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研究生： 陳致勤
Chih-Chin Chen
論文名稱： 一種人類促腎上腺皮質激素免疫分析新方法的開發
Development of a new immunoassay for human adrenocorticotropic hoemone
指導教授： 許宗雄
Dr. Tzong-Hsiung Hseu
口試委員:
學位類別： 碩士
Master
系所名稱： 生命科學暨醫學院 - 生命科學系
Department of Life Sciences
論文出版年： 2003
畢業學年度： 91
語文別： 中文
論文頁數： 80
中文關鍵詞： 單株抗體 、促腎上腺皮質激素 、綠色螢光蛋白質 、融合蛋白質 、表面薄層共振 、競爭性酵素連接免疫吸附分析
外文關鍵詞： monoclonal antibody, Adrenocorticotropic Hormone (ACTH), Enhanced Green Fluorescent Protein (EGFP), fusion protein, Surface Plasmon Resonance (SPR), Competitive Enzyme-Linked Immunosorbent Assay (CELISA)
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    • 本研究旨在開發一種以單株抗體為基礎，用來分析人類血清中促腎上腺皮質激素(Adrenocorticotropic Hormone, ACTH)的免疫分析新方法。這種免疫分析的原理是利用人類ACTH和ACTH-EGFP (Enhanced Green Fluorescent Protein綠色螢光蛋白質)融合蛋白質之間對抗-ACTH單株抗體的競爭，造成信號強弱的改變而來。為此，我們建構了一個帶有ACTH-EGFP基因的質體，並利用鎳離子樹脂從大腸桿菌中純化這個His˙tag融合蛋白質。利用表面薄層共振(Surface Plasmon Resonance, SPR)來測定ACTH-EGFP、Human ACTH 1-39和抗-ACTH單株、多株抗體的親和力。抗-ACTH單株抗體的親和力較高，對ACTH-EGFP為1.94×10-12 M、對Human ACTH 1-39為1.36×10-8 M。最後，這種競爭性酵素連接免疫吸附分析(Competitive Enzyme-Linked Immunosorbent Assay, CELISA)能測量4~10 ng/ml範圍內的人類ACTH。

    An immunoassay based on a monoclonal antibody for the analysis of adrenocorticotropic hormone (ACTH) in human serum has been developed. The principle of this immunoassay is the competition between human ACTH and ACTH-EGFP (Enhanced Green Fluorescent Protein) fusion protein. We constructed a prokaryotic vector expressing ACTH-EGFP gene and purified this His˙tag fusion protein from Escherichia coli cultures using nickel resin.
    Surface plasmon resonance (SPR) was used as an independent means of determining the affinity of ACTH-EGFP, human ACTH 1-39 and anti-ACTH monoclonal, polyclonal antibodies. The interaction between anti-ACTH monoclonal antibody and ACTH-EGFP, Human ACTH 1-39 was demonstrated to be of high affinity (1.94×10-12 M and 1.36×10-8 M, respectively).

    Finally, human ACTH concentration determined by this competitive enzyme-linked immunosorbent assay (CELISA) ranged from 4~10 ng/ml.

