摘要
目的筛选新的肿瘤特异性抗原,为探索新的肿瘤诊断标志物和肿瘤疫苗奠定基础。方法将人宫颈癌细胞系Hela细胞免疫BALB/c小鼠,制备杂交瘤,应用杂交瘤培养上清对人结肠癌细胞系Colo205、人Burkitt淋巴瘤细胞系Raji等细胞进行红细胞花环形成实验以筛选阳性杂交瘤。应用Colo205、Raji、人肺癌细胞系PLA801、人胃癌细胞系7901、人T细胞白血病细胞系Jurkat、小鼠骨髓瘤细胞系SP2/0等肿瘤细胞系、胚胎细胞系人胚肾上皮细胞系293T、人脐静脉内皮细胞系ECV304和健康人外周血单个核细胞(PBMC)等对所筛选的单克隆抗体进行活细胞免疫荧光染色和流式细胞仪鉴定单克隆抗体对多种细胞系膜表面天然抗原的识别情况,同时应用免疫沉淀法和Western blotting检测所筛单克隆抗体对细胞膜抗原的识别。结果采用红细胞花环形成实验从制备的抗体中成功筛选出3株单克隆抗体,流式分析显示其可同时识别Hela、293T、ECV304、Raji和Colo205细胞,但却不能识别Jur-kat、PLA801、7901、SP2/0细胞以及静止或活化的人PBMC。Western blotting证实这3株抗体可识别表达于Hela、Raji细胞膜的分子量约为47kD的膜蛋白。结论成功地制备了3株可识别肿瘤抗原候选分子的mAb,为筛选和鉴定肿瘤特异性抗原提供了有力手段。
Objective To screen the tumor specific antigens, and explore new methods for diagnosis and therapy of tumor. Methods Hela cells, the human cervical carcinoma cell line, were used to immunize the BALB/C mice, and the positive hybridomas were screened by rosette formation screening system in human carcinoma cell line Colo205, Burkitt lymphoma cell line, Raji, etc. The immunofluorescence staining and flow cytometry analysis were performed to characterize if the prepared monoclonal antibodies （mAb） could reorganize the natural membrane antigen expressed on Hela, Colo205, Raji, PLA801, 7901, Jurkat, 293T, ECV304 and resting or activated human petipheral blood mononuclear cells （PBMC）. Furthermore, the membrane protein of Hela, Raji and Sp2/0 cells was segregated by ultraeen- trifugation and the mAbs were cross-linked with the Sepharose CL-4B. In order to identify the molecules which could be recognized by the prepared mAbs, immunoprecipitation was performed to purify the tumor antigen candidates with the cell membrane protein from Hela, Raji and SP2/0 （as negative control） through Sepharose CL-4B cross-linked with the prepared mAbs. Finally, western blotting was performed to detect the molecular weight of the products from the immunoprecipitation. Results Three mAbs were obtained which could recognize the tumor antigen candidates expressed in several human carcinoma cell lines, such as Hela, Raji and Colo205, and some embryonic cell lines, such as 293T and ECV304, but they could not bind to Jurkat, PLA801, 7901, SP2/0, resting or activated human PBMC. Western blotting results indicated that the three mAbs could recognize a 47kD molecule which expressed in Hela and Raji but not on SP2/0 cell membrane. Conclusion Three mAbs, which can recognize tumor membrane antigen candidates, are obtained, providing useful tools for screening and identify the tumor specific antigens.
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2008年第11期1348-1351,共4页
Medical Journal of Chinese People's Liberation Army
基金
国家重点基础研究发展规划(973)资助项目(2001CB510004)
关键词
抗体
单克隆
免疫沉淀法
印记法
蛋白质
抗原
肿瘤
antibodies, monoclonal
immunoprecipitation
blotting, westem
antigens, neoplasm
