請用此 Handle URI 來引用此文件：
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/34706完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 林?輝(Feng-Huei Lin) | |
| dc.contributor.author | Hao-Lin Chen | en |
| dc.contributor.author | 陳豪麟 | zh_TW |
| dc.date.accessioned | 2021-06-13T06:23:48Z | - |
| dc.date.available | 2012-07-29 | |
| dc.date.copyright | 2011-07-29 | |
| dc.date.issued | 2011 | |
| dc.date.submitted | 2011-07-26 | |
| dc.identifier.citation | 1. Kern, D. and E.R.P. Zuiderweg, The role of dynamics in allosteric regulation. Current Opinion in Structural Biology, 2003. 13(6): p. 748-757.
2. Cantrell, D., T cell antigen receptor signal transduction pathways. Annual Review of Immunology, 1996. 14: p. 259-274. 3. Davies, D.R., E.A. Padlan, and S. Sheriff, Antibody-Antigen Complexes. Annual Review of Biochemistry, 1990. 59: p. 439-473. 4. Wasserman, J.D., S. Urban, and M. Freeman, A family of rhomboid-like genes: Drosophila rhomboid-1 and roughoid/rhomboid-3 cooperate to activate EGF receptor signaling. Genes & Development, 2000. 14(13): p. 1651-1663. 5. Zhang, H.T., et al., ErbB receptors: from oncogenes to targeted cancer therapies. Journal of Clinical Investigation, 2007. 117(8): p. 2051-2058. 6. Goodey, N.M. and S.J. Benkovic, Allosteric regulation and catalysis emerge via a common route. Nature Chemical Biology, 2008. 4(8): p. 474-482. 7. Kohler, G. and C. Milstein, Continuous Cultures of Fused Cells Secreting Antibody of Predefined Specificity. Nature, 1975. 256(5517): p. 495-497. 8. Chambers, R.S., High-throughput antibody production. Current Opinion in Chemical Biology, 2005. 9(1): p. 46-50. 9. Goding, J.W., Antibody production by hybridomas. J Immunol Methods, 1980. 39(4): p. 285-308. 10. Kohler, G. and C. Milstein, Derivation of Specific Antibody-Producing Tissue-Culture and Tumor Lines by Cell-Fusion. European Journal of Immunology, 1976. 6(7): p. 511-519. 11. Herzenberg, L.A., et al., The history and future of the fluorescence activated cell sorter and flow cytometry: A view from Stanford. Clinical Chemistry, 2002. 48(10): p. 1819-1827. 12. Molday, R.S., S.P.S. Yen, and A. Rembaum, Application of Magnetic Microspheres in Labeling and Separation of Cells. Nature, 1977. 268(5619): p. 437-438. 13. Bonner, W.A., et al., Fluorescence Activated Cell Sorting. Review of Scientific Instruments, 1972. 43(3): p. 404-&. 14. Moldavan, A., Photo-Electric Technique for the Counting of Microscopical Cells. Science, 1934. 80(2069): p. 188-9. 15. Davey, H.M. and D.B. Kell, Flow cytometry and cell sorting of heterogeneous microbial populations: the importance of single-cell analyses. Microbiol Rev, 1996. 60(4): p. 641-96. 16. Fu, A.Y., et al., A microfabricated fluorescence-activated cell sorter. Nat Biotechnol, 1999. 17(11): p. 1109-11. 17. Miltenyi, S., et al., High-Gradient Magnetic Cell-Separation with Macs. Cytometry, 1990. 11(2): p. 231-238. 18. Herzenberg, L.A. and R.G. Sweet, Fluorescence-activated cell sorting. Sci Am, 1976. 234(3): p. 108-17. 19. Wang, T.W., et al., Application of highly sensitive, modified glass substrate-based immuno-PCR on the early detection of nasopharyngeal carcinoma. Biomaterials, 2008. 29(33): p. 4447-4454. 20. Dong, G.C., et al., A study on grafting and characterization of HMDI-modified calcium hydrogenphosphate. Biomaterials, 2001. 22(23): p. 3179-89. 