甲型H1N1流感病毒血凝素蛋白单抗杂交瘤细胞株的制备与初步鉴定

目的:建立具有高特异、高效价的甲型H1N1流感病毒血凝素蛋白(HA)单抗的杂交瘤细胞株。方法:以纯化的昆虫杆状病毒表达的甲型H1N1流感病毒HA蛋白为免疫原免疫BALB/c小鼠,取脾细胞与Sp2/0小鼠骨髓瘤细胞融合,通过有限稀释法筛选阳性克隆,经ELISA和Western blot分析单抗的特性和特异性。结果:获得6株甲型H1N1流感HA抗原特异单克隆抗体杂交瘤细胞株,抗原肽库ELISA检测结果表明其中3株(1E12,3F12,1C11)单抗只与甲型H1N1流感HA抗原肽库反应,不与H5N1病毒HA抗原肽库反应;Western blot分析表明,单抗1B3只特异识别甲型H1N1流感HA抗原,而与其他季节性甲流病毒(H1,H3)及人禽流感H5N1病毒不反应。结论:所获杂交瘤细胞株特异性强,效价高,分泌抗体性能稳定,为分析甲型H1N1流感病毒抗原性位点、建立诊断试剂奠定了基础。

Establishment and Characterization of Hybridoma Cell Secreting Monoclonal Antibody Against Hemagglutinin of the Pandemic Swine-Origin Influenza Virus

Objective：To prepare hybridoma cell secreting monoclonal antibody（McAb）against hemagglutinin（HA） of swine-origin influenza virus（S-OIV）.Methods：BALB/c mice were immunized twice at regular intervals of 2 weeks with 50 and 25μg of recombinant HA antigen of S-OIV derived from Bac-to-Bac system.Thereafter,mice were boosted intravenously with 12.5μg of recombinant antigen 3 days before the fusion of splenocytes with Sp2/0 cells.Hybridoma culture supernatants were screened and evaluated by ELISA.Positive clones were checked for isotype and McAbs were confirmed by Western blotting analysis using several seasonal influenza virus stocks and ELISA coating HA peptides of S-OIV or H5N1.Results：We obtained six strains of stable hybridoma cells secreting McAb against HA of S-OIV.Three strains（1E12,3F12 and 1C11）of which recognized to HA peptides of S-OIV by ELISA,not to that of H5N1.Furthermore,when analyzed by Western blotting,McAb 1B3 only reacted with HA of S-OIV,not with that of seasonal H1 and H3 influenza viruses.Conclusion：Six strains of specific and stable hybridoma cells secreting McAb against HA of S-OIV have been obtained,which provide useful tools for diagnosis and analyzing of pandemic S-OIV H1N1 infection.