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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 林峰輝(Feng-Huei Lin) | |
| dc.contributor.author | Yu-Chung Chang | en |
| dc.contributor.author | 張毓忠 | zh_TW |
| dc.date.accessioned | 2021-06-15T01:37:47Z | - |
| dc.date.available | 2014-07-23 | |
| dc.date.copyright | 2009-07-23 | |
| dc.date.issued | 2009 | |
| dc.date.submitted | 2009-07-16 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/43117 | - |
| dc.description.abstract | 單株抗體的製備在1975年由George Kohler和Cesar Milstein成功利用細胞融合的技術製造出來後，廣泛被運用，然而在操作流程上較為繁瑣，尤其是單株化選殖的過程，不僅需花費大量時間，同時也必須消耗很多的人力，而操作者的熟練度也會影響結果，因此往往實驗之成敗決定於此步驟。
因此，本研究之主要目的在於希望開發出一組高專一性的細胞篩選器(high-through-put cell sorter)，期望不僅能夠縮短單株化選殖之時間，也能夠讓單株抗體的製備步驟單一化(one step)，以利操作者的使用並節省單株抗體的生產時間。 細胞篩選器利用本實驗室開發出來的虹吸式生物反應器(cytoflow-bioreactor)為主體，配合carrier的選用，希望能讓具特異性的融合瘤細胞製備步驟單一化。 為了讓細胞篩選器能夠穩定的操作，在carrier的選用上，本研究論文選擇竹炭(bamboo charcoal)作為carrier，屏除過去常用的磁珠、玻璃珠…等，主要是由於這些材料進行表面改質接枝生物分子時，常利用其表面的氫氧基(hydroxyl group)來與生物分子相接，因此往往使用HMDI或是aminosilane來進行生物分子的接枝。而HMDI對水敏感，容易與水反應，因此，接枝過程必須保持乾燥，一旦水分進入反應槽，反應即終止；至於利用aminosilane進行接枝後，產物在水溶液中有水解斷鍵的疑慮，因此材料並不夠穩定。此外，這些材料的比表面積均不若竹炭來的多。 至於竹炭表面具有豐富的C=C鍵，容易進行酸處理產生(carboxyl group)而與生物分子(purified human EGFR)穩定以醯胺鍵結(amide bond)相接；另外竹炭表面也具有高度孔洞，可以提供相當高的比表面積接枝生物分子(human EGFR)，進而增進細胞篩選器的效能，提高細胞篩選器的靈敏度，並且希望藉由孔洞的形狀而能讓B細胞與骨髓瘤(myeloma)細胞有更靠近的機率，而增加細胞融合成功的比率，以產生更多專一性的融合瘤細胞(specific hybridoma cell)，進而分泌單株抗體。 由Raman spectrum可了解藉由硝酸處理能夠使竹炭結構中的C=C鍵氧化而被破壞；並進一步利用FT-IR圖譜可得知利用硝酸處理能讓竹炭表面產生酸基(carboxyl group)；而免疫螢光分析(IFA)中可證實經由硝酸處理的竹炭可以藉由EDC/NHS來與purified human EGFR以共價鍵(amide bond)穩定相接；此部份之結果亦可經由熱重分析儀(TGA)來獲得佐證。 由SEM以及OM圖可以推測本研究欲開發出來之「高專一性細胞篩選器」確實能夠抓到B細胞，並進一步能夠與骨髓瘤細胞融合成融合瘤細胞；再利用ELISA來分析”有抓到B細胞之竹炭”是否能夠分泌IgG以及定量其分泌的含量；而在免疫螢光分析(IFA)之數據則可得之融合瘤細胞(有抓到B細胞之竹炭)能夠於培養過程中分泌Mouse anti-human EGFR antibody。 在未來，除了希望讓本研究開發出來之細胞篩選裝置能夠更加穩定操作外，更希望能夠有機會應用於人類單株抗體的製造上。 | zh_TW |
| dc.description.abstract | George Kohler and Cesar Milstein employed fusion theory to produce monoclonal antibody in 1975. An attempt was made in order to increase the mAb production by a high-through-out cell sorter in subcloning (selection step). A siphon bioreactor (cytoflow-bioreactor) and the new material (bamboo charcoal) combined as a cell sorter called “high-through-put cell sorter”. The advantage of the high-through-put cell sorter was reduced time-comsuming, labor-intensive, cost-effective and animal-reducing for monoclonal antibody production.
