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學校名稱輔仁大學
系所名稱生命科學系
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研究生(中)吳思諭
研究生(英)Wu, Szu-yu
論文名稱(中)使用混合抗原策略建立人類血清蛋白之單株抗體庫的生產平台
論文名稱(英)Establishing the production platform of monoclonal antibody bank for human serum proteome : the poly-antigen strategy
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指導教授(中)陳翰民
指導教授(英)Chen, Han-Min
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畢業學年度94
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關鍵字(中)血液 蛋白質表現側寫圖譜 單株抗體庫 抗體晶片
關鍵字(英)blood protein expression profile monoclonal antibody bank antibody chips
摘要(中)在臨床診斷上,疾病的檢測常以血液中特定的蛋白質為標的,若能發現專一性與靈敏度高的疾病標的物,則可以實踐早期發現,早期治療的醫學鐵律。近年來,本實驗室已建立了篩選疾病特異性之血液標的蛋白質的作業平台,也成功的自數種疾病中,篩選出幾個可能的血液蛋白質標的物。然而,許多實驗結果顯示,單一蛋白質標的物,有時不能作為準確的診斷依據,而藉由判斷一組蛋白質表現量的蛋白質表現側寫圖譜 (protein expression profile),可能更適合做為臨床診斷的依據。 本實驗室之長程目標在於人類血液蛋白質的抗體晶片開發,除了可產生血液蛋白質表現側寫圖譜,以供臨床診斷分析外,也可鑑定出疾病特定之血液標的蛋白身份,作為研究致病機轉的依據。由於抗體晶片為微型化之酵素免疫連結分析法 (ELISA),因此要生產晶片,必先取得各血液相關抗原蛋白質之單株或多源抗體,其中又以單株抗體之專一性為佳,因此單株抗體之獲得是開發抗體晶片的關鍵步驟。然而利用傳統免疫學方法,需先取得個別抗原分別免疫小鼠,進而以融合瘤技術來篩選所需抗體分泌細胞株,若要獲得數百種以上的單株抗體,此過程不僅花費大量的人力與經費,亦曠日廢時,實不符合研究與應用之需求。因此,本論文主要的目的,就是為了開發出一個高效率、低成本的單株抗體產生方式,我們稱之為單株抗體庫生產平台。 此平台使用混合抗原來免疫一隻小鼠,一次細胞融合的手續,理論上可篩得數十至上百種不同抗原之相對應單株抗體。此法可大幅降低生產成本與時程,非常符合現今高產出的研究特性。然而,傳統單株抗體的生產方式,卻無法直間應用於此抗體庫,單株抗體庫之建構仍必須突破數個技術瓶頸,包括了 (1) 提昇融合瘤細胞存活率、(2) 增加篩選方式之靈敏度,以及 (3) 高效能抗體身分之鑑定平台,才能順利完成。研究過程中,我們已發現了提升融合瘤細胞存活率的最佳條件,其細胞存活率較傳統細胞培養條件高20倍以上。此外,最適化後的篩選方式,可以大幅增進獲得微弱反應細胞株的機率。而上述融合瘤細胞株的身份,則可藉由胜?圖譜結合質譜分析方式來鑑定。藉由上述單株抗體生產平台,我們已獲得抗人類血液蛋白質之單株抗體 153 株,部分抗體之相對應抗原之身份已鑑定出,包括人類血液主要蛋白 transferrin、albumin、fibronectin、IgA、?-1 antitrypsin、haptoglobin、IgG kappa light chain 等,其餘抗體身份陸續鑑定中。 此單株抗體生產平台之建立,不僅可以用來產生人類血液蛋白的單株抗體庫,亦可直接應用於所有生物材料的抗體庫置備。現有的單株抗體,將先開發用來去除人類血液主要蛋白之用的免疫親和性吸附管柱,後續將用來生產人類血液蛋白之單株抗體晶片。此單株抗體庫的建構與抗體晶片的開發成功,對於血液蛋白質體學的研究動能,及臨床檢驗之發展,應有非常正面的助益。
摘要(英)In clinical application, the diagnosis of disease often targets specific proteins in blood. The golden rule, early detection, early treatment, will be successfully accomplished if high specific and sensitive disease markers are identified. In these years, we have established the proteomic platform to screen specific protein markers for disease. Possible blood protein markers have been identified for studied case, such as Parkinson’s disease. However, more and more reports have shown that single protein markers sometimes can not be a reliable and accurate indicator for diagnosing disease. It is more proper to use “protein expression profile”, which represents the expression patterns of a set of proteins, as the criteria for clinical diagnosis. Our ultimate goal is to develop the antibody chips for human blood proteins, which can be used for generating the protein expression profile for clinical diagnosis and elucidating the possible pathological mechanism of diseases. Basically, antibody chip is a miniature ELISA assays, it is necessary, in advance, to obtain all required antibodies for the chip production. In other words, the production