【摘要】： 目的研制抗人肺癌細(xì)胞(A549)單克隆抗體,對其生物學(xué)特性進(jìn)行鑒定,并對其所識別的抗原進(jìn)行免疫親和層析純化。方法采用B淋巴細(xì)胞雜交瘤技術(shù),以人肺癌細(xì)胞系A(chǔ)549免疫Balb/c小鼠,常規(guī)融合,以間接ELISA方法篩選陽性克隆,有限稀釋法亞克隆篩選單克隆細(xì)胞株,并對其進(jìn)行核型、亞類、效價、特異性等特性鑒定,免疫組織化學(xué)法研究單克隆抗體在各種組織中的表達(dá)。裂解A549細(xì)胞,提取細(xì)胞總蛋白,采用ELISA方法篩選合適的細(xì)胞裂解液和洗脫液,將制備的人肺癌相關(guān)單克隆抗體與溴化氰活化的Sepharose 4B交聯(lián),進(jìn)行免疫親和層析,從體外培養(yǎng)的人肺癌A549細(xì)胞裂解液中純化該單抗所識別的特異抗原,進(jìn)行SDS-PAGE和Western-blot鑒定。結(jié)果成功獲得了一株能夠穩(wěn)定分泌抗人肺癌相關(guān)抗原單克隆抗體的雜交瘤細(xì)胞株2B9,所分泌單克隆抗體的亞類為IgM,利用親和層析純化提取了其識別的肺癌相關(guān)抗原,分子量約為38KD。結(jié)論抗人肺癌相關(guān)抗原單克隆抗體2B9對肺癌的診斷有一定價值,親和層析純化得到的2B9識別抗原有可能成為一種新的肺癌腫瘤標(biāo)志物,有望進(jìn)一步用于肺癌的實(shí)驗室診斷。
[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.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R392

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