    目錄 摘要 1 Abstract 2 謝誌 3 目錄 4 表目錄 8 圖目錄 9 第一章 序論 11 第二章 材料與方法 16 材料 16 儀器 17 方法 17 質體的抽取 17 DNA電泳 17 質體的切割與建構 17 DNA的切割 17 DNA的分離 17 DNA的連接 18 轉型入大腸桿菌中 18 引子的設計 18 EGFP PCR 19 His˙Tag融合蛋白質(ACTH-EGFP)的表現 19 Ni-NTA Agarose管柱的製備 19 ACTH-EGFP的純化 20 SDS-PAGE分離蛋白質 20 Western blot 20 定量蛋白質 21 濃縮蛋白質 21 抗-ACTH多株抗體的純化 21 GST融合蛋白質(GST A3-1M)的表現 21 GST A3-1M的純化 22 CNBr-activated Sepharose 4B的製備 22 GST A3-1M的聯結 22 用GST A3-1M的親和力純化抗-ACTH多株抗體 23 SPR 23 螢光免疫分析(Fluoroimmunoassay, FIA) 23 三明治型ELISA (Sandwich ELISA, SELISA) 24 競爭性FIA (Competitive FIA, CFIA) 24 競爭性ELISA (Competitive ELISA, CELISA) 25 第三章 結果 26 pEGFP-N1 H/B的分離 26 EGFP PCR 26 表現ACTH-EGFP勝任細胞的選擇 26 ACTH-EGFP表現條件的選擇 26 GST A3-1M的表現 27 GST A3-1M的純化 27 利用GST A3-1M的親和力純化抗-ACTH多株抗體 27 SPR 28 選擇固定鍵合物的pH值 28 Amine Coupling 28 親和力分析 29 FIA 29 SELISA 29 CFIA 30 CELISA 30 第四章 討論 31 第五章 結論 35 參考文獻 36 附錄 75 表目錄 表一、GFP和EGFP的光譜學特性比較表。 41 表二、用SPR分析ACTH-EGFP、Human ACTH 1-39和抗-ACTH多株、單株抗體結合的動力學和親和力資料表。 42 圖目錄 圖一、糖皮質類固醇對ACTH和CRH的負回饋控制圖。 43 圖二、POMC水解成ACTH和其他荷爾蒙示意圖。 44 圖三、質體pEGFP-N1特徵圖。 45 圖四、用HindIII和BamHI切割質體pEGFP-N1產物電泳圖。 46 圖五、EGFP PCR產物電泳圖。 47 圖六、建構好的質體pET15b-ACTH-EGFP特徵圖。 48 圖七、在BL21(DE3)用1 mM IPTG誘導ACTH-EGFP表現的SDS-PAGE圖。 49 圖八、在變性的情況下純化ACTH-EGFP的SDS-PAGE圖。 50 圖九、在自然的情況下純化ACTH-EGFP的SDS-PAGE和Western blot圖。 51 圖十、ACTH-EGFP光譜圖。 52 圖十一、質體GST A3-1M特徵圖。 53 圖十二、在Top10用1 mM IPTG誘導GST A3-1M表現的SDS-PAGE圖。 54 圖十三、純化GST A3-1M的SDS-PAGE圖。 55 圖十四、用抗-ACTH血清當第一抗體的ACTH-EGFP Western blot圖。 56 圖十五、純化抗-ACTH多株抗體的SDS-PAGE圖。 57 圖十六、用抗-ACTH多株抗體當第一抗體的ACTH-EGFP Western blot圖。 58 圖十七、用不同pH值緩衝液固定ACTH-EGFP感應圖。 59 圖十八、用不同pH值緩衝液固定Human ACTH 1-39感應圖。 60 圖十九、用Amine Coupling Kit將ACTH-EGFP固定在Sensor Chip CM5上感應圖。 61 圖二十、用Amine Coupling Kit將Human ACTH 1-39固定在Sensor Chip CM5上感應圖。 62 圖二十一、不同濃度的抗-ACTH多株抗體和ACTH-EGFP結合感應圖。 63 圖二十二、不同濃度的抗-ACTH單株抗體和ACTH-EGFP結合感應圖。 64 圖二十三、不同濃度的抗-ACTH多株抗體和Human ACTH 1-39結合感應圖。 65 圖二十四、不同濃度的抗-ACTH單株抗體和Human ACTH 1-39結合感應圖。 66 圖二十五、50 μg/ml抗-ACTH多株、單株抗體和Human ACTH 1-39結合比較圖。67 圖二十六、FIA數據線性迴歸圖。 68 圖二十七、SELISA數據線性迴歸圖。 69 圖二十八、典型的CELISA標準曲線圖和CFIA數據趨勢圖。 70 圖二十九、典型的CELISA半對數標繪圖和CFIA數據半對數圖。 71 圖三十、典型的CELISA用log-logit變化成線性分析圖和CFIA數據用log-logit變化成線性迴歸圖。 72 圖三十一、CELISA數據趨勢圖、半對數圖和用log-logit變化成線性迴歸圖。 74

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