博碩士論文 993204015 詳細資訊




以作者查詢圖書館館藏 以作者查詢臺灣博碩士 以作者查詢全國書目 勘誤回報 、線上人數:7 、訪客IP:3.233.239.102
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)
相關論文
★ 量子點表面改質與動物細胞標定★ 以螢光光譜觀測蛋白質吸附於疏水表面後之構型變化與吸附位向
★ 利用雙功能吸附基材進行蛋白復性-蛋白吸附狀態對復性的影響★ 界面聚合之奈米過濾膜的抗氯性研究
★ 以螢光光譜探討Indolicidin及其類似物與微脂粒之交互作用★ 負電性奈米過濾膜之排鹽特性
★ 金奈米粒子親水化及與DNA一對一鍵結之探討★ 以雙重電性表面改質方式製作抗生物吸附之超過濾與奈米過濾膜
★ 以表面修飾之材料控制間葉幹細胞貼附及對其往軟骨分化之影響★ 金奈米粒子與DNA一對一鍵結及其在檢測單一核苷酸變異的應用
★ 以三聚氰氯為單體的抗氯型奈米過濾膜★ 鹼性胜肽抗生素indolicidin及其類似物之溶血作用機制探討
★ 蛋白質特定方向固定化-以α-amylase為例★ Indolicidin及其類似物與微脂粒交互作用之熱力學研究
★ 位向性固定化葡萄糖氧化酶之新方法★ Indolicidin 及其類似物與微脂粒交互作用之焓測 量
檔案 [Endnote RIS 格式]    [Bibtex 格式]    [相關文章]   [文章引用]   [完整記錄]   [館藏目錄]   至系統瀏覽論文 ( 永不開放)
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
參考文獻 [1] P.S. Mead, L. Slutsker, V. Dietz, L.F. McCaig, J.S. Bresee, C. Shapiro, P.M. Griffin, R.V. Tauxe, Food-related illness and death in the United States, Emerg Infect Dis 5 (1999) 607-625.
[2] B. Lu, M.R. Smyth, R. Okennedy, Oriented immobilization of antibodies and its applications in immunoassays and immunosensors, Analyst 121 (1996) R29-R32.
[3] U. Bilitewski, Protein-sensing assay formats and devices, Analytica Chimica Acta 568 (2006) 232-247.
[4] F. Rusmini, Z.Y. Zhong, J. Feijen, Protein immobilization strategies for protein biochips, Biomacromolecules 8 (2007) 1775-1789.
[5] S.P. Silvia Ferretti, David A. Russell*, Kim E. Sapsford, Self-assembled monolayers: a versatile tool for the formulation of bio-surfaces, trends in analytical chemistry 19 (2000) 530-540.
[6] R.T. Karin Busch, Single molecule research on surfaces: from analytics to construction and back, Reviews in Molecular Biotechnology 82 (2001) 3-24.
[7] N.K. Jo Tominagaa, Satoshi Doia, Hirofumi Ichinoseb, Masahiro Gotoa, An enzymatic strategy for site-specific immobilization of functional proteins using microbial transglutaminase, Enzyme and Microbial Technology 35 (2004) 613-618.
[8] Z. Pei, H. Anderson, A. Myrskog, G. Dunér, B. Ingemarsson, T. Aastrup, Optimizing immobilization on two-dimensional carboxyl surface: pH dependence of antibody orientation and antigen binding capacity, Anal Biochem 398 (2010) 161-168.
[9] G.F.n.-L. Cesar Mateo, Olga Abian, Roberto Ferna´ndez-Lafuente, and, J.M. Guisa´n*, Multifunctional Epoxy Supports: A New Tool To Improve the Covalent Immobilization of Proteins. The Promotion of Physical Adsorptions of Proteins on the Supports before Their Covalent Linkage, Biomacromolecules 1 (2000) 739-745.
[10] U. Bilitewski, Protein-sensing assay formats and devices, Anal Chim Acta 568 (2006) 232-247.
