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目的:对制备的5株抗BCG Hsp65单克隆抗体(Hsp65 mAb)做进一步鉴定和应用。方法:采用ELISA方法对mAb的效价、亚类和结合抗原表位特性进行鉴定,应用mAb对Hsp65融合蛋白进行了Western blot和免疫荧光检测。结果:5株mAb的类型均为IgG,其中ⅠC1和ⅡC3株mAb为IgG1亚类,ⅠA5、ⅠC3和ⅠD6株mAb为IgG2a亚类。抗原表位竞争结合分析显示,在配对的不同亚类mAb中,存在着结合Hsp65表面不同抗原表位的mAb。通过Western blot检测证实,Hsp65 mAb对Hsp65-MUC1检测结果,与MUC1 mAb的一致。细胞免疫荧光检测显示,制备的mAb能够识别通过蛋白装载进入树突状细胞内的Hsp65-PSA。结论:获得的Hsp65 mAb可做为检测工具,用于研究BCG Hsp65及其融合蛋白的功能和表达。
OBJECTIVE: To further identify and apply the prepared 5 anti-BCG Hsp65 monoclonal antibodies (Hsp65 mAb). Methods: The titer, subclass and antigenic epitope of mAb were identified by ELISA. The anti-Hsp65 fusion protein was detected by Western blot and immunofluorescence with mAb. Results: The five mAb types were IgG. The mAb of ⅠC1 and ⅡC3 were IgG1 subclass, and the ⅠA5, ⅠC3 and ⅠD6 mAb were IgG2a subclass. Antigenic epitope binding analysis revealed that there are mAbs that bind to different epitopes on the surface of Hsp65 in the paired different subclass mAbs. Western blot analysis confirmed that Hsp65 mAb on Hsp65-MUC1 test results, consistent with MUC1 mAb. Immunofluorescence assays showed that the prepared mAb was able to recognize Hsp65-PSA that had been loaded into dendritic cells via protein loading. Conclusion: The obtained Hsp65 mAb can be used as a detection tool to study the function and expression of BCG Hsp65 and its fusion protein.