21. Lin, C.C., et al., A cell sorter with modified bamboo charcoal for the efficient selection of specific antibody-producing hybridomas. Biomaterials, 2010. 31(32): p. 8445-53. 22. Hoogenboom, H.R., Selecting and screening recombinant antibody libraries. Nature Biotechnology, 2005. 23(9): p. 1105-1116. 23. Rajewsky, K., Clonal selection and learning in the antibody system. Nature, 1996. 381(6585): p. 751-758. 24. Nossal, G.J., The double helix and immunology. Nature, 2003. 421(6921): p. 440-4. 25. Jacobs, H. and L. Bross, Towards an understanding of somatic hypermutation. Current Opinion in Immunology, 2001. 13(2): p. 208-218. 26. Cambier, J.C., et al., B-cell anergy: from transgenic models to naturally occurring anergic B cells? Nature Reviews Immunology, 2007. 7(8): p. 633-643. 27. Kuby, J., Immunology. 3rd ed. 1997, New York: W.H. Freeman. xxiv, 664 p. 28. Stavnezer, J., Immunoglobulin class switching. Current Opinion in Immunology, 1996. 8(2): p. 199-205. 29. Kindt, T.J., et al., Kuby immunology. 6th ed. 2007, New York: W.H. Freeman. xxii, 574, A-31, G-12, AN-27, I-27 p. 30. Ollila, J. and M. Vihinen, B cells. International Journal of Biochemistry & Cell Biology, 2005. 37(3): p. 518-523. 31. Jastrow, H., M.A. VonMach, and L. Vollrath, Adaptation of the disector method to rare small organelles in TEM sections exemplified by counting synaptic bodies in the rat pineal gland. Journal of Anatomy, 1997. 191: p. 399-405. 32. Davies, D.R. and S. Chacko, Antibody Structure. Accounts of Chemical Research, 1993. 26(8): p. 421-427. 33. Federation of European Biochemical Societies., J. Gergely, and G.A. Medgyesi, Antibody structure and molecular immunology. Its Proceedings of the ninth FEBS meeting [6]. 1975, Amsterdam New York: North-Holland ; American Elsevier. vi, 170 p. 34. Sampathu, D.M., et al., Pathological heterogeneity of frontotemporal lobar degeneration with ubiquitin-positive inclusions delineated by ubiquitin immunohistochemistry and novel monoclonal antibodies. Am J Pathol, 2006. 169(4): p. 1343-52. 35. Chuntharapai, A., et al., Isotype-dependent inhibition of tumor growth in vivo by monoclonal antibodies to death receptor 4. Journal of Immunology, 2001. 166(8): p. 4891-8. 36. Grassi, J., et al., Screening of Monoclonal-Antibodies Using Antigens Labeled with Acetylcholinesterase - Application to the Peripheral Proteins of Photosystem-1. Analytical Biochemistry, 1988. 168(2): p. 436-450. 37. Bomford, R., Adjuvants for Anti-Parasite Vaccines. Parasitology Today, 1989. 5(2): p. 41-46. 38. Kishiro, Y., et al., A Novel Method of Preparing Rat-Monoclonal Antibody-Producing Hybridomas by Using Rat Medial Iliac Lymph-Node Cells. Cell Structure and Function, 1995. 20(2): p. 151-156. 39. Mirza, I.H., et al., A Comparison of Spleen and Lymph-Node Cells as Fusion Partners for the Raising of Monoclonal-Antibodies after Different Routes of Immunization. Journal of Immunological Methods, 1987. 105(2): p. 235-243. 40. Roitt, I.M., J. Brostoff, and D.K. Male, Immunology. 1985, London ; New York St. Louis: Gower Medical Pub. ; C.V. Mosby. 41. Vienken, J. and U. Zimmermann, An Improved Electrofusion Technique for Production of Mouse Hybridoma Cells. Febs Letters, 1985. 182(2): p. 278-280. 42. Wilmut, I., et al., Viable offspring derived from fetal and adult mammalian cells. Nature, 1997. 