[11] A.A. Karyakin, G.V. Presnova, M.Y. Rubtsova, A.M. Egorov, Oriented immobilization of antibodies onto the gold surfaces via their native thiol groups, Analytical Chemistry 72 (2000) 3805-3811.
[12] K. Bonroy, F. Frederix, G. Reekmans, E. Dewolf, R. De Palma, G. Borghs, P. Declerck, B. Goddeeris, Comparison of random and oriented immobilisation of antibody fragments on mixed self-assembled monolayers, Journal of Immunological Methods 312 (2006) 167-181.
[13] W.C. Tsai, P.J.R. Pai, Surface plasmon resonance-based immunosensor with oriented immobilized antibody fragments on a mixed self-assembled monolayer for the determination of staphylococcal enterotoxin B, Microchimica Acta 166 (2009) 115-122.
[14] W. Lee, B.K. Oh, W.H. Lee, J.W. Choi, Immobilization of antibody fragment for immunosensor application based on surface plasmon resonance, Colloids Surf B Biointerfaces 40 (2005) 143-148.
[15] B.G. Mathias Uhlen$QlI, Bjorn NilssonSStTeni, Gatenbeck$, LennarPt hilipsonQII, and, M. Lindberg$*, Complete Sequence of the Staphylococcal Gene EncodinPg rotein A, THE JOURNAOFL BIOLOGICAL CHEMISTRY 259 (1984) 1695-1702.
[16] P. Gagnon, Technology trends in antibody purification, Journal of Chromatography A 1221 (2012) 57-70.
[17] A. Kausaite-Minkstimiene, A. Ramanaviciene, J. Kirlyte, A. Ramanavicius, Comparative Study of Random and Oriented Antibody Immobilization Techniques on the Binding Capacity of Immunosensor, Analytical Chemistry 82 (2010) 6401-6408.
[18] G. Bergstrom, C.F. Mandenius, Orientation and capturing of antibody affinity ligands: Applications to surface plasmon resonance biochips, Sensors and Actuators B-Chemical 158 (2011) 265-270.
[19] Y. Yuan, H.Y. He, J. Lee, Protein A-Based Antibody Immobilization Onto Polymeric Microdevices for Enhanced Sensitivity of Enzyme-Linked Immunosorbent Assay, Biotechnol Bioeng 102 (2009) 891-901.
[20] T. Ikeda, Y. Hata, K. Ninomiya, Y. Ikura, K. Takeguchi, S. Aoyagi, R. Hirota, A. Kuroda, Oriented immobilization of antibodies on a silicon wafer using Si-tagged protein A, Anal Biochem 385 (2009) 132-137.
[21] Z.H. Youngeun Kwon, Ece Karatan, Milan Mrksich, and Brian K. Kay, Antibody Arrays Prepared by Cutinase-Mediated Immobilization on Self-Assembled Monolayers, Anal. Chem. 76 (2004) 5713-5720.
[22] C.D. Kang, S.W. Lee, T.H. Park, S.J. Sim, Performance enhancement of real-time detection of protozoan parasite, Cryptosporidium oocyst by a modified surface plasmon resonance (SPR) biosensor, Enzyme and Microbial Technology 39 (2006) 387-390.
[23] X. Wen, H. He, L.J. Lee, Specific antibody immobilization with biotin-poly(l-lysine)-g-poly(ethylene glycol) and protein A on microfluidic chips, Journal of Immunological Methods 350 (2009) 97-105.
[24] W.-C.W. Hsiu-Mei Chen, and Sheng-Horng Chen, A Metal-Chelating Piezoelectric Sensor Chip for Direct Detection and Oriented Immobilization of PolyHis-Tagged Proteins, Biotechnol. Prog. 20 (2004) 1237-1244.
[25] S.C. Naoufel Haddour, * and Chantal Gondran, Electrogeneration of a Poly(pyrrole)-NTA Chelator Film for a Reversible Oriented Immobilization of Histidine-Tagged Proteins, J. AM. CHEM. SOC. 127 (2005) 5752-5753.
