抗结核杆菌耐热抗原单克隆抗体杂交瘤细胞的建立及其特性的初步鉴定

目的：建立针对结核杆菌耐热抗原（Mtb-HAg）的单克隆杂交瘤细胞株,并对其产生的单克隆抗体的特异性和生物学活性作初步鉴定。方法：BALB/C小鼠用Mtb-HAg免疫3次后,取脾细胞与小鼠骨髓瘤细胞SP2/0以10∶1比例在50%PEG作用下细胞融合,随后在HAT培养液中进行选择性培养,常规有限稀释法筛选克隆和亚克隆化;用间接ELISA法测定单抗效价和特异性鉴定。结果：在细胞融合2次,筛选杂交瘤细胞3次和亚克隆化后,获得了3株杂交瘤细胞株,ⅠC12、ⅡE4、ⅡG8,效价分别为1∶32 000、1∶16 000、1∶16 000。其中ⅠC12杂交瘤株分泌的抗体仅与Mtb-HAg的蛋白峰Ⅰ有阳性反应,但ⅠC12单抗对Mtb-HAg激活人γδT细胞的活性没有明显影响。ⅡE4和ⅡG8杂交瘤株分泌的抗体与Mtb-HAg蛋白峰Ⅰ、峰Ⅱ、峰Ⅲ均无反应。结论：应用杂交瘤技术获得了三株能稳定分泌高滴度抗Mtb-HAg的单克隆抗体细胞株,其中ⅠC12对蛋白峰I有特异性。

Primary characterization of hybridoma cells secreting monoclonal antibody against Mycobacterium tuberculosis heat-resistant antigen

Objective：To develop the hybridoma cells secreating the monoclonal antibody（McAb） against Mycobatcteium tuberculosis heat-resistant antigen（Mtb-HAg）,and identify the specificity of the McAb and its biological activity.Methods：BALB/C mice were immunized 3 times with Mtb-HAg,and the spleen cells of immunized mice were fused with myeloma cells SP2/0 at the ratio of 10∶1 by 50% PEG,and cultured in the HAT medium,followed by selection and subclone procedure by routine limited dilution method.The specificity and titer of the McAb were detected by ELISA method.Results：After fused 2 times,and selected and cloned 3 times,three hybridoma cell lines,Ⅰ C12,ⅡE4,and Ⅱ G8 were generated,and titers of their McAb were 1：32 000,116 000,and 116 000,respectively.ⅠC12 only had positive reaction with the Mtb-HAg protein peak Ⅰ,whereas ⅠC12 had no inhibitory effect on the Mtb-HAg activity to stimulate human γδ T cells.ⅡE4 andⅡG8 showed no positive reaction with Mtb-HAg protein peak Ⅰ,peak Ⅱ and peak III.Conclusions：By the hybridoma technique,three hybridoma cell lines have been generated and the hybridoma Ⅰ C12 cells produce the specific McAb against Mtb-HAg protein peak I.