Yamagata系乙型流感病毒血凝素蛋白单克隆抗体制备及双抗体夹心ELISA检测方法的建立  

Preparation of monoclonal antibodies against Yamagata-lineage influenza B virus hemagglutinin protein and establishment of a dual antibody sandwich ELISA detection method

作　　者：程林芳[1] 杨帆 刘福民[1] 姚航平[1] 吴南屏[1] 吴海波[1] Cheng Linfang;Yang Fan;Liu Fumin;Yao Hangping;Wu Nanping;Wu Haibo(State Key Laboratory for Diagnosis and Treatment of Infectious Diseases,National Clinical Research Center for Infectious Diseases,Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases,The First Affiliated Hospital,School of Medicine,Zhejiang University,Hangzhou 310003,China)

机构地区：[1]浙江大学医学院附属第一医院传染病重症诊治全国重点实验室,国家感染性疾病临床医学研究中心,感染性疾病诊治协同创新中心,杭州310003

出　　处：《国际流行病学传染病学杂志》2023年第5期331-336,共6页International Journal of Epidemiology and Infectious Disease

基　　金：国家自然科学基金(32273092);中央高校基本科研业务费(2022ZFJH003)。

摘　　要：目的制备Yamagata系乙型流感病毒血凝素蛋白(hemagglutinin,HA)的特异性单克隆抗体,建立检测Yamagata系乙型流感病毒的双抗体夹心ELISA方法。方法用Yamagata系乙型流感病毒(B/Phuket/3073/2013)抗原免疫BALB/c小鼠,取免疫小鼠脾细胞与骨髓瘤SP2/0细胞进行融合,通过4次有限稀释获得阳性杂交瘤细胞并建株。将单克隆抗体1F7和2H6分别作为捕获抗体和检测抗体,建立一种检测Yamagata系乙型流感病毒的双抗体夹心ELISA法。结果该方法中捕获抗体1F7在96孔板的包被量为40 ng/孔,检测抗体2H6工作浓度为100 ng/孔;该方法仅能与Yamagata系乙型流感病毒发生反应,具有较强特异性。灵敏度分析显示,该方法对病毒尿囊液和病毒纯化HA蛋白最低检测值分别为2-1 HAU/100μL和1.22 ng/mL,具有较高灵敏度。重复性试验表明,该方法批内和批间重复性变异系数均小于5%。结论本研究建立的ELISA方法特异性强、灵敏度高、重复性好,为Yamagata系乙型流感病毒快速诊断及定量检测提供技术支持。Objective To prepare specific monoclonal antibodies against Yamagata-lineage influenza B virus hemagglutinin(HA)and establish a dual antibody sandwich ELISA method for detecting Yamagata-lineage influenza B virus.Methods BALB/c mice were immunized with Yamagata-lineage influenza B virus(B/Phuket/3073/2013)antigen.The spleen cells of the immunized mice were fused with myeloma SP2/0 cells,and positive hybridoma cells were obtained through four limited dilutions and established.A dual antibody sandwich ELISA method for detecting Yamagata-lineage influenza B virus was established by using monoclonal antibodies 1F7 and 2H6 as capture and detection antibodies,respectively.Results In this method,the encapsulation amount of antibody 1F7 captured on a 96 well plate was 40 ng/well,and the working concentration of antibody 2H6 detected was 100 ng/well.This method showed strong specificity for only Yamagata-lineage influenza B virus.Sensitivity analysis showed high sensitivity with the minimum detection values of 2-1 HAU/100μL and 1.22 ng/mL for virus allantoic fluid and virus purified HA protein,respectively.The repeatability test showed that the coefficients of variation in intra-and inter-assay were less than 5%.Conclusions The ELISA method established in this study has strong specificity,high sensitivity,and good repeatability,providing technical support for the rapid diagnosis and quantitative detection of Yamagata-lineage influenza B virus.

关 键 词：流感病毒B型 Yamagata系 病毒检测方法 ELISA 单克隆抗体 特异性 

分 类 号：R446.6[医药卫生—诊断学] R511.7[医药卫生—临床医学]