人肺癌相关抗原单克隆抗体的制备及其识别抗原的亲和层析纯化
发布时间：2018-09-11 06:12
【摘要】： 目的研制抗人肺癌细胞(A549)单克隆抗体,对其生物学特性进行鉴定,并对其所识别的抗原进行免疫亲和层析纯化。方法采用B淋巴细胞杂交瘤技术,以人肺癌细胞系A549免疫Balb/c小鼠,常规融合,以间接ELISA方法筛选阳性克隆,有限稀释法亚克隆筛选单克隆细胞株,并对其进行核型、亚类、效价、特异性等特性鉴定,免疫组织化学法研究单克隆抗体在各种组织中的表达。裂解A549细胞,提取细胞总蛋白,采用ELISA方法筛选合适的细胞裂解液和洗脱液,将制备的人肺癌相关单克隆抗体与溴化氰活化的Sepharose 4B交联,进行免疫亲和层析,从体外培养的人肺癌A549细胞裂解液中纯化该单抗所识别的特异抗原,进行SDS-PAGE和Western-blot鉴定。结果成功获得了一株能够稳定分泌抗人肺癌相关抗原单克隆抗体的杂交瘤细胞株2B9,所分泌单克隆抗体的亚类为IgM,利用亲和层析纯化提取了其识别的肺癌相关抗原,分子量约为38KD。结论抗人肺癌相关抗原单克隆抗体2B9对肺癌的诊断有一定价值,亲和层析纯化得到的2B9识别抗原有可能成为一种新的肺癌肿瘤标志物,有望进一步用于肺癌的实验室诊断。
[Abstract]:Objective to prepare monoclonal antibody against human lung cancer cell (A549), identify its biological characteristics and purify its recognized antigen by immunoaffinity chromatography. Methods Balb/c mice were immunized with human lung cancer cell line A549 by B lymphocyte hybridoma technique. Positive clones were screened by indirect ELISA method and monoclonal cell lines were screened by limited dilution method. The expression of monoclonal antibody in various tissues was studied by immunocytochemistry. A549 cells were lysed, total protein was extracted, suitable cell lysate and eluate were screened by ELISA method, and the prepared monoclonal antibody was crosslinked with Sepharose 4B activated by cyanide bromide, and then immunoaffinity chromatography was performed. The specific antigen recognized by the monoclonal antibody was purified from the lysate of human lung cancer cell line A549 in vitro and identified by SDS-PAGE and Western-blot. Results A hybridoma cell line 2B9 was successfully obtained, which could stably secrete monoclonal antibody against human lung cancer associated antigen. The subclass of monoclonal antibody secreted was IgM, which was purified by affinity chromatography and its molecular weight was about 38 KD. Conclusion the monoclonal antibody 2B9 against human lung cancer associated antigen has certain value in the diagnosis of lung cancer. The 2B9 recognition antigen purified by affinity chromatography may become a new tumor marker of lung cancer, and it is expected to be further used in laboratory diagnosis of lung cancer.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392
本文编号：2235845
[Abstract]:Objective to prepare monoclonal antibody against human lung cancer cell (A549), identify its biological characteristics and purify its recognized antigen by immunoaffinity chromatography. Methods Balb/c mice were immunized with human lung cancer cell line A549 by B lymphocyte hybridoma technique. Positive clones were screened by indirect ELISA method and monoclonal cell lines were screened by limited dilution method. The expression of monoclonal antibody in various tissues was studied by immunocytochemistry. A549 cells were lysed, total protein was extracted, suitable cell lysate and eluate were screened by ELISA method, and the prepared monoclonal antibody was crosslinked with Sepharose 4B activated by cyanide bromide, and then immunoaffinity chromatography was performed. The specific antigen recognized by the monoclonal antibody was purified from the lysate of human lung cancer cell line A549 in vitro and identified by SDS-PAGE and Western-blot. Results A hybridoma cell line 2B9 was successfully obtained, which could stably secrete monoclonal antibody against human lung cancer associated antigen. The subclass of monoclonal antibody secreted was IgM, which was purified by affinity chromatography and its molecular weight was about 38 KD. Conclusion the monoclonal antibody 2B9 against human lung cancer associated antigen has certain value in the diagnosis of lung cancer. The 2B9 recognition antigen purified by affinity chromatography may become a new tumor marker of lung cancer, and it is expected to be further used in laboratory diagnosis of lung cancer.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392
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