抗H5N1亚型禽流感病毒血凝素单克隆抗体的制备及鉴定

Preparation and Characterization of Monoclonal Antibody Specific to Hemagglutinin of Subtype H5 Avian Influenza Virus

目的建立稳定分泌抗H5N1亚型禽流感病毒血凝素单克隆抗体的杂交瘤细胞系,为进一步研究禽流感诊断技术奠定基础。方法以纯化的H5亚型禽流感病毒按常规方法免疫BALBc小鼠,最后一次免疫后第3天取其脾细胞与SP20细胞在聚乙二醇作用下融合,用选择性培养、有限稀释法克隆和血凝抑制试验进行筛选,对获得阳性克隆株用ELISA方法进行亚型鉴定,并用37株H5、H7、H9亚型AIV测定其特异性、覆盖性。结果最后获得了3株分泌特异性抗体的杂交瘤细胞,命名为1E5、4A4、4B1,经长期体外培养和冻存后复苏能稳定地分泌抗体。经鉴定,其亚型均为IgG1、kappa链。腹水HI效价1∶210～1∶216,细胞培养上清HI效价1∶26～1∶28。3株杂交瘤所分泌的单克隆抗体均能与本中心保存的全部20株H5亚型禽流感病毒分离株发生反应,而与15株H9亚型禽流感病毒分离株、2株H7亚型禽流感病毒分离株以及H1H4、H6H15亚型禽流感病毒标准毒株均不反应,与鸡新城疫病毒、鹅新城疫病毒、鹅腺病毒和鸡产蛋下降综合征病毒等均无交叉反应。结论所获3株单克隆抗体可用于禽流感病毒特异性诊断试剂的研制。

Objective To develop and characterize monoclonal antibody (McAb) against AIVH5 for related research work.Methods BALB/c mice were immunized conventionally with the purified AIVH5 On the third day after the final immunization, spleen cells of mice fused with myeloma cells SP2/0 were filtered by culturing selectively, diluting finitely and hemagglutination_inhibition (HI) test. The subtypes were obtained from the clone line by ELISA test and their characteristics and mantles were determined by H5, H7 and H9 subtypes of 37 AIV lines. Results The three cell lines secreting McAbs stably by culturing in vitro and recovery tests were obtained and named as 1E 5 , 4A 4 , and 4B 1 respectively. The subtypes of the three McAbs were identified as IgG1, Kappa light chain. The HI titers of culture supernatants and ascites were 1∶2 6 -1∶2 8 and 1∶2 10 -1∶2 16 respectively. The three McAbs reacted specifically with only AIVH5 but not with AIVH9, AIVH7, NDV in chickens, NDV in geese, EDS76 and so on. Conclusion The three McAbs will be very useful as specific reagents in diagnosing and controlling avian influenza.