[26] E.L. Schmid, T.A. Keller, Z. Dienes, H. Vogel, Reversible oriented surface immobilization of functional proteins on oxide surfaces, Analytical Chemistry 69 (1997) 1979-1985.
[27] C. Boozer, J. Ladd, S. Chen, Q. Yu, J. Homola, S. Jiang, DNA Directed Protein Immobilization on Mixed ssDNA/Oligo(ethylene glycol) Self-Assembled Monolayers for Sensitive Biosensors, Analytical Chemistry 76 (2004) 6967-6972.
[28] C. Boozer, J. Ladd, S.F. Chen, S.T. Jiang, DNA-directed protein immobilization for simultaneous detection of multiple analytes by surface plasmon resonance biosensor, Analytical Chemistry 78 (2006) 1515-1519.
[29] Y. Jung, J.M. Lee, H. Jung, B.H. Chung, Self-Directed and Self-Oriented Immobilization of Antibody by Protein G−DNA Conjugate, Analytical Chemistry 79 (2007) 6534-6541.
[30] H. Yang, P.V. Gurgel, R.G. Carbonell, Hexamer peptide affinity resins that bind the Fc region of human immunoglobulin G, J Pept Res 66 Suppl 1 (2005) 120-137.
[31] G. Fassina, A. Verdoliva, M.R. Odierna, M. Ruvo, G. Cassini, Protein a mimetic peptide ligand for affinity purification of antibodies, Journal of Molecular Recognition 9 (1996) 564-569.
[32] W.L. DeLano, M.H. Ultsch, A.M. de Vos, J.A. Wells, Convergent solutions to binding at a protein-protein interface, Science 287 (2000) 1279-1283.
[33] Y. Jung, H.J. Kang, J.M. Lee, S.O. Jung, W.S. Yun, S.J. Chung, B.H. Chung, Controlled antibody immobilization onto immunoanalytical platforms by synthetic peptide, Anal Biochem 374 (2008) 99-105.
[34] O.A. Cesar Mateo, Roberto Fernandez-Lafuente, Jose M. Guisan, Reversible Enzyme Immobilization via a Very Strong and Nondistorting Ionic Adsorption on Support– Polyethylenimine Composites, BIOTECHNOLOGY AND BIOENGINEERING, 68 (2000).
[35] B.M. Inbal Halperin, HaimWolfson, and Ruth Nussinov, Principles of Docking: An Overviewof Search Algorithms and a Guide to Scoring Functions, PROTEINS: Structure, Function, and Genetics 47 (2002) 409-443.
[36] 林榮信, 計算統計物理與藥物設計, 物理雙月刊 廿九卷 (2007) 920-928.
[37] B.G.a.A. Goldblum*, High quality binding modes in docking ligands to proteins, Proteins 71 (2008) 1373–1386.
[38] B.J. Alder, T.E. Wainwright, Studies in Molecular Dynamics. I. General Method, The Journal of Chemical Physics 31 (1959) 459-466.
[39] F.H. Stillinger, A. Rahman, Improved simulation of liquid water by molecular dynamics, The Journal of Chemical Physics 60 (1974) 1545-1557.
[40] J.A. McCammon, B.R. Gelin, M. Karplus, Dynamics of folded proteins, Nature 267 (1977) 585-590.
[41] B.R. Brooks, C.L. Brooks, A.D. Mackerell, L. Nilsson, R.J. Petrella, B. Roux, Y. Won, G. Archontis, C. Bartels, S. Boresch, A. Caflisch, L. Caves, Q. Cui, A.R. Dinner, M. Feig, S. Fischer, J. Gao, M. Hodoscek, W. Im, K. Kuczera, T. Lazaridis, J. Ma, V. Ovchinnikov, E. Paci, R.W. Pastor, C.B. Post, J.Z. Pu, M. Schaefer, B. Tidor, R.M. Venable, H.L. Woodcock, X. Wu, W. Yang, D.M. York, M. Karplus, CHARMM: The Biomolecular Simulation Program, Journal of Computational Chemistry 30 (2009) 1545-1614.
