生物传感器对抗戊型肝炎病毒单克隆抗体部分特性的研究

Characterazation of the anti-HEV ORF2 monoclonal antibodies by biosensor

目的 应用生物传感器对新制备的抗戊型肝炎病毒单克隆抗体 (mAb)的亲和力、Ig亚类 (型 )及抗原结合的动力学进行研究。方法 用HEVORF2区基因工程重组蛋白NE2 ,免疫BALB/c小鼠 ,经杂交瘤技术制备mAb ,采用ELISA、Westernblot和生物传感器鉴定其有关特性。结果获得 12株可稳定分泌抗NE2mAb的杂交瘤细胞系。用ELISA及生物传感器等鉴定各株mAb ,分别为IgM和IgG1、IgG2a,轻链均为κ型。其中在用ELISA法对mAb3F5亚类鉴定过程中 ,发现其与HRP GAMIgG2a和HRP GAMIgM均有反应 ,生物传感器鉴定其为IgM。Westernblot验证各株mAb的特异性 ,同时各株mAb对于不同聚合形式的NE2蛋白的反应性有一定的差别。应用生物传感器测定了 4株mAb的KD 值 ,mAb 8C11为 4 .36× 10 7,mAb8H3为 1.5 3× 10 5,mAb 13D8为 1.2 1× 10 6,mAb 16D7为8.0 3× 10 7。从生物传感器测得的各株mAb与NE2的结合、解离过程中发现 ,mAb 8H3与NE2可快速结合、快速解离 ,其它 3株mAb结合稳定。结论 经多种方法鉴定 ,所获得的 12株杂交瘤细胞分泌的抗体 。

Aim To prepare the anti HEV ORF2 monoclonal antibodies (mAbs), and idantify their partial properties by biosensor. Methods The BALB/c mice were immunized with purified recombinant protein NE2 of HEV ORF2. Anti HEV ORF2 mAbs were prepared by hybridoma technique, and the relative properties of the mAbs were identified by ELISA, Western blot and biosensor. Results 12 hybridoma cell lines steadily secreting the anti NE2 mAbs were obtained. Of these mAbs, mAb 3F5 was IgM, and the others were IgG (IgG1, IgG2a or IgG2b), and the isotype of their light chains were kappa (κ). MAb 3F5 cross reacted to HRP GAM IgG2a and HRP GAM IgM in ELISA, but this mAb was proved to be IgM by biosensor. The results of the Western blot showed that 12 mAbs reacted to NE2 protein. Reactivities of mAbs 1F6, 8C10, 10D4, 12G8, 2C9, 8C11, 8H3 and 13D8 to NE2 dimer and polymer were stronger than that to NE2 monomer, while mAbs 3F5, 7E8, 15B2, and 16D7 had the same reactivities to monomer, dimer or polymer of NE2. The K D of four mAbs (8C11,8H3,13D8 and 16D7) were calculated through binding curve of coulpled NE2 to varying concentritions of mAbs. And from the curve of the binding and dissociation, we found that the binding of mAb 8H3 to NE2 and dissociation from it were much faster than that of the other three. Conclusion Twelve hybodoma cell lines secreting mAbs to recombinant HEV ORF2 protein were obtained and characterized.