【摘要】 目的制备抗脊髓灰质炎病毒单克隆抗体,为ELISA检测方法的建立奠定基础。方法将Ⅰ型脊髓灰质炎减毒疫苗原液和灭活疫苗原液经超滤、超离纯化,以纯化的减毒脊髓灰质炎病毒D抗原免疫BALB/c小鼠,得到的脾细胞与SP2/0-A14小鼠骨髓瘤细胞融合,以纯化的灭活脊髓灰质炎病毒D抗原建立间接ELISA法,筛选分泌特异性单抗的阳性细胞株,诱生小鼠腹水,通过正辛酸-硫酸铵法纯化抗体,并进行各项鉴定。结果获得两株稳定分泌抗脊髓灰质炎病毒单抗的细胞株7G5及5E4。纯化的7G5和5E4株单抗分别可与脊髓灰质炎病毒D抗原的VP2、VP3衣壳蛋白结合;在不同温度下放置均比较稳定;pH4不利于7G5株单抗的保存;5E4株单抗的相对亲和力大于7G5株;7G5株单抗的亚类为IgM,5E4株单抗的亚类为IgG2a;两株单抗可识别相似表位;7G5株和5E4株单抗的中和效价分别为1:64和1:128。结论已成功制备了两株分泌抗脊髓灰质炎病毒的单抗。更多还原

【Abstract】 Objective To prepare the monoclonal antibody(McAb)against poliovirus and lay a foundation of developing ELISA for the virus.Methods The bulks of live attenuated and inactivated poliovirus typeⅠ were purified by ultrafiltration and ultracentrifugation.The purified antigen of live attenuated poliovirus was used for immunization of BALB /c mice,and the obtained splenic cells were fused with SP2 /0-A14 cells.An indirect ELISA was developed with the purified antigen of inactivated poliovirus and used for screening of positive cell strains secreting specific McAb.The secreted McAb was purified from the ascites of mice by ncaprylic acid-ammonium sulfate method and identified.Results Two cell strains stably secreting McAb against poliovirus,7G5 and 5E4,were obtained.The purified McAbs 7G5 and 5E4 were bound to the capsid proteins VP2 and VP3 of D antigen of poliovirus respectively,and showed high stability after storage at various temperatures.McAb 7G5 was not suitable for storage at pH4.The relative affinity of 5E4 was higher than that of 7G5.McAb 7G5 was identified as IgM,while 5E4 as IgG2a.McAbs 7G5 and 5E4 recognized similar epitopes,with neutralizing titers of 1:64 and 1:128 respectively.Conclusion The McAbs 7G5 and 5E4 against poliovirus were successfully prepared.更多还原