抗DNA单克隆抗体杂交瘤细胞株的建立及其抗体的鉴定

目的制备抗DNA单克隆抗体,用以建立特异、灵敏、简便的外源性DNA残留量测定方法。方法将小牛胸腺DNA与阳离子化的牛血清白蛋白通过Mannich反应连接成为完全抗原,免疫BALB/c小鼠得到的脾细胞与SP2/0小鼠骨髓瘤细胞融合,获得稳定分泌抗DNA单抗的杂交瘤细胞株,对抗体进行纯化、浓缩及鉴定。结果得到3株可稳定分泌抗DNA单抗的杂交瘤细胞株,单抗的ELISA间接效价分别为1∶4×103、1∶8×104和1∶1×103;亚型分别为IgM/κ、IgM/λ和IgM/κ;亲和力常数分别为3.96×108、4.04×109和1.26×108L/mol;3株细胞分泌的单抗与ctDNA、DH5αDNA、GS115DNA和H.S.DNA均有较高的结合能力,而对RNA、BSA和酪蛋白结合较弱或不能结合;3株细胞分泌的单抗识别3个不同的抗原表位;可检出4ng/ml以上的DNA。结论成功制备了3株细胞分泌的抗DNA单抗,为外源性DNA残留量免疫测定方法的深入研究奠定了基础。

Establishment of Hybridoma Cell Secreting McAb Against DNA and Characterization of The Secreted McAb

Objective To prepare the McAb against DNA and use for the development of a specific,sensitive and simple method for the determination of residual exogenous DNA.Methods DNA-bovine serum albumin(BSA) antigen was prepared by coupling calf thymus DNA fragment with cationized BSA by Mannish reaction and used for the immunization of BALB/c mice.The hybridoma cell strains stably secreting the McAb against DNA was obtained by the fusion of murine splenocytes with SP2/0 plasmacytoma cells,and the secreted McAb was purified,concentrated and characterized.Results Three hybridoma cell strains of subtypes IgM/κ,IgM/λ and IgM/κ respectively were obtained.The indirect ELISA titers of the three strains were 1∶4×103,1∶8×104 and 1∶1×103,and the affinity constants of them were 3.96×108,4.04×109 and 1.26×108 L/mol,respectively.All the three strains showed strong binding capacity to ctDNA,DH5αDNA,CS115 DNA and H.S.DNA but showed weak or no binding capacity to RNA,BSA and casein.The three strains recognized 3 different epitopes.The detection limit of DNA with the prepared McAb by sandwich ELISA was 4 ng/ml.Conclusion Three hybridoma cells secreting McAb against DNA was successfully established.It laid a foundation of development of immunological method for determination of residual exogenous DNA.