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dc.contributorChiou-Lin Chenen_US
dc.contributor.advisorHappy K. Shiehen_US
dc.contributor.authorLi, Tung-Hauen_US
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dc.description.abstract自從1997年至今,高病原性禽類流行性感冒 (Avian Influenza,AI) 病毒H5N1造成家禽工業重大的經濟損失及危害動物生命安全,H5N1目前經證實有318人感染,有192人因此死亡。高病原性禽類流行性感冒,已成為獸醫界或全球最關注的一個問題。本研究目的主要是以禽類流行性感冒病毒H5N3為目標,製備單株抗體,以進行快速診斷。BALB/c小白鼠免疫不活化H5N3全病毒,之後取脾臟細胞和骨髓瘤細胞進行融合。融合瘤篩選以血球凝集抑制作用、免疫墨點法、西方轉漬法和間接免疫螢光抗體分析。篩選出兩株對流感病毒血球凝集素有特異性反應,歷經了五次融合瘤製備,篩選了三千孔的上清液。藉由極限稀釋 (limiting dilution) 進行單株化。藉由單株抗體分型可知分別為IgA和IgG2a。zh_TW
dc.description.abstractThe outbreaks of highly pathogenic H5N1 influenza A virus from 1997 to the present day,The virus cancause economical losses to the poultry industry and have devastating consequences for animal health,H5N1 infections have resulted in 318 laboratory-confirmed infected people,including 192 deaths。HPAI receives immense attention in the veterinary world and is globally treated as a disease immediately notifiable on suspicion to the authorities。The purpose of this study is to prepare monoclonal antibodies against H5N3 AIV and to use these monoclonal antibodies to develop a rapid method for detection of AIV。BALB/c mice were immunized with H5N3 virus and then the splenocytes from the immunized mouse were collected and fused with NS-1 myeloma cells。 The hybridomas secreting antibodies against AIV were screened by hemagglutination inhibition、dot immunoblotting、Western blotting and immunofluorescence assay。Two hybridomas which secreted specific antibodies to AIV,were selected among 3000 hybridomas in five fusion experiments。Cloning of these established hybridomas was carried by the limiting dilution method in order to confirm their monoclonality。After two times cloning,two monoclonal antibodies were purified.Hybridomas C2-8G and D1-11G,secreting IgA and IgG2a respectively。zh_TW
dc.description.tableofcontents目錄 口試論文通過書 Ⅱ 中文摘要 Ⅲ 英文摘要 Ⅳ 致謝 Ⅴ 目錄 Ⅶ 第一章 緒 言 1 第二章 文獻探討 3 2-1 前言 3 2-2 病毒特徵 3 2-3 病毒結構與功能 5 2-3.1 血球凝集素 5 2-3.2 神經胺酸酶 6 2-4 病毒複製 7 2-5 自然宿主 7 2-6 高病原性禽類流行性感冒之發病 9 2-7 病毒結構與毒力 9 2-8 臨床症狀 10 2-9 高病原性禽流感之病理學變化 10 2-10 實驗診斷 11 2-10.1 禽流感病毒感染的直接檢驗 11 2-10.2 禽流感病毒感染的間接檢驗 13 2-11 感染 13 2-11.1 禽鳥間的感染 13 2-11.2 對哺乳類動物的感染 14 2-12 