H1亚型禽流感病毒单克隆抗体的制备及夹心ELISA检测H1抗原方法的建立  

Production of monoclonal antibodies and development of a sandwich ELISA for detection of H1 subtype avian influenza virus antigen

作　　者：谢志勤 谢芝勋 张艳芳 范晴 张民秀 罗思思 谢丽基 黄娇玲 王盛 曾婷婷 李孟 李丹 邓显文 韦悠 万丽军 阮志华 李小凤 任红玉 刘加波 XIE Zhi-qin;XIE Zhi-xun;ZHANG Yan-fang;FAN Qing;ZHANG Min-xiu;LUO Si-si;XIE Li-ji;HUANG Jiao-ling;WANG Sheng;ZENG Ting-ting;LI Meng;LI Dan;DENG Xian-wen;WEI You;WAN Li-jun;RUAN Zhi-hua;LI Xiao-feng;REN Hong-yu;LIU Jia-bo(Guangxi Key Laboratory of Veterinary Biotechnology/Guangxi Veterinary Research Institute,Nanning 530001,China)

机构地区：[1]广西壮族自治区兽医研究所,广西兽医生物技术重点实验室,广西南宁530001

出　　处：《中国兽医科学》2021年第9期1106-1112,共7页Chinese Veterinary Science

基　　金：广西科技重大专项(桂科AA17204057);广西科技基地和人才专项(桂科AD17195083);中央引导地方专项(桂科ZY19183013);国家“万人计划”领军人才专项(W02060083);“广西八桂学者”专项(2019A50)。

摘　　要：用禽流感病毒(AIV)A/Chicken/Guangxi/90C/2011(H1N2)毒株制备单克隆抗体(McAb)并建立夹心ELISA检测H1亚型AIV。AIV 90C/2011(H1N2)毒株经纯化灭活后免疫BALB/c小鼠,取第5次免疫3 d后的脾细胞与骨髓瘤细胞SP2/0进行融合。用4 U的H1抗原经血凝抑制试验(HI)筛选效价高的杂交瘤细胞株并经3次亚克隆,最终获得抗体分泌高且稳定的杂交瘤细胞株。分泌的抗体经H1 HI、Western-blot和间接免疫荧光检测进行验证,并用于建立夹心ELISA方法检测H1 AIV。经融合细胞筛选,获得4株稳定分泌抗体的杂交瘤细胞株,用其中一株7B9制备单克隆抗体,分泌抗体效价达1∶512,其抗体类型经鉴定为Ig G1。用制备的单抗建立夹心ELISA方法检测H1 AIV,测定其阴阳判定值为0.2714。特异性试验显示,检测的H1 AIV毒株D450值均大于0.2714,对照的其他AIV毒株以及新城疫病毒、产蛋下降综合征病毒毒株的D450值均小于0.2714。敏感性测试最低能检测出10^(-5)稀释的HA效价的H1 AIV。本试验建立的基于McAb的H1 AIV夹心ELISA检测方法特异性好,敏感。To develop a sandwich ELISA for detection of H1 subtype avian influenza virus(AIV)based on monoclonal antibody(McAb)prepared using AIV A/Chicken/Guangxi/90 C/2011(H1 N2),BALB/c mice were immunized five times with the purified AIV 90 C/2011(H1 N2)strain after inactivation.Splenocytes was taken and fused with myeloma cells SP2/0 after the last immunization 3 days.The positive hybridoma cell lines were screened by hemagglutination inhibition(HI)test after subcloning 3 times with 4 U H1 antigen.The Mc Abs secreted from hybridoma cell lines were verified by H1 HI test,Western-blot,and indirect immunofluorescence assay.A sandwich ELISA method based on the Mc Ab for detecting H1 AIV was established.The results showed that 4 hybridoma cell lines stably secreting antibodies were obtained by screening fusion cells.The Mc Ab secreted from 7 B9 which was one of 4 hybridoma cell lines was prepared and the titer was 1∶512.The Mc Ab type was identified as Ig G1 by identification kit.A sandwich ELISA based on Mc Ab 7 B9 was established to detect H1 AIV antigen.The cutoff D450 value was 0.2714.The D450 value of all H1 strains were greater than the cutoff value.The D450 values of other virus strains as a contract were less than the cutoff value.The detection limit was 10^(-5) dilution virus(1∶28 HA titer).The sandwich ELISA based on Mc Ab has good specificity and sensitivity.

关 键 词：H1亚型禽流感病毒 单克隆抗体 夹心ELISA 检测 

分 类 号：S852.659.5[农业科学—基础兽医学]