385(6619): p. 810-813. 43. Davidson, R.L., K.A. Omalley, and T.B. Wheeler, Polyethylene Glycol-Induced Mammalian-Cell Hybridization - Effect of Polyethylene-Glycol Molecular-Weight and Concentration. Somatic Cell Genetics, 1976. 2(3): p. 271-280. 44. Norwood, T.H., C.J. Zeigler, and G.M. Martin, Dimethyl-Sulfoxide Enhances Polyethylene Glycol-Mediated Somatic-Cell Fusion. Somatic Cell Genetics, 1976. 2(3): p. 263-270. 45. Destgroth, S.F. and D. Scheidegger, Production of Monoclonal-Antibodies - Strategy and Tactics. Journal of Immunological Methods, 1980. 35(1-2): p. 1-21. 46. Feit, C., et al., Monoclonal-Antibodies to Human Sarcoma and Connective-Tissue Differentiation Antigens. Cancer Research, 1984. 44(12): p. 5752-5756. 47. Lane, R.D., R.S. Crissman, and M.F. Lachman, Comparison of Polyethylene Glycols as Fusogens for Producing Lymphocyte-Myeloma Hybrids. Journal of Immunological Methods, 1984. 72(1): p. 71-76. 48. Littlefield, J.W., Selection of Hybrids from Matings of Fibroblasts in Vitro + Their Presumed Recombinants. Science, 1964. 145(363): p. 709-&. 49. Szybalska, E.H. and W. Szybalski, Genetics of Human Cell Lines .4. DNA-Mediated Heritable Transformation of a Biochemical Trait. Proceedings of the National Academy of Sciences of the United States of America, 1962. 48(12): p. 2026-&. 50. M-Y, C., 機能性竹炭之研製. National Pingtung University of Science an Technology, 2006. 51. Wu, K.H., et al., Preparation and characterization of bamboo charcoal/Ni0.5Zn0.5Fe2O4 composite with core-shell structure. Materials Letters, 2006. 60(21-22): p. 2707-2710. 52. Wang, J.Z., et al., Hydro-spinning: a novel technology for making alginate/chitosan fibrous scaffold. J Biomed Mater Res A, 2010. 93(3): p. 910-9. 53. Barbieri, A., et al., Magnetite Fe3o4(111) - Surface-Structure by Leed Crystallography and Energetics. Surface Science, 1994. 302(3): p. 259-279. 54. Wang, H., et al., Magnetoresistance and magnetic properties of Fe3O4 nanoparticle compacts. Chinese Physics, 2002. 11(2): p. 178-182. 55. Lee, F.N., A Method to Eliminate Solution Trapping in Applying Progressive Optimality Principle to Short-Term Hydrothermal Scheduling. Ieee Transactions on Power Systems, 1989. 4(3): p. 935-941. 56. Siano, D.B. and J. Bock, A Micro-Emulsion Method for Polymer Molecular-Weight Determination. Journal of Polymer Science Part C-Polymer Letters, 1982. 20(3): p. 151-158. 57. Vijayakumar, R., et al., Sonochemical synthesis and characterization of pure nanometer-sized Fe3O4 particles. Materials Science and Engineering a-Structural Materials Properties Microstructure and Processing, 2000. 286(1): p. 101-105. 58. Rahmi, D., et al., An in-syringe La-coprecipitation method for the preconcentration of oxo-anion forming elements in seawater prior to an ICP-MS measurement. Anal Sci, 2008. 24(9): p. 1189-92. 59. Baranov, V.S., et al., [The possibility of the incorporation of macromolecules, including exogenous DNA, into the germ cells of male mice. The liposome method and Ca-P coprecipitation method]. Tsitol Genet, 1990. 24(2): p. 52-5. 60. Kim, Y.J., et al., Surface characterization and in vitro blood compatibility of poly(ethylene terephthalate) immobilized with insulin and/or heparin using plasma glow discharge. Biomaterials, 2000. 21(2): p. 121-130. 61. Plueddemann, E.P., Reminiscing on Silane Coupling Agents. Journal of Adhesion Science and Technology, 1991. 