立武 赵光伟 FENG Xudong;WU Linnan;CHEN Tianze;ZHAO Mengru;LIU Yanyan;ZHOU Xuehui;YANG Xiaowei;YU Lei;ZHANG Liwu;ZHAO Guangwei(College of Veterinary Medicine,Southwest University,Rongchang,Chongqing 402460,China;Animals,Plants and Food Inspection and Quarantine Technical Center of Shanghai Customs,Shanghai 200135,China;Chongqing Sanjiezhongxin Bioengineering Co.,Ltd.,Rongchang,Chongqing 402460,China)

机构地区：[1]西南大学动物医学院,重庆荣昌402460 [2]上海海关动植物与食品检验检疫技术中心,上海200135 [3]重庆三杰众鑫生物工程有限公司,重庆荣昌402460

出　　处：《中国兽医学报》2024年第12期2556-2563,2578,共9页Chinese Journal of Veterinary Science

基　　金：农牧高新技术产业研发专项资助项目;贵州省科技支撑资助项目(黔科合支撑2023一般022)。

摘　　要：为实现鸭甲型肝炎病毒3型(DHAV-3)的快速检测,本试验首先对DHAV-3的非结构蛋白3D进行原核表达,纯化后免疫BALB/c小鼠,四免后取小鼠脾细胞与骨髓瘤细胞(SP2/0)融合制备单克隆抗体,进而利用单克隆抗体建立双抗夹心ELISA(DAS-ELISA)检测方法,评估其灵敏性、特异性和重复性,最后将所建方法应用于临床样本的检测,并与RT-PCR方法进行符合性验证。结果显示:DHAV-33D蛋白在BL21(DE3)中高效表达,经Western blot验证,纯化后的蛋白其特异性良好;细胞融合后经3次亚克隆,成功筛选到6株杂交瘤细胞,分别命名为1A3、1B6、1C7、1D9、2A1和3A9,抗体亚型鉴定结果显示1A3为IgG2b,1B6为IgG2a,3A9为IgG3,1C7、1D9和2A1均为IgG1;经筛选,将1B6和1A3两株亲和性高的单抗分别作为捕获抗体与检测抗体,建立DAS-ELISA检测方法,优化反应条件后确定捕获抗体1B6最佳包被质量浓度为1×10^(-3) g/L,检测抗体1A3的最佳稀释度为1∶1000,阴阳临界值(cut-off)为0.256;灵敏性试验显示该方法对3D蛋白的最低检测限度为4.0×10^(-4) g/L;重复性试验显示,该方法批内、批间变异系数(Cv)均小于9%,重复性好;特异性试验显示,该方法对鸭腺病毒(DAdV)、番鸭细小病毒(MDPV)、鸭圆环病毒(DuCV)、鸭瘟病毒(DPV)、鸭呼肠孤病毒(DRV)、鸭疫里默氏杆菌(RA)病原无特异性反应,但与鸭甲型肝炎病毒1型(DHAV-1)具有交叉反应,可以同时检测出DHAV-3与DHAV-1病原;应用该试验建立的DASELISA方法与RT-PCR检测方法同时对186份临床样本进行检测,DAS-ELISA方法可同时识别DHAV-1和DHAV-3,且与RT-PCR检测方法的符合率为98.9%。结果表明,本试验建立的DAS-ELISA方法重复性好、灵敏性高,可用于DHAV-1与DHAV-3的诊断,为我国鸭甲型肝炎的流行病学调查及防控提供了技术支撑。In order to achieve rapid detection of duck hepatitis A virus type 3(DHAV-3),this experiment initially performed prokaryotic expression of the non-structural protein 3Dof DHAV-3,followed by immunization of BALB/c mice with the purified protein.After immunization,mouse spleen cells were fused with myeloma cells(SP2/0)to prepare monoclonal antibodies.Subsequently,a double-antibody sandwich ELISA(DAS-ELISA)detection method was established using the monoclonal antibodies,and its sensitivity,specificity,and repeatability were evaluated.Finally,the established method was applied to the detection of clinical samples and validated for compliance with the RT-PCR method.The results showed that the DHAV-33Dprotein was efficiently expressed in BL21(DE3),and its specificity was confirmed by Western blot after purification.After cell fusion and three rounds of subcloning,six hybridoma cells were successfully screened and named 1A3,1B6,1C7,1D9,2A1,and 3A9.The subtype identification of the antibodies showed that 1A3belonged to IgG2b,1B6belonged to IgG2a,3A9belonged to IgG3,and 1C7,1D9,and 2A1belonged to IgG1.After screening,the high-affinity monoclonal antibodies 1B6and 1A3were selected as the capture antibody and detection antibody,respectively,and use to establish the DAS-ELISA detection method.After optimizing the reaction conditions,the optimal coating concentration of the capture antibody 1B6was determined to be 1×10^(-3) g/L,and the optimal dilution of the detection antibody 1A3was 1∶1000.The cut-off value was established as 0.256.The sensitivity test showed that the method had a minimum detection limit of 4.0×10^(-4) g/L for the 3Dprotein.The repeatability test showed that the within-batch and between-batch coefficients of variation were both less than 9%,indicating good repeatability.The specificity test showed that the method did not show specific reactions with duck adenovirus(DAdV),muscovy duck parvovirus(MDPV),duck circovirus(DuCV),duck plague virus(DPV),duck reovirus(DRV),or Riemerella anatipestifer(RA),but cros

关 键 词：DHAV-3 单克隆抗体 DAS-ELISA 3D蛋白 检测方法 

分 类 号：S852.65[农业科学—基础兽医学]