[42] P. England, F. Bregegere, H. Bedouelle, Energetic and kinetic contributions of contact residues of antibody D1.3 in the interaction with lysozyme, Biochemistry 36 (1997) 164-172.
[43] Z.N. Wu, K.W. Johnson, Y. Choi, T.L. Ciardelli, LIGAND-BINDING ANALYSIS OF SOLUBLE INTERLEUKIN-2 RECEPTOR COMPLEXES BY SURFACE-PLASMON RESONANCE, Journal of Biological Chemistry 270 (1995) 16045-16051.
[44] J. Ladd, C. Boozer, Q.M. Yu, S.F. Chen, J. Homola, S. Jiang, DNA-directed protein immobilization on mixed self-assembled monolayers via a Streptavidin bridge, Langmuir 20 (2004) 8090-8095.
[45] P. Ansorena, A. Zuzuarregui, E. Perez-Lorenzo, M. Mujika, S. Arana, Comparative analysis of QCM and SPR techniques for the optimization of immobilization sequences, Sensors and Actuators B-Chemical 155 (2011) 667-672.
[46] A. Otto, Excitation of nonradiative surface plasma waves in silver by the method of frustrated total reflection, Zeitschrift für Physik 216 (1968) 398-410.
[47] E. Kretschmann, H. Raether, Radiative decay of non radiative surface plasmons excited by light, Radiative Decay of Nonradiative Surface Plasmons Excited by Light (1968) 2135-2136.
[48] K. Inamori, M. Kyo, Y. Nishiya, Y. Inoue, T. Sonoda, E. Kinoshita, T. Koike, Y. Katayama, Detection and quantification of on-chip phosphorylated peptides by surface plasmon resonance imaging techniques using a phosphate capture molecule, Analytical Chemistry 77 (2005) 3979-3985.
[49] K.V. Gobi, H. Iwasaka, N. Miura, Self-assembled PEG monolayer based SPR immunosensor for label-free detection of insulin, Biosens Bioelectron 22 (2007) 1382-1389.
[50] A.J. Haes, L. Chang, W.L. Klein, R.P. Van Duyne, Detection of a biomarker for Alzheimer’’s disease from synthetic and clinical samples using a nanoscale optical biosensor, Journal of the American Chemical Society 127 (2005) 2264-2271.
[51] S. Cimitan, M.T. Lindgren, C. Bertucci, U.H. Danielson, Early absorption and distribution analysis of antitumor and anti-AIDS drugs: Lipid membrane and plasma protein interactions, Journal of Medicinal Chemistry 48 (2005) 3536-3546.
[52] T.R.K. Reddy, R. Mutter, W. Heal, K. Guo, V.J. Gillet, S. Pratt, B. Chen, Library design, synthesis, and screening: Pyridine dicarbonitriles as potential prion disease therapeutics, Journal of Medicinal Chemistry 49 (2006) 607-615.
[53] P.P. Dillon, S.J. Daly, A.J. Killard, R. O’’Kennedy, Development and use of antibodies in surface plasmon resonance-based immunosensors for environmental monitoring, International Journal of Environmental Analytical Chemistry 83 (2003) 525-543.
[54] K.V. Gobi, H. Tanaka, Y. Shoyama, N. Miura, Highly sensitive regenerable immunosensor for label-free detection of 2,4-dichlorophenoxyacetic acid at ppb levels by using surface plasmon resonance imaging, Sensors and Actuators B-Chemical 111 (2005) 562-571.
[55] W. Haasnoot, R. Verheijen, A direct (Non-Competitive) immunoassay for gentamicin residues with an optical biosensor, Food and Agricultural Immunology 13 (2001) 131-134.
[56] W. Haasnoot, K. Olieman, G. Cazemier, R. Verheijen, Direct biosensor immunoassays for the detection of nonmilk proteins in milk powder, Journal of Agricultural and Food Chemistry 49 (2001) 5201-5206.