5(4): p. 261-277. 62. Arslan, G., et al., Surface modification of glass beads with an aminosilane monolayer. Turkish Journal of Chemistry, 2006. 30(2): p. 203-210. 63. Kuan, H.C., et al., Synthesis, thermal, mechanical and rheological properties of multiwall carbon nano tube/waterborne polyurethane nanocomposite. Composites Science and Technology, 2005. 65(11-12): p. 1703-1710. 64. Saito, T., K. Matsushige, and K. Tanaka, Chemical treatment and modification of multi-walled carbon nanotubes. Physica B-Condensed Matter, 2002. 323(1-4): p. 280-283. 65. Zhang, G.X., et al., The surface analytical characterization of carbon fibers functionalized by H2SO4/HNO3 treatment. Carbon, 2008. 46(2): p. 196-205. 66. Jiang, K.Y., et al., Protein immobilization on carbon nanotubes via a two-step process of diimide-activated amidation. Journal of Materials Chemistry, 2004. 14(1): p. 37-39. 67. Tseng, C.L., et al., Development of gelatin nanoparticles with biotinylated EGF conjugation for lung cancer targeting. Biomaterials, 2007. 28(27): p. 3996-4005. 68. Desai, S.M. and R.P. Singh, Surface modification of polyethylene. Long-Term Properties of Polyolefins, 2004. 169: p. 231-293. 69. Clegg, W., Crystal structure analysis : principles and practice. International Union of Crystallography texts on crystallography. 2001, Chester, England New York: International Union of Crystallography ; Oxford University Press. xiv, 265 p. 70. Bretcanu, O., et al., The influence of crystallised Fe3O4 on the magnetic properties of coprecipitation-derived ferrimagnetic glass-ceramics. Acta Biomater, 2005. 1(4): p. 421-9. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/34706 | - |
| dc.description.abstract | 單株抗體的製備在1975年由George Kohler和Cesar Milstein成功地利用細胞融合的技術製造出來後就開始被廣泛的運用，然而其在操作流程上較為繁瑣，尤其在選殖融合瘤細胞(hybridoma cell)單株化的過程時，不僅需要花費大量的時間、步驟繁瑣、同時也必須消耗很多的人力，且操作者的熟練度往往對結果影響很大，由於以上諸多缺點，使單株抗體的製備一直無法有效地大量生產。先前，在本實驗室中，我們開發出一組高產量的細胞篩選器，成功的縮短單株化選殖的時間，此種細胞篩選器是利用本實驗室開發出來的虹吸式生物反應器(cytoflow-bioreactor)配合竹炭作為載體(carrier)。但是目前此細胞篩選器還是有一些問題，如載體(carrier)的顆粒大小受到限制、非專一性吸附的問題嚴重、專一性鍵結(specific binding)上carrier的細胞數量太少、專一性融合瘤細胞(specific hybridoma cell)的生產效率差以及無法有效地分離carrier和細胞…等等問題。
因此，本研究主要目的在於希望能改善此細胞篩選器(Cytoflow-Bioreactor base Cell Sorter, CBCS)的種種問題，提高細胞篩選器的分選效率與專一性融合瘤細胞(specific hybridoma cell)的生產效率，同時避免非專一性鍵結(non-specific binding)的可能性和降低操作上的困難，發展出高效率的虹吸式細胞篩選器(High-efficiency Cytoflow-Bioreactor base Cell Sorter, HCBCS)，最終達到單株抗體大量生產的目的。 | zh_TW |
| dc.description.abstract | George Kohler and Cesar Milstein employed fusion theory to produce monoclonal antibody in 1975. Monoclonal antibodies (mAbs) have been proven useful in research and clinical applications. However, the generation of mAbs by conventional hybridoma technology is time-, cost- and labor-consuming. Before, a siphon bioreactor and the carrier(bamboo charcoal) combined as a cell sorter called : Cytoflow-bioreactor base cell sorter(CBCS). Our design allows the production of mAbs while avoiding time-consuming steps, such as large numbers of limiting dilutions and screening assays, and demonstrates that the CBCS could be a powerful tool for monoclonal antibody production. But the CBCS still had some problems : the particle size of carrier is limited、non-specific binding、low-efficiency cell sorting and operational difficulties.
