甲型流感病毒H1N1单克隆抗体特性鉴定和应用

Characterization and application of monoclonal antibodies against H1N1 influenza A virus

目的:制备抗甲型流感病毒单克隆抗体(mAbs)并分析抗体反应特性,评价mAbs在免疫荧光(IFA)和细胞免疫化学染色中的应用,初步建立H1N1流感病毒抗原检测的双抗体夹心ELISA方法。方法:取A/PR/8/34(H1N1)毒种接种于鸡胚尿囊腔,培养72 h后收获尿囊液,甲醛灭活,蔗糖密度梯度超离心法纯化,用于mAbs免疫原开发。病毒颗粒免疫BALB/c小鼠,取小鼠脾细胞与骨髓瘤细胞Sp2/0进行融合,ELISA和IFA筛选稳定分泌抗甲型流感病毒mAbs的杂交瘤细胞株。ELISA、IFA、血凝抑制试验(HI)和中和试验(MN)分析抗体反应特性。依据mAbs特性建立细胞IFA试验、HI试验和双抗体夹心ELISA方法。结果:共制备13株mAbs,其中6株(PR8-10、PR8-19、PR8-20、PR8-26、PR8-28、PR8-30)具有较高血凝抑制活性,血凝抑制滴度≤3μg/ml。PR8-13和PR8-15无中和活性,其他11株均具有中和活性。部分mAbs可用于IFA试验和细胞免疫化学染色。依据PR8-24和PR8-25抗体反应特性初步建立H1N1流感病毒抗原检测的双抗体夹心ELISA法。结论:制备出可用于IFA试验和细胞免疫化学染色的A/PR/8/34(H1N1)mAbs,初步建立了快速H1N1检测的ELISA方法,为H1N1流感病毒或病毒血凝素蛋白快速定量方法建立提供依据。

Objective:To prepare monoclonal antibodies(mAbs)against influenza A virus,and to analyze characterization of mAbs.To assess application of mAbs in immunofluorescence assay(IFA)and cell-based immunohistochemistry,and to establish double antibody sandwich ELISA for detection H1 N1 based on characterization of mAbs to H1 N1.Methods:A/PR/8(34)H1 N1 influenza A virus was prepared from egg allantoic fluid produced and allantoic fluid harvested after 72 h post infection,inactivated with formalin,purified by sucrose density gradient ultracentrifugation,which were used as immunogen for development of mAbs.BALB/c mice were immunized with virus particles,splenic cells of mice were fused with myeloma cells Sp2/0,and hybridioma cell lines secreted stably against influenza A virus mAbs were screened by ELISA and IFA.ELISA,IFA,Hemagglutination Inhibition Test(HI)and Microneutralization assay(MN)were performed to identify characterization of mAbs.According to characterization of mAbs to H1 N1,mAbs were used in IFA and cell-based immunohistochemistry respectively,and double antibody sandwich ELISA was also established.Results:13 mAbs were obtained and characterized,in which 6 mAbs(PR8-10,PR8-19,PR8-20,PR8-26,PR8-28,PR8-30)have hemagglutination inhibition activity,whose HI titer were≤3μg/ml.11 mAbs have neutralizing activity.A part of mAbs could be used in IFA and cell-based immunohistochemistry.Double antibody sandwich ELISA was developed utilizing PR8-24 and PR8-25,which could be available for detection of H1 N1 viral particles.Conclusion:mAbs to A/PR/8/34(H1 N1)were prepared,which could be used in IFA and cell-based immunohistochemistry respectively.Double antibody sandwich ELISA for H1 N1 viral particles was developed,which provide basis for quantitative analysis of H1 N1 or hemagglutinin antigen.