[57] C. Mader, C. Huber, D. Moll, U.B. Sleytr, M. Sara, Interaction of the crystalline bacterial cell surface layer protein SbsB and the secondary cell wall polymer of Geobacillus stearothermophilus PV72 assessed by real-time surface plasmon resonance biosensor technology, Journal of Bacteriology 186 (2004) 1758-1768.
[58] B.K. Oh, W. Lee, B.S. Chun, Y.M. Bae, W.H. Lee, J.W. Choi, Surface plasmon resonance immunosensor for the detection of Yersinia enterocolitica, Colloids and Surfaces a-Physicochemical and Engineering Aspects 257-58 (2005) 369-374.
[59] S. Dhesingh Ravi, M. Norio, Trends in interfacial design for surface plasmon resonance based immunoassays, Journal of Physics D: Applied Physics 40 (2007) 7187.
[60] L.J. Harris, S.B. Larson, K.W. Hasel, A. McPherson, Refined Structure of an Intact IgG2a Monoclonal Antibody†,‡, Biochemistry 36 (1997) 1581-1597.
[61] A.D. MacKerell, D. Bashford, M. Bellott, R.L. Dunbrack, J.D. Evanseck, M.J. Field, S. Fischer, J. Gao, H. Guo, S. Ha, D. Joseph-McCarthy, L. Kuchnir, K. Kuczera, F.T.K. Lau, C. Mattos, S. Michnick, T. Ngo, D.T. Nguyen, B. Prodhom, W.E. Reiher, B. Roux, M. Schlenkrich, J.C. Smith, R. Stote, J. Straub, M. Watanabe, J. Wiorkiewicz-Kuczera, D. Yin, M. Karplus, All-atom empirical potential for molecular modeling and dynamics studies of proteins, Journal of Physical Chemistry B 102 (1998) 3586-3616.
[62] D.R. Shankaran, K.V. Gobi, N. Miura, Recent advancements in surface plasmon resonance immunosensors for detection of small molecules of biomedical, food and environmental interest, Sensors and Actuators B: Chemical 121 (2007) 158-177.
[63] G.B. Sigal, M. Mrksich, G.M. Whitesides, Effect of surface wettability on the adsorption of proteins and detergents, Journal of the American Chemical Society 120 (1998) 3464-3473.
[64] F. Frederix, K. Bonroy, W. Laureyn, G. Reekmans, A. Campitelli, W. Dehaen, G. Maes, Enhanced Performance of an Affinity Biosensor Interface Based on Mixed Self-Assembled Monolayers of Thiols on Gold, Langmuir 19 (2003) 4351-4357.
[65] M. Piliarik, M. Bockova, J. Homola, Surface plasmon resonance biosensor for parallelized detection of protein biomarkers in diluted blood plasma, Biosens Bioelectron 26 (2010) 1656-1661.
[66] W.-P. Hu, L.-Y. Huang, T.-C. Kuo, W.-W. Hu, Y. Chang, C.-S. Chen, H.-C. Chen, W.-Y. Chen, Optimization of DNA-directed immobilization on mixed oligo(ethylene glycol) monolayers for immunodetection, Anal Biochem 423 (2012) 26-35.
[67] D.R. Shankaran, K. Matsumoto, K. Toko, N. Miura, Development and comparison of two immunoassays for the detection of 2,4,6-trinitrotoluene (TNT) based on surface plasmon resonance, Sensors and Actuators B-Chemical 114 (2006) 71-79.
指導教授 阮若屈(Ruoh-chyu Ruaan) 審核日期 2012-7-31
推文 facebook   plurk   twitter   funp   google   live   udn   HD   myshare   reddit   netvibes   friend   youpush   delicious   baidu   
網路書籤 Google bookmarks   del.icio.us   hemidemi   myshare   

若有論文相關問題,請聯絡國立中央大學圖書館推廣服務組 TEL:(03)422-7151轉57407,或E-mail聯絡  - 隱私權政策聲明