of polyclonal or monoclonal antibodies, therefore, is the key step in the industrial mass production of antibody chips. Generally, it is thought that monoclonal antibodies are more specific than polyclonal antibodies. And in most circumstance, the application of using monoclonal antibodies is more reproducible than polyclonal antibodies. Currently, the production of monoclonal antibodies begins from the immunization of mice by specific antigen, generation of hybridoma cells and screen for the positive clones. It has no problem for laboratory if only one or a few antigens are used. However, if monoclonal antibodies against hundreds to thousands of antigen are required, the traditional way would be very labour-, time- and money- consuming. The reduction of cost, both in labour and cost, for generation of large quantity antibodies, is the motivation of our research. In this thesis, we have established a workflow, namely the production platform for antibody bank, for high efficient and low cost production of antibody in large amount. This platform employs a simultaneous immunization of mixtures of different antigens on one mouse. In theory, by only one fusion with myeloma cell with the elicited spleen cells, hundreds of different antibody producing cell lines can be obtained. This process reduces greatly both in labour and cost, and is hence a very applicable method for current high throughput purpose. However, the traditional strategy for monoclonal production can not be adapted directly on our platform. It is required to optimize the condition for (1) increase the survival rate of obtaining hybridoma cell lines, (2) increasing the sensitivity of screening methods, and (3) identifying the obtaining antibodies by high throughput method. In our study, we have optimized the hybridoma growth condition which can increase up to 20 times more viable colonies. We also optimized a sensitive screen procedure that enables us to identify very weak hybridoma clones. The identity of produced hybridoma can be validated by utilization of peptide mapping and mass spectrometry. By employing the established platform, we have identified 153 clones of hybridoma which produce monoclonal antibodies against different human plasm proteins, including transferrin, albumin, fibronectin, IgA, ?-1 antitrypsin, haptoglobin, IgG kappa light chain, and so forth. The identification of other hybridomas is in progress. The established platform for generating monoclonal antibody bank can apply to all biological samples, including human plasma proteins. The obtained monoclonal antibodies against the major proteins in human blood will be used for developing immuno-depletion chromatograph media. In the coming future, it will be also used for fabricate the antibody chip. The success in the development of the production platform for the monoclonal antibody bank and the antibody chip will input tremendous energy into the research of sero-proteomics and clinical diagnosis.
