【摘要】 目的获得抗HBsAg单链抗体(HBScFv)的单克隆抗体,并初步研究其在抗HBsAg类小分子抗体及其融合蛋白质的纯化、活性鉴定方面的应用。方法利用杂交瘤技术,以大肠杆菌表达的重组抗HBScFv为抗原,免疫Balb/c小鼠,再将小鼠脾细胞与小鼠骨髓瘤细胞SP2/0进行细胞融合。通过ELISA法、Western-blot鉴定特异性单克隆抗体,通过免疫双扩散法鉴定单抗亚型,并将筛选到的高效价单抗用于抗HBsAg Fab抗体等重组蛋白质的亲和色谱及ELISA鉴定。结果建立了9株能够稳定分泌抗HBScFv特异性单克隆抗体的细胞株,其细胞上清效价为1∶160~1∶12 800,腹水效价为1∶50 000~1∶400 000;单克隆抗体的亚类分析结果显示有6株为IgG1,其余3株为IgG2a。初步应用结果表明制备的单克隆抗体14F7可用于抗HBsAg Fab抗体等重组蛋白质的亲和色谱及活性鉴定。结论成功建立了能够稳定分泌HBScFv特异性单克隆抗体的细胞株。更多还原

【Abstract】 PurposeTo obtain Monoclonal Antibody against anti-HBs single-chain Fv(HBScFv),and to study their application in the purification and activity detection of anti-HBs small molecule antibody and their fusion proteins.MethodsThe Balb/c mice were immunized with purified HBScFv expressed in E.coli,and then the spleen cells from immunized mice were fused with SP2/0 myeloma cells.The specific McAb were screened by ELISA and identified by western-blot,and the Ig subclass of MCAb were detected by double immunodiffusion method,the high titer McAb were used in the affinity chromatography and activity detection of anti-HBs Fab and other recombinant proteins.Results9 lines of hybridoma cells which could stably produce monocloned antibodies against HBScFv were obtained.The titers of the supernants were 1∶160-1∶12 800,and the ascites were 1∶50 000-1∶400 000.The Ig subclass of six McAb were IgG1 and others were IgG2a.The McAb 14F7 was successfully used in the purification and activity detection of anti-HBs small molecule antibody and their fusion proteins.Conclusion9 lines of hybridoma cells which could stably produce monoclonal antibodies against HBScFv were established.更多还原