臺灣博碩士論文加值系統

日本腦炎病毒 (Japanese Encephalitis Virus, JEV) 是感染神經系統的重要病源之一，然而感染日本腦炎病毒大多沒有明顯症狀，只有少數患者會出現急性腦炎的臨床症狀，且伴隨著高死亡率。而在存活病例中，也可能造成中樞神經系統後遺症。目前台灣現有的日本腦炎疫苗為活性減毒嵌合型 (Live, attenuated chimeric Japanese encephalitis vaccine) 及以Vero細胞培養 (Vero cell culture-derived Japanese encephalitis vaccine) 的死毒疫苗。對疫苗的製程而言，快速檢測疫苗的半成品及成品的抗原含量是非常重要的，因此本論文目的是建立一個檢測日本腦炎病毒抗原含量的平台。
本實驗先以無血清培養基培養日本腦炎病毒，且將純化及滅活後的JEV抗原免疫兔子製備出JEV兔源多株抗體，以免疫螢光染色法 (Immunofluorescence assay, IFA) 確認此兔源多株抗體能辨認到JEV病毒。同時將純化的JEV抗原免疫小鼠，並取其脾臟細胞製備融合瘤細胞 (Hybridoma) ，最後篩選出3株抗日本腦炎病毒的單株抗體。此三株分別以酵素連接免疫吸附法 (Enzyme-linked immunosorbent assay, ELISA) 和西方轉漬法 (Western blot) 確認其具有良好的結合能力外，也將其進行抗體亞型分析，得知此三株單株抗體的重鏈皆為IgG1，輕鏈皆為kappa。此外，將純化的單株抗體搭配多株抗體建立抗原定量ELISA (Quantification ELISA, Q-ELISA) 檢測平台。最後，本論文建立出最佳條件是以純化的單株抗體作為捕抓抗體，兔源多株抗體為偵測抗體，標定HRP的山羊抗兔子抗體為二級抗體，並以純化後的JEV抗原作為標準品，其檢測範圍為2500 ng/ mL至156 ng/ mL (R2=0.99) ，且利用此平台可成功檢測出病毒在產製過程的抗原含量。

Japanese encephalitis virus (JEV) is one of the important pathogenic viruses causing infection of the central nervous system. Although symptoms of Japanese encephalitis are rare, the case-fatality rate among those with encephalitis is high. Furthermore, survivors suffer severe permanent neurologic or psychiatric sequelae. Currently, two types of vaccines have been used in Taiwan. One is live-attenuated chimeric Japanese encephalitis vaccine and the other is Vero cell culture-derived inactivated Japanese encephalitis vaccine. In the vaccine manufacturing process, rapid test of the amount of antigen in bulk and finished product is a key parameter for vaccine formulation. Therefore, we established a Q-ELISA platform that could detect and quantify JEV antigen.
JEV was cultured in serum-free medium, then purified and inactivated in order to prepare antigen to immunize rabbit for polyclonal antibodies (pAbs). The pAbs were verified to recognize JEV by immunofluorescence assay (IFA). Simultaneously, mice were immunized with purified antigen for hybridoma preparation from splenocytes. Three clones of hybridoma were selected after screening, and their binding affinity was confirmed by enzyme-linked immunosorbent assay (ELISA) and Western blotting. Isotype analysis by mouse monoclonal antibody isotyping test kit showed that the hybridoma isotypes were IgG1 with kappa light chain. In addition, the anti-JEV mAb and rabbit anti-JEV pAbs were used for the development of quantification ELISA (Q-ELISA) to quantify JEV antigen. In this study, the best range of antigen detection was from 2500 ng/ mL to 156 ng/ mL (R2 values 0.99) when using anti-JEV mAb as capture, rabbit anti-JEV pAbs as detector and goat anti rabbit conjugated HRP as secondary antibody. We have shown that this platform can be successfully applied to detect JEV antigen from cultivated viral fluid.