The feasibility of the high-through-put cell sorter was tested with A549 cell as the model antigen, due to the high expression of the “epidermal growth factor receptor, EGFR” on the membrane of the A549 cell. The results of Raman spectrum revealed the oxidation of bamboo charcoal might be because of the usage of nitric acid or acid mixture (nitric acid/sulfuric acid: 1/3). From the FT-IR, we could further analyze the introduction of carboxyl group on the surface of bamboo charcoal. The IFA confirmed that we could conjugate purified human EGFR on the surface of the acid-treated bamboo charcoal by forming an amide bond using EDC/NHS and those results were further confirmed by TGA. In SEM, the conjugation of purified human EGFR on the acid-treated bamboo charcoal surface captured the splenocytes (B cells) from immunized mice. The results of ELISA monitored the IgG secretion from the supernatant of the bamboo charcoal binding with hybridoma cell cultured medium. Thus it was confirmed that it could secrete anti-human EGFR antibody in the supernatant of the cultured bamboo charcoal binding with hybridoma cell from IFA. In the future, this “high-through-put cell sorter” could be also employed to harvest antibody-secreting B cells from the peripheral blood without sacrificing the animals and also expected to produce humanized monoclonal antibody. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T01:37:47Z (GMT). No. of bitstreams: 1 ntu-98-R96548034-1.pdf: 4292384 bytes, checksum: e5405990db47b2e74b5efb36f68f3c07 (MD5) Previous issue date: 2009 | en |
| dc.description.tableofcontents | 口試委員會審定書 I
誌謝 II 中文摘要 III Abstract V 目錄 VII 圖索引 XII 表索引 XVII 第一章 導論 1 1-1 前言 (Introduction) 1 1-2 抗體的生產 (Antibody Production) 3 1-3 細胞篩選器 (Cell Sorter) 5 1-3-1 FACS (Fluorescence Activated Cell Sorter) 5 1-3-2 MACS (Magnetic Activated Cell Sorter) 6 1-4 其他篩選方式 (Other Screening Mehtods ) 8 1-5 研究目的 (The Purpose of Study) 9 第二章 理論基礎 (Theoretical Basis) 10 2-1 B細胞的活化 (B Cell Development) 10 2-1-1 Clonal Selection Theory 10 2-1-2 Memory B Cell 12 2-1-3 Plasma Cell 13 2-1-4 抗體的構造 (The Structure of Antibody) 14 2-2 單株抗體製造原理 (The Theory of Monoclonal Antibody Production) 16 2-2-1 脾臟細胞取得 (Immunization) 16 2-2-2 細胞融合技術 (Cell Fusion Techniques) 16 2-2-3 HAT培養基 (HAT Selection Medium) 18 2-2-4 限數稀釋 (Limiting Dilution) 19 2-2-5 擴大增殖 (Expand) 20 2-3 虹吸式反應器 (Cytoflow-bioreactor, Siphon-bioreactor) 21 2-4 竹炭材料 (Bamboo Charcoal) 24 2-4-1 竹材 (Bamboo) 24 2-4-2 炭化反應 (The Carbonization of Bamboo) 25 2-4-3 竹炭 (Bamboo Charcoal) 27 2-5 材料表面改質與接枝生物分子 31 2-5-1 ㄧ般材料 31 2-5-2 碳質材料 32 第三章 材料與方法 (Materials and Methods) 36 3-1 實驗儀器 (Experimental Apparatus) 36 3-2 實驗藥品及配方 37 3-3 實驗流程圖 (Experimental Procedure) 42 3-4 材料製備 (Preparation of the Material) 44 3-4-1 竹炭顆粒 (Bamboo Charcoal Particle) 44 3-4-2 表面改質 (Surface Modification) 44 3-4-3 接枝purified human Epidermal Growth Factor Receptor (purified human EGFR) 45 3-5 高專一性細胞篩選裝置設計 (The Design of the High-through-put Cell Sorter) 46 3-6 細胞 (Cell) 47 3-6-1 抗原 (Antigen) 及脾臟細胞 (Spleen Cell) 47 3-6-2 骨髓瘤細胞 (Myeloma Cell) 48 3-7 專一性融合瘤細胞製造 (Specific Hybridoma Cell Production) 49 3-8 單株抗體之產生 (The Production of the Monoclonal Antibody) 51 3-9 實驗分析方法 (Analysis) 52 3-9-1 材料 (Carrier) 52 3-9-1-1 拉曼光譜儀 (Raman Spectroscopy) 52 3-9-1-2 FT-IR (Fourier Transform Infrared Spectroscopy) 52 3-9-1-3 熱重分析儀 (Thermal Gravimetric Analyzer, TGA) 53 3-9-1-4 免疫螢光染色分析 (Immunofluorescence Assay, IFA) 54 3-9-1-5 竹炭於培養基中之情況 56 3-9-2 老鼠體內之抗體產生 (The Antibody Production from Mouse) 57 3-9-2-1 免疫染色分析 (Immunostaining Assay) 57 3-9-2-2 免疫螢光染色分析 (Immunofluorescence Assay, IFA) 58 3-9-3 掃描式電子顯微鏡分析 (Scanning Electron Microscopy, SEM) 60 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) 60 3-9-4 抗體分析 (Antibody Assay) 61 3-9-4-1 酵素免疫分析儀 (Enzyme-linked Immunosorbent Assay, ELISA) 61 3-9-4-2 免疫螢光染色分析 (Immunofluorescence Assay, IFA) 62 第四章 結果 (Results) 63 4-1 竹炭表面改質及接枝生物分子分析 63 4-1-1 初步篩選之酸處理條件 63 4-1-2 拉曼光譜儀 (Raman Spectroscopy) 66 4-1-3 傅立葉轉換紅外線光譜儀 (Flourier-transform Infrared Spectroscopy, FT-IR) 68 4-1-4 熱重分析儀 (Thermal Gravimetric Analysis, TGA) 70 4-1-5 免疫螢光分析 (Immunofluorescence Assay, IFA) 75 4-1-6 竹炭於培養液中之情況 77 4-2 老鼠體內誘發免疫反應產生之抗體 (The Antibody Production from Immunized mouse) 79 4-2-1 免疫染色分析 (Immunostaining Assay) 79 4-2-2 免疫螢光染色分析 (Immunofluorescence Assay, IFA) 81 4-3 掃描式電子顯微鏡 (Scanning Electron Microscopy, SEM) 82 4-3-1 專一性B細胞篩選分析 (Specific B Cell Assay) 82 4-3-2 專一性融合瘤細胞分析 (Specific Hybridoma Cell Assay) 83 4-4 單株抗體分析 (Antibody Assay) 84 4-4-1 酵素免疫分析儀 (Enzyme-linked Immunosorbent Assay, ELISA) 84 4-4-2 免疫螢光分析 (Immunofluorescence Assay, IFA) 85 第五章 討論 (Discussions) 86 5-1 竹炭性質之討論 (Discussions of the Bamboo Charcoal) 86 5-1-1 酸處理對竹炭的影響 (The Effects of Bamboo Charcoal by Using Acid-treatment) 87 5-1-2 高壓滅菌對竹炭之影響 (The Effects of Bamboo Charcoal by Using Autoclave) 88 5-2 竹炭接枝生物分子(glycine and human EGFR) 89 5-3 老鼠體內誘發之抗體 (Immunization from Mouse) 90 5-4 Specific Hybridoma Cell Production 92 第六章 結論 (Conclusions) 94 第七章 未來展望 (Future) 95 參考文獻 (References) 96 附錄A (Appendix A): 個人簡歷(Resume) 107 附錄B (Appendix B): 個人著作(Publication) 109 | |
| dc.language.iso | zh-TW | |
| dc.subject | 竹炭 | zh_TW |
| dc.subject | 虹吸式生物反應器 | zh_TW |
| dc.subject | 生物反應器 | zh_TW |
| dc.subject | 表面改質 | zh_TW |
| dc.subject | 單株抗體 | zh_TW |
| dc.subject | 融合瘤細胞 | zh_TW |
| dc.subject | 上皮細胞生長因子接受器 | zh_TW |
| dc.subject | 抗上皮細胞生長因子接受器之單株抗體 | zh_TW |
| dc.subject | 細胞篩選器 | zh_TW |
| dc.subject | 細胞分流器 | zh_TW |
| dc.subject | surface modification | en |
| dc.subject | bioreactor | en |
| dc.subject | monoclonal antibody | en |
| dc.subject | cytoflow-bioreactor | en |
| dc.subject | bamboo charcoal | en |
| dc.subject | cell sorter | en |
| dc.subject | anti-EGFR antibody | en |
| dc.subject | EGFR | en |
| dc.subject | epidermal growth factor receptor | en |
| dc.subject | hybridoma cell | en |
| dc.subject | siphon bioreactor | en |
| dc.title | 利用經表面修飾之竹炭設計高專一性之細胞篩選器應用於單株抗體製造之研究 | zh_TW |
| dc.title | Surface Modification of Bamboo Charcoal as a Template of High-through-put Cell Sorter for Monoclonal Antibody Production | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 97-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 郭宗甫(Tzong-Fu Kuo),楊禎明(Jen-Ming Yang),S. Savillia(S. Savillia) | |
| dc.subject.keyword | 竹炭, 虹吸式生物反應器, 生物反應器, 表面改質, 單株抗體, 融合瘤細胞, 上皮細胞生長因子接受器, 抗上皮細胞生長因子接受器之單株抗體, 細胞篩選器, 細胞分流器, | zh_TW |
| dc.subject.keyword | bamboo charcoal, cytoflow-bioreactor, siphon bioreactor, bioreactor, surface modification, monoclonal antibody, hybridoma cell, epidermal growth factor receptor, EGFR, anti-EGFR antibody, cell sorter, | en |
| dc.relation.page | 110 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2009-07-16 | |
| 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 | |
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