對抗高病原性禽流感的控制措施 14 2-13 免疫 15 2-14 疾病全國流行的風險 17 2-15 單株抗體之歷史背景 18 2-15.1 細胞融合的原理 18 2-15.2 單株抗體的特性與應用 19 第三章 材料和方法 21 3-1 病毒來源及紅血球懸浮液之製備 21 3-1.1 病毒來源 21 3-1.2 紅血球懸浮液之製備 21 3-2 紅血球凝集試驗 21 3-3 血球凝集抑制試驗 22 3-4 病毒增值 22 3-5 病毒純化 22 3-5.1 病毒初步純化 22 3-5.2 病毒純化 23 3-6 全病毒不活化 23 3-7 單株抗體之製備 23 3-8 骨髓瘤細胞 (Myeloma cell) 之培養 24 3-9 融合細胞 25 3-10 HAT藥物篩選之機制 26 3-11 融合瘤細胞之培養 26 3-12 血球凝集抑制試驗 27 3-13 融合瘤細胞單株化 27 3-14 單株化之融合瘤細胞對其他流感病毒亞型血球凝集抑 制試驗 27 3-15 西方墨點法 28 3-16 西方墨漬法 28 3-17 抗體亞型 (Isotype) 之測定 29 3-18 間接式免疫螢光抗體試驗 30 第四章 結果 31 4-1 病毒純化之結果 31 4-2 病毒之不活化 31 4-3 小鼠之免疫計畫 31 4-4 融合瘤細胞之單株化 32 4-5 血球凝集抑制試驗 32 4-6 免疫墨點法 33 4-7 西方墨漬法 33 4-8 間接式免疫螢光抗體染色法 (IFA) 33 4-9 單株抗體亞型分析 33 表1. 證實與亞洲爆發高病原性H5N1禽類流行性感冒病毒提高哺乳類動物致病性有關的基因位點概要 34 圖1. A型流行性感冒病毒受體偏好示意圖 35 圖2. 融合瘤製備之卡通示意圖 36 圖3. 核酸代謝路徑 37 圖4. 以超高速離心機(Beckman)進行離心,28000 rpm離心三小時之產物 38 圖5. 小白鼠行斷尾採血後,分離血清,血清稀釋10倍,以4 HAU /25 μL進行血球凝集抑制試驗 39 圖6. 以H5病毒4 HAU/25μL進行融合瘤力價分析 40 圖7. 以H6和H7病毒4 HAU/25μL進行融合瘤力價分析 41 圖8. D1-11G以免疫墨點法進行全病毒H5N3、H6N1及H7N1行Native和Denature 分析,使用病毒HA titer為26,2抗為Goat anti-mouse IgG 42 圖9. D1-11G以免疫墨點法進行全病毒H5N3、H6N1及H7N1行Native和Denature 分析,使用病毒HA titer為26,2抗為Goat anti-mouse IgA 43 圖10. 以SDS-PAGE進行全病毒和表現蛋白之分析 44 圖11. 圖14轉印至NC paper上,一抗為免疫之老鼠陽性血清,二抗為goat anti-mouse IgG 45 圖12. 圖14轉印至NC paper上,一抗為C2-8G培養之上清液,二抗為goat anti-mouse IgG 46 圖13. 圖14轉印至NC paper上,一抗為D1-11G培養之上清液,二抗為goat anti-mouse IgG 47 圖14. 單株抗體之上清液進行間接螢光免疫抗體分析,細胞為未攻毒之MDCK細胞,A圖一抗為C2-8G,B圖一抗為D1-11G 48 圖15. 單株抗體之上清液進行間接螢光免疫抗體分析,細胞為攻H5N3之MDCK細胞,A圖一抗為C2-8G,B圖一抗為D1-11G 49 圖16. 單株抗體之上清液進行間接螢光免疫抗體分析,細胞為攻H6N1之MDCK細胞,A圖一抗為C2-8G,B圖一抗為D1-11G 50 圖17. 單株抗體之上清液進行間接螢光免疫抗體分析,細胞為攻H7N1之MDCK細胞,A圖一抗為C2-8G,B圖一抗為D1-11G 51 圖18. 單株抗體之上清液進行間接螢光免疫抗體分析,細胞為未攻毒之VERO細胞,A圖一抗為C2-8G,B圖一抗為D1-11G 52 圖19. 單株抗體之上清液進行間接螢光免疫抗體分析,細胞為攻H5N3之VERO細胞,A圖一抗為C2-8G,B圖一抗為D1-11G 53 圖20. 單株抗體之上清液進行間接螢光免疫抗體分析,細胞為攻H6N1之VERO細胞,A圖一抗為C2-8G,B圖一抗為D1-11G 54 圖21. 單株抗體之上清液進行間接螢光免疫抗體分析,細胞為攻H7N1之VERO細胞,A圖一抗為C2-8G,B圖一抗為D1-11G 55 圖22. C2-8G細胞培養上清液之抗體分型 56 第五章 討論 57 參考文獻 61zh_TW
dc.subjectavian influenza virusen_US
dc.subjectmonoclonal antibodiesen_US
dc.titleProduction and characterization of monoclonal antibodies of H5 avian influenza virusen_US
dc.typeThesis and Dissertationzh_TW
item.openairetypeThesis and Dissertation-
item.fulltextno fulltext-
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