摘要 I
Abstract II
誌謝 III
目錄 IV
圖目錄 VI
表目錄 VII
第一章、 前言 1
第二章、 文獻探討 2
第一節、 日本腦炎的簡介 2
第二節、 日本腦炎病毒結構及基因體介紹 2
第三節、 日本腦炎病毒的複製機制 3
第四節、 日本腦炎診斷方法 4
一、 病毒分離 4
二、 血清學 4
三、 分子生物學 4
第五節、 日本腦炎疫苗 4
一、 不活化鼠腦疫苗 4
二、 活性減毒嵌合型日本腦炎疫苗 5
三、 Vero細胞培養不活化之日本腦炎疫苗 5
第六節、 日本腦炎病毒的產製 5
第七節、 日本腦炎病毒單株抗體的研製 6
第八節、 酵素連接免疫吸附法 (ELISA) 在日本腦炎病毒的應用 6
第三章、 實驗策略 7
第四章、 材料與方法 8
第一節、 分別以850 cm2轉瓶及潮汐式生物反應器產製日本腦炎病毒抗原 8
一、 以850 cm2轉瓶製備JEV抗原 8
二、 以潮汐式BelloCell製備JEV抗原 8
三、 JEV抗原的純化 8
四、 膠體過濾管柱純化後日本腦炎病毒抗原的分析 9
第二節、 日本腦炎病毒單株抗體的製備 10
一、 製備具有分泌日本腦炎病毒抗體之小鼠B淋巴細胞 10
二、 小鼠骨髓瘤NS-1細胞的培養 10
三、 融合瘤細胞的製備 11
第三節、 融合瘤細胞篩選及細胞單株化 11
一、 以酵素連接免疫吸附法 (ELISA) 篩選具分泌抗體能力的融合瘤細胞 11
二、 融合瘤細胞放大培養 12
三、 以免疫螢光染色法 (IFA) 檢測融合瘤細胞分泌的抗體 12
四、 融合瘤細胞進行極限稀釋 12
第四節、 單株抗體特性分析 13
一、 以酵素連接免疫吸附法 (ELISA) 分析單株抗體的專一性 13
二、 以西方墨點法 (Western blotting) 分析單株抗體的專一性 13
三、 單株抗體的抗體亞型分析 14
四、 單株抗體中和能力分析 14
五、 單株抗體分子之互補決定區分析 14
第五節、 日本腦炎病毒多株抗體的製備 17
第六節、 日本腦炎病毒單株抗體及多株抗體的純化 17
一、 日本腦炎病毒單株抗體的製備與純化 17
二、 日本腦炎病毒多株抗體的純化 18
第七節、 日本腦炎病毒三明治ELISA抗原檢測試劑的開發 18
一、 抗體標定Horseradish Peroxidase酵素 18
二、 以三明治ELISA檢測JEV抗原 19
第八節、 三明治ELISA法應用於JEV抗原之製程 20
一、 JEV收穫病毒液的製備 21
二、 測定不同時間點的病毒液力價 21
三、 以單株抗體作為捕捉抗體，兔源多株抗體為偵測抗體，以山羊抗兔子標定HRP抗體為二級抗體，檢測不同時間點的日本腦炎病毒液 21
第五章、 實驗結果 22
第一節、 以不同系統產製出的日本腦炎病毒抗原之分析 22
一、 以Dot blot分析純化後之JEV 22
二、 以SDS-PAGE及西方墨點法 (Western blotting) 分析純化且濃縮後之JEV 22
第二節、 日本腦炎病毒單株抗體的製備 22
一、 以純化後的日本腦炎病毒抗原免疫小鼠 22
二、 融合瘤細胞篩選及細胞單株化 22
三、 以酵素連接免疫吸附法 (ELISA) 分析單株抗體的專一性 23
四、 以西方墨點法 (Western blotting) 分析單株抗體的專一性 23
五、 單株抗體的抗體亞型分析 23
六、 單株抗體中和能力分析 23
七、 單株抗體分子之互補決定區分析 23
第三節、 日本腦炎病毒多株抗體的製備 24
第四節、 日本腦炎病毒單株抗體及多株抗體的純化 24
一、 單株抗體的純化 24
二、 多株抗體的純化 24
第五節、 日本腦炎病毒三明治ELISA抗原檢測試劑的開發 24
一、 設計三明治ELISA檢測JEV抗原之最佳抗體配對 24
第六節、 三明治ELISA法應用於JEV抗原之製程 26
第六章、 討論 27
第七章、 附錄 30
第八章、 參考文獻 56

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