論文目次目錄 I 謝誌 IV 中文摘要 V ABSTRACT VI 第一章 概論 - 1 - 1.1血液蛋白質體學之特點 - 1 - 1.1.1 以蛋白質為臨床檢驗標的物之優點 - 1 - 1.1.2 血液為一良好之臨床檢驗樣品 - 2 - 1.1.3 鑑定疾病相關之血液蛋白質標的物 - 4 - 1.2 蛋白質標的物於臨床篩檢之應用 - 5 - 1.2.1 單一標的物於應用上之限制 - 6 - 1.2.2蛋白質表現側寫 (protein expression profile) - 7 - 1.2.3 SELDI 分析原理與其應用 - 8 - 1.3 血液蛋白質之抗體晶片 - 10 - 1.3.1 抗體晶片之原理與應用 - 10 - 1.3.2 人類血液蛋白質晶片之開發 - 12 - 1.3.3 建立人類血液蛋白之單株抗體庫 - 12 - 1.4 單株抗體庫相關研究之現況 - 13 - 1.4.1 國內部分 - 13 - 1.4.2 國外部分 - 14 - 1.5 研究動機與目的 - 15 - 第二章 材料與方法 - 16 - 2.1 血漿白蛋白去除 - 16 - 2.2 血漿免疫球蛋白去除 - 17 - 2.3 蛋白質沉澱與濃縮 - 18 - 2.3.1 蛋白質沉澱 (protein precipitation) - 18 - 2.3.2 蛋白質濃縮 (protein concentration) - 19 - 2.4 蛋白質定量 - 20 - 2.5 第一次元電泳 - 22 - 2.5.1第一次元電泳 (Isoelectric focusing, IEF) - 22 - 2.5.2 IPG 膠體平衡反應 (IPG strip equilibration) - 24 - 2.6 第二次元電泳 - 25 - 2.7 膠體染色 - 28 - 2.8 影像擷取與分析 - 32 - 2.9 蛋白?水解 - 34 - 2.10 質譜儀樣品處理 - 37 - 2.11 製備式電泳與電泳溶離 - 39 - 2.12 單株抗體製備 - 40 - 2.12.1 動物免疫 - 41 - 2.12.2 尾部採血 - 43 - 2.12.3 細胞融合 - 44 - 2.12.4 細胞保存法 - 49 - 2.12.5 單株抗體生產 - 50 - 2.12.6 免疫球蛋白純化 - 51 - 2.12.7 免疫球蛋白身分鑑定 - 52 - 2.13 酵素免疫分析法 - 54 - 2.14 蛋白質免疫轉印法 - 56 - 2.14.1 蛋白質電泳轉印法 - 56 - 2.14.2 酵素免疫染色 - 57 - 第三章 實驗結果 - 61 - 3.1 以人類血清蛋白免疫小鼠以建立單株抗體庫 - 61 - 3.1.1 以親和性吸附法去除血清中的大量蛋白質 - 62 - 3.1.2 誘導小鼠產生免疫反應 - 62 - 3.2 建立單株抗體庫之所需之關鍵技術 - 62 - 3.2.1 提昇融合瘤細胞存活率 - 63 - 3.2.1.1 改以人類血漿培養融合瘤細胞 - 63 - 3.2.1.2 添加介白質 6 - 64 - 3.2.1.3 調整融合細胞之比例 - 65 - 3.2.1.4 改變培養初融合瘤細胞之密度 - 66 - 3.2.2 改進篩選方式之靈敏度 - 67 - 3.2.2.1 以 SDS 破壞抗原的三度空間結構 - 67 - 3.2.2.2 改變 pH 值並以尿素破壞抗原立體結構 - 68 - 3.2.2.3 HRP 與 AP 可見光基質呈色之比較 - 68 - 3.2.2.4 培養基組成對 ELISA 呈色之影響 - 69 - 3.2.2.5 冷光免疫分析 (LIA) - 69 - 3.2.2.6 16 槽快速篩選器應用於免疫染色 - 70 - 3.2.2.7 人類血漿濃度對於免疫染色之影響 - 70 - 3.2.2.8 填充反應之比較 - 71 - 3.2.2.9 冷光試劑之比較 - 71 - 3.2.2.10 Tween 20 濃度之比較 - 71 - 3.2.3抗體身份鑑定 - 72 - 3.3現有之小鼠抗人類血清蛋白單株抗體庫內容 - 72 - 3.3.1 單株抗體庫之免疫染色圖譜 - 72 - 3.3.2 單株抗體庫之融合瘤細胞身份一覽表 - 72 - 3.3.3單株抗體庫之免疫沈澱分析 - 73 - 第四章 討論 - 74 - 4.1以混合抗原建構單株抗體庫是可行的 - 74 - 4.2融合瘤細胞培養條件有助於提昇存活率 - 74 - 4.3 篩選方式條件之最適化 - 75 - 4.4 未來展望 - 75 - 第五章 圖表集 - 76 - 第六章 參考資料 - 109 - 答問錄 - 114 -
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異動記錄M admin Y2008.M7.D3 23:18 61.59.161.35