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姓名	鄭祥龍(Siang-long Jheng)  查詢紙本館藏	畢業系所	化學工程與材料工程學系
論文名稱	老鼠免疫球蛋白IgG2a之位向性固定法—Fc區域的親和性配體設計 (Peptide Ligand Design for Oriented Immobilization of Mouse IgG2a through Its Fc Region)
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摘要(中)	免疫生物感測器是藉由偵測在生物晶片表面上抗體(antibody)與抗原 (antigen)之間的辨識行為，以檢測溶液相中之有害物、病原體或是特定的標 示物(marker)，而偵測的靈敏度會受到下列因素的影響：儀器感測訊號的機 制、抗體在晶片表面的數量、抗體固定化的位向性。而抗體在晶片表面上 隨機的固定化會造成偵測訊號下降，因此為了提升抗體抗原之間的辨識效 率，抗體於基材表面的位向性固定化方式被廣為研究。 我們提出一個策略設計與抗體 Fc 區域有高結合親和力(binding affinity)的 胜肽配體(peptide ligand)。結合分子嵌合及分子動態模擬(molecular dynamics simulation，MD simulation)成功地設計、篩選出一條與Fc 區域有高親和力 的胜肽配體，以此作為理想配體之主要序列並改質於金片表面上，使用表 面電漿共振儀(surface plasmon resonance，SPR)量測Mouse IgG2a 在金片表面 的吸附量，以及PSA 與Mouse IgG2a 的二抗(secondary antibody，2nd Ab)被 辨識到的量，經計算後可得到Mouse IgG2a 的抗原辨識效率及位向因子。根 據我們提出之設計策略，可設計出用於抗體位向性固定之胜肽配體，並改 善抗體於表面之位向性，提升抗原辨識效率。
摘要(英)	Immunosensors utilize specific bio-affinity interaction between antibody and antigen to detect toxicants, pathogens, or specific markers in complex mixtures. Antibodies need to be immobilized on the detection surface before targeted antigen can be captured. The recognition sensitivity depends on the sensitivity of signal sensing mechanism, the amount of immobilized antibody, and the orientation of antibody immobilized. Signal can be significantly reduced due to the random orientation of antibody on chip surface. Therefore, the methods for oriented immobilization of antibody were studied by many research groups in order to improve the recognition efficiency. We propose a strategy to design a small peptide ligand which has high binding affinity to the Fc region of antibody. It is expected that antibodies can be oriented immobilized on surface through the affinity between the desired Fc region and peptide ligand. Human prostate specific antigen (PSA) and PSA specific antibody derived from Mouse IgG2a are selected as the targeted antigen and antibody in this study. A peptide with a high Fc binding affinity is successfully designed and selected by combining the molecular docking and MD simulation. The recognition efficiency and orientation factor are measured by the adsorption amounts of PSA and the secondary antibody of Mouse IgG2a iii through the help of surface plasmon resonance (SPR). Our approach provided a new strategy for oriented immobilization of antibody by small ligands, which can significantly improve the recognition efficiency.
關鍵字(中)	★ 表面電漿共振 ★ 分子動態模擬 ★ 分子嵌合 ★ 位向性固定 ★ 抗體	關鍵字(英)	★ SPR ★ MD simulation ★ molecular docking ★ antibody ★ oriented immobilization
論文目次	摘要 I Abstract III 誌謝 V 目錄 VI 圖目錄 IX 表目錄 XIII 第一章 緒論 1 1.1研究動機 1 1.2研究目的 3 第二章 文獻回顧 4 2.1蛋白質固定化 4 2.1.1共價鍵結(Covalent coupling) 6 2.1.2生物親和性(Bio-affinity) 10 2.1.3物理吸附(Physical adsorption) 18 2.2分子嵌合(Molecular docking) 20 2.2.1分子嵌合介紹 20 2.2.2 AutoDock 22 2.3分子動態模擬 (Molecular Dynamic Simulations) 25 2.3.1分子動態模擬計算方法 26 2.3.2 CHARMM力場(force field) 28 2.4表面電漿共振 (Surface Plasmon Resonance，SPR ) 29 2.4.1表面電漿共振原理 29 2.4.2表面電漿共振生物感測器(SPR-based biosensor) 32 第三章 實驗藥品、儀器、方法 35 3.1實驗藥品 35 3.2實驗儀器 37 3.3實驗方法 38 3.3.1抗體位向性固定化之策略 38 3.3.2抗體表面性質分析 39 3.3.3 Peptide Ligand之設計與篩選 40 3.3.4 SPR金片表面改質 41 3.3.5負電荷表面鑑定 44 3.3.6 EDC/NHS表面改質鑑定 46 3.3.7正電荷表面電性鑑定 47 3.3.8表面電漿共振(surface plasmon resonance，SPR)實驗 48 第四章 結果與討論 51 4.1抗體表面性質分析 51 4.1.1 Fc區域表面疏水性質分析 53 4.1.2 Ligand可能結合位置分析 55 4.2 Peptide Ligand之設計 57 4.3 Peptide Ligand之篩選 58 4.4 RRGW於Fc區域底部結合之專一性 66 4.5 SPR金片改質(Mixed SAM) 69 4.5.1負電荷表面鑑定 69 4.5.2 EDC/NHS表面改質鑑定 73 4.6正電荷表面電性鑑定 74 4.7表面電漿共振(surface plasmon resonance，SPR)實驗 75 4.7.1 Mouse IgG2a辨識抗原能力分析 77 4.7.2 Mouse IgG2a於表面之位向性分析 86 第五章 結論 93 第六章 參考文獻 95
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指導教授	阮若屈(Ruoh-chyu Ruaan)	審核日期	2012-7-31
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