The purpose of study is improving cytoflow-bioreactor base cell sorter, promote cell sorting efficiency, avoid the possibility of non-specific binding and operational difficulties, create a high-efficiency cytoflow-bioreactor base cell sorter(HCBCS) could be employed to harvest antibody-secreting B cells from the peripheral blood without sacrificing the animal. In the future, we hope this method not only produce animals monoclonal antibody but also produce humanized monoclonal antibody, to achieve the purpose of monoclonal antibody mass production. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-13T06:23:48Z (GMT). No. of bitstreams: 1 ntu-100-R98548033-1.pdf: 5747424 bytes, checksum: 899b36fa6c0af50ca0e1998a42786b8c (MD5) Previous issue date: 2011 | en |
| dc.description.tableofcontents | 目錄
Acknowledgements --------------------------------------------Ⅰ 中文摘要 ----------------------------------------------------Ⅳ Abstract -----------------------------------------------------Ⅴ 目錄 --------------------------------------------------------Ⅵ 圖次 --------------------------------------------------------Ⅹ 表次 ------------------------------------------------------ⅩⅣ 第一章 導論 ---------------------------------------------------1 1-1前言 ----------------------------------------------------- 1 1-2抗體的生產(Antibody Production) ------------------------------2 1-3細胞篩選器(Cell Sorter) --------------------------------------4 1-3-1 FACS(Fluorescence Activated Cell Sorter) ------------------- 4 1-3-2 MACS(Magnetic Activated Cell Sorter) ----------------------5 1-3-3 CBCS(Cytoflow-Bioreactor base Cell Sorter) ----------------- 6 1-4其他篩選方式(Other Screening Methods) ------------------------9 1-5研究目的(The Purpose of Study) ------------------------------10 第二章 理論基礎(Theoretical Basis) ------------------------------ 12 2-1 B細胞的活化(B Cell Development) --------------------------- 12 2-1-1 Clonal Selection Theory --------------------------------- 12 2-1-2 Memory B Cell ---------------------------------------- 15 2-1-3 Plasma Cell ------------------------------------------- 16 2-1-4 抗體的構造(The Structure of Antibody) --------------------17 2-2單株抗體製造原理(The Theory of Monoclonal Antibody Production) -18 2-2-1 脾臟細胞取得(Immunization) ----------------------------18 2-2-2 細胞融合技術(Cell Fusion Techniques) --------------------20 2-2-3 HAT培養基(HAT Selection Medium) ---------------------- 22 2-2-4 篩選步驟(Selection Step) ------------------------------- 24 2-2-5 擴大增值(Expand) -------------------------------------25 2-3 虹吸式反應器(Cytoflow-Bioreactor,Siphon-Bioreactor) -----------26 2-4 竹炭材料(Bamboo Charcoal) -------------------------------- 28 2-4-1 竹材(Bamboo) ---------------------------------------- 29 2-4-2 炭化反應(The Carbonization of Bamboo) -------------------30 2-4-3 竹炭性質之討論(Discussions of Bamboo Charcoal) ---------- 32 2-5 磁性材料(Magnetic Material) --------------------------------35 2-6 材料表面改質與接枝生物分子 ------------------------------40 2-6-1 碳質材料 --------------------------------------------42 第三章 材料與方法(Materials and Methods) -----------------------46 3-1 實驗儀器(Experimental Apparatus) --------------------------- 46 3-2 實驗藥品及配方 ------------------------------------------47 3-3 實驗流程圖(Experimental Procedure) ------------------------- 51 3-4 材料製備(Preparation of the Material) ------------------------- 53 3-4-1 竹炭顆粒(Bamboo Charcoal) ---------------------------- 53 3-4-2 表面改質(Surface Modification) ------------------------- 53 3-4-3 接枝purified human Epidermal Growth Factor Receptor -------55 3-5 高效率虹吸式細胞篩選裝置設計(The Design of the High-efficiency Cytoflow-Bioreactor base Cell Sorter) ------------------------- 56 3-6 