摘要
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。
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.
出处
《蚌埠医学院学报》
CAS
2010年第10期976-978,共3页
Journal of Bengbu Medical College
基金
国家科技重大专项基金资助(2008ZX10003-011)
关键词
结核杆菌
抗原
Mtb-HAg
杂交瘤细胞株
单克隆抗体
Mycobatcteium tuberculosis heat-resistant antigen
hybrdoma cell line
monoclonal antibody