細胞(cell) ------------------------------------------------57 3-6-1 抗原(Antigen)及脾臟細胞(Spleen Cell) --------------------57 3-6-2 骨髓瘤細胞(Myeloma Cell) ----------------------------- 58 3-7 專一性融合瘤細胞製造(Specific Hybridoma Cell Production) ------58 3-8 單株抗體之產生(The Production of the Monoclonal Antibody) -----59 3-9 實驗分析方法(Analysis) ------------------------------------60 3-9-1 材料(Carrier) -----------------------------------------60 3-9-1-1 傅立葉轉換紅外線光譜儀(Fourier Transform Infrared Spectroscopy, FT-IR) ------------------------------60 3-9-1-2 掃描式電子顯微鏡(Scanning Electron Microscopy) ---- 61 3-9-1-3 X光粉末繞射儀(X-ray Diffraction, XRD) ------------ 62 3-9-1-4 免疫染色分析(Immunostaining Assay, IFA) -----------64 3-9-2 老鼠體內之抗體產生 ----------------------------------67 3-9-2-1 免疫螢光染色分析(Immunofluorescence Assay, IFA) ---67 3-9-3 掃描式電子顯微鏡分析(Scanning Electron Microscopy, SEM) - 68 3-9-3-1 B細胞篩選及專一性融合瘤細胞分析(The Assay of the B cell and Specific Hybridoma cell) --------------------69 3-9-4 抗體分析(Antibody Assay) ------------------------------69 3-9-4-1 酵素免疫分析儀(Enzyme-linked Immunosorbent Assay, ELISA) ---------------------------------------- 69 3-9-4-2 免疫螢光染色分析(Immunofluorescence Assay, IFA) ---71 第四章 結果與討論(Results and Discussions) ---------------------- 72 4-1 竹炭表面改質與接枝生物分子分析 --------------------------72 4-1-1 酸處理條件 ------------------------------------------72 4-1-1-1 傅立葉轉換紅外線光譜儀(Flourier-transform Infrared Spectroscopy, FT-IR) ----------------------------- 75 4-1-2 氧化鐵(Fe3O4)共沉條件 --------------------------------76 4-1-2-1 掃描式電子顯微鏡(Scanning Electron Microscopy) ---- 78 4-1-2-2 能量散佈分析儀(Energy Dispersive Spectrometer, EDS)-81 4-1-2-3 X光粉末繞射儀(X-ray Diffraction, XRD) -------------82 4-1-3 免疫染色分析(Immunostaining Assay, IFA) -----------------86 4-2 老鼠體內之抗體產生 --------------------------------------92 4-2-1 免疫螢光染色分析(Immunofluorescence Assay, IFA) ---------92 4-3 掃描式電子顯微鏡分析(Scanning Electron Microscopy, SEM) ----- 94 4-3-1 專一性B細胞篩選分析(Specific B cell Assay) -------------- 94 4-3-2 專一性融合瘤細胞分析(Specific Hybridoma cell Assay) ------ 96 4-4 抗體分析(Antibody Assay) ----------------------------------98 4-4-1 酵素免疫分析儀(Enzyme-linked Immunosorbent Assay, ELISA) 98 4-4-2 免疫螢光染色分析(Immunofluorescence Assay, IFA) ---------99 第五章 結論(Conclusions) -------------------------------------102 第六章 未來展望(Future Work) --------------------------------103 參考文獻(References) ----------------------------------------- 104 圖次 圖 1-1 酵素之異位結合上inhibitor或activator來抑制或活化酵素之活性位的活性位而造成酵素活性位之構形改變 ----------------------------2 圖 1-2 流式細胞分選儀(FACS)示意圖 -----------------------------5 圖 1-3 磁性細胞分選儀(MACS)示意圖 ----------------------------6 圖 1-4 CBCS的使用方法示意圖 ----------------------------------7 圖 1-5 培養液殘留在生物反應器底部之情況(虛線圓圈處) ------------9 圖 1-6 HCBCS的操作流程示意圖 ------------------------------- 11 圖 2-1 Clonal Selection Theory ----------------------------------- 13 圖 2-2 Stages of B cell development ------------------------------- 14 圖 2-3 血清中抗體濃度隨外來抗原刺激次數關係圖 ----------------15 圖 2-4 記憶B細胞(Memory B cell)的膜外結構 -------------------- 16 圖 2-5 漿細胞(plasma cell)的電子顯微鏡圖 ----------------------- 17 圖 2-6 抗體的構造 --------------------------------------------18 圖 2-7 低濃度的抗原只能和親和力最強的IgG結合 ---------------- 20 圖 2-8 兩種細胞(NS-1 cell和myeloma cell)融合後可能的情形 ------- 22 圖 2-9 The pathways of the DNA synthesis --------------------------23 圖 2-10 細胞融合後之融合瘤細胞的染色體對 ---------------------25 圖 2-11 Cytoflow-Bioreactor結構示意圖 -------------------------- 27 圖 2-12 Cytoflow-Bioreactor 設計實際圖 --------------------------27 圖 2-13 (A)、(B)、(C)為Cytoflow-Bioreactor操作示意圖，(D)為管口處放大圖 -------------------------------------------------------- 28 圖 2-14 孟宗竹碳化前橫切面圖 ---------------------------------30 圖 2-15 竹材熱處理時內部結構變化 -----------------------------31 圖2-16 (a)竹炭多孔結構之SEM圖 (b)骨髓瘤細胞貼附於竹炭孔洞之SEM圖 ---------------------------------------------------------- 32 圖 2-17 The Raman spectrum result of the bamboo charcoal ------------ 34 圖 2-18 The XRD pattern of the bamboo charcoal ---------------------34 圖 2-19 氧化鐵(Fe3O4)之結構示意圖 ------------------------- 36 圖 2-20 水熱法的操作原理示意圖 -------------------------------37 圖 2-21 The Raman spectrum of the multi-wall carbon nanotube -----42 圖 2-22 奈米碳管經酸處理之後利用EDC/NHS的催化與蛋白質接枝 --43 圖 2-23 利用EDC保護羧基和胺基接合的機制 -------------------44 圖 2-24 具carboxyl group之竹炭利用EDC/NHS接枝生物分子時可能的反應 -------------------------------------------------------- 45 圖3-1 The flow chart of the surface modification of bamboo charcoal ----- 52 圖 3-2 The flow chart of the conjugation of the biomolecule (purified human EGFR) on the surface of the bamboo charcoal ------------------------ 52 圖 3-3 The flow chart of the specific splenocytes and specific hybridoma cell production ---------------------------------------------------- 53 圖 3-4 HCBCS的實際操作示意圖 ------------------------------- 56 圖 3-5 Bragg’s Law之幾何圖 ------------------------------------63 圖 3-6 The immunofluorescence assay’s step ------------------------ 67 圖 3-7 The ELISA assay’s step from Hybridomas Supernatant(for mouse IgG) ------------------------------------------------------------- 70 圖 3-8 The ELISA assay’s step from Hybridoma Supernatant(for anti-human EGFR antibody) ------------------------------------------------71 圖 4-1 The FT-IR spectra of the bamboo charcoal, acid-treated bamboo charcoal . (a)未經過酸處理的竹炭；(b)經酸處理的竹炭 ---------------75 圖 4-2 磁化竹炭的製備過程與參數條件圖 ------------------------77 圖 4-3 (a)經酸處理之竹炭的SEM圖 -----------------------------79 圖 4-4 (b)經酸處理之竹炭的SEM圖 -----------------------------79 圖 4-5 (a)磁化竹炭的低倍率SEM圖 ----------------------------- 80 圖 4-6 (b)磁化竹炭的高倍率SEM圖 ----------------------------- 80 圖 4-7 磁化竹炭之氧化鐵沉積圖 ------------------------------- 81 圖 4-8 磁化竹炭上氧化鐵之能量散佈分析圖譜-------------------- 82 圖 4-9 經前處理後的竹炭XRD pattern ---------------------------84 圖 4-10 經過前處理與酸處理知竹炭XRD pattern ------------------84 圖 4-11 市售之氧化鐵奈米粒子 -------------------------------- 85 圖 4-12與磁化竹炭同時形成之氧化鐵奈米粒子XRD pattern --------- 85 圖 4-13 磁化竹炭之XRD pattern --------------------------------86 圖 4-14 (1)The Immunofluorescence assay of bamboo charcoal and acidic-treated bamboo charcoal conjugated with purified human EGFR ---- 88 圖 4-14 (2) The Immunofluorescence assay of acidic-treated bamboo charcoal and bamboo charcoal deposited iron oxide conjugated with purified human EGFR ------------------------------------------------------- 89 圖 4-14 (3) The Immunofluorescence assay of bamboo charcoal deposited iron oxide and Magnetic bamboo charcoal conjugated with purified human EGFR ------------------------------------------------------------- 90 圖 4-15 The immunofluorescence images of the Magnetic Bamboo Charcoal conjugated with purified EGFR ------------------------------------91 圖 4-16 The immunofluorescence 3D-merged images of the Magnetic Bamboo Charcoal conjugated with purified EGFR ----------------------------91 圖 4-17 The immunofluorescence images of the mouse serum --------92、93 圖 4-18 被磁化竹炭表面catch之B cell(俯視圖) ------------------94 圖 4-19 被磁化竹炭表面catch之B cell(側視圖) ------------------95 圖 4-20 被磁化竹炭表面catch之B cell(低倍圖) ------------------95 圖 4-21 骨髓瘤細胞(myeloma cell)與磁化竹炭上B cell融合之情形 -96 圖 4-22 兩個骨髓瘤細胞(myeloma cell)與磁化竹炭上B cell三個細胞融合之情形 -------------------------------------------------- 97 圖 4-23 骨髓瘤細胞(myeloma cell)與磁化竹炭上B cell融合之情形-97 圖 4-24 培養融合瘤細胞(hybridoma cell)上清液之ELISA分析(n=6, 組別可參考表 3-14) ------------------------------------------------98 圖 4-25 利用CBCS流程生產之融合瘤細胞上清液ELISA分析 --------99 圖 4-25 The immunofluorescence images of the hybridoma supernatant (a) Non-immunized mouse serum (b)hybridoma supernatant --------101 表次 表 2-1 竹炭與木炭結構特性比較 --------------------------------29 表 3-1 The table of the experimental instruments and apparatus ----------46 表 3-2 酸處理部分 --------------------------------------------47 表 3-3 氧化鐵共沉部分 ----------------------------------------47 表 3-4 接枝與分析部分 -------------------------------------47、48 表 3-5 Kit部分 ----------------------------------------------- 48 表 3-6 細胞培養與融合分析部分 --------------------------------48 表 3-7 10X PBS 配方 ------------------------------------------49 表 3-8 0.1M MES buffer配方 ------------------------------------49 表 3-9 Culture medium of the myeloma cell --------------------- 49、50 表 3-10 Culture medium of the A549 cell --------------------------- 50 表 3-11 1X HAT selection medium protocol ----------------------50、51 表 3-12 融合試劑配方 (fusion reagent protocol) -------------------- 51 表 3-13 酸處理實驗組別 ---------------------------------------55 表 3-14 The experimental groups of the specific hybridoma cells production ------------------------------------------------------------- 59 表 3-15 FT-IR absorption band of the common functional group --------- 61 表 3-16 觀測SEM的實驗組別 ----------------------------------62 表 3-17 X-ray Diffraction分析實驗組別 ---------------------------64 表 3-18 The Immunofluorescence assay groups of the magnetic bamboo charcoal conjugated with purified human EGFR ---------------------- 66 表 3-19 The immunofluorescence assay groups of the mice serum from tail-bleeding ---------------------------------------------------68 表 3-20 The ELISA group of the antibody secreted from hybridoma cell ---70 表 3-21 The IFA groups of the antibody secreted from specific hybridoma cell ------------------------------------------------------------- 71 表 4-1 利用硝酸/硫酸(1/3)混合酸溶液進行竹碳酸處理 ------------- 72 表 4-2 利用硝酸溶液於60℃下進行竹碳酸處理 ------------------- 73 表 4-3 利用硝酸溶液於70℃下進行竹碳酸處理 ------------------- 73 表 4-4 利用硝酸溶液於80℃下進行竹碳酸處理 ------------------- 74 表 4-5 利用硝酸溶液於90℃下進行竹碳酸處理 ------------------- 74 表 4-6 波長吸收峰對應的官能基 --------------------------------76 表 4-7 能量散佈分析圖譜之元素分布 --------------------------- 82 表 4-8 X-ray Diffraction分析實驗組別 ----------------------------83 | |
| dc.language.iso | zh-TW | |
| dc.subject | 單株抗體 | zh_TW |
| dc.subject | 表面改質 | zh_TW |
| dc.subject | 磁化竹炭 | zh_TW |
| dc.subject | 生物反應器 | zh_TW |
| dc.subject | 融合瘤 | zh_TW |
| dc.subject | 虹吸式反應器 | zh_TW |
| dc.subject | Magnetic Bamboo charcoal | en |
| dc.subject | Surface modification | en |
| dc.subject | Bioreactor | en |
| dc.subject | Hybridomas | en |
| dc.subject | Cytoflow reactor | en |
| dc.subject | Monoclonal antibody | en |
| dc.title | 製備磁化竹炭發展高效率的細胞篩選器應用於單株抗體生產之研究 | zh_TW |
| dc.title | The Preparation of Magnetic Bamboo Charcoal as a Template for High-efficiency Cytoflow-bioreactor Base Cell Sorter(HCBCS): Monoclonal Antibody Production | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 99-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 陳克紹(Ke-Shau Chen),王盈錦(Ying-Jin Wang),吳信志(Shin-Jr Wu),陳天牧(Tian-Mu Chen) | |
| dc.subject.keyword | 生物反應器,融合瘤,虹吸式反應器,單株抗體,磁化竹炭,表面改質, | zh_TW |
| dc.subject.keyword | Bioreactor,Hybridomas,Cytoflow reactor,Monoclonal antibody,Magnetic Bamboo charcoal,Surface modification, | en |
| dc.relation.page | 112 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2011-07-26 | |
| dc.contributor.author-college | 工學院 | zh_TW |
| dc.contributor.author-dept | 醫學工程學研究所 | zh_TW |
| 顯示於系所單位： | 醫學工程學研究所 | |
文件中的檔案：
| 檔案 | 大小 | 格式 | |
|---|---|---|---|
| ntu-100-1.pdf 未授權公開取用 | 5.61 MB | Adobe PDF |
系統中的文件，除了特別指名其著作權條款之外，均受到著作權保護，並且保留所有的權利。