臺灣博碩士論文加值系統

為檢測病毒的感染，本研究發展了一個結合微珠 (磁珠、以及塑膠珠) 為輔助的流式細胞儀分析平台。在此研究裡，欲偵測的病毒 (登革病毒、腸病毒71型、或流行性感冒病毒) 首先經由表面結合具病毒專一性抗體、或唾液酸 (sialic acid) 的微珠抓下，接著使用另一株已被標幟螢光、且同樣具病毒專一性的“呈色”抗體 (developing antibody) 來辨認被抓下的病毒，最後使用流式細胞儀上來分析此樣品。此平台偵測登革病毒的靈敏度為1x105 個感染力價 (PFU)；腸病毒71型則同樣為1x105 個感染力價；其中以偵測流感病毒最為靈敏，最低可測得1個單位的血球凝集力價 (HAU)。由於實驗結果已証實結合唾液酸的塑膠珠可被用來抓下不同A型流感病毒的亞型 (H1N1、H3N2) 及B型流感，因此未來若使用具不同亞型特異性的“呈色”抗體來偵測已抓下的病毒，便可進一步的診斷出所感染的流感病毒亞型。除此之外已抓住病毒的磁珠也可利用來偵測血清裡的抗登革病毒或抗腸病毒71型的抗體IgG及IgM。而使用流式細胞儀所分析出來IgG及IgM的數據，能分辨出初次或二次以上的登革病毒感染，並提供患者所感染的病毒血清型資訊。本研究結果顯示，結合了微珠作為輔助的流式細胞儀分析平台，除了可偵測病毒顆粒以外也可分析血清裡具病毒特異性的抗體 (IgM/IgG)，在病毒感染的檢測上也能夠成為一個具專一性及靈敏度的免疫分析方法。

An immunodetection system by applying magnetic or polystyrene beads on flow cytometry is developed for the detection of various viral infections. In this study, the targeted virus (influenza virus, EV 71 or dengue virus) was first captured by viral specific antibody- or sialic acid-coated beads. The captured virus was then detected by viral specific developing antibody which labeled with fluorescence for flow cytometry analysis. The specificity was warranted by the viral specific antibody. The sensitivity to detect dengue virus or EV 71 was around 1x105 plaque forming unit, and to detect influenza virus was around 1 hemagglutination units. Since sialic acid-coated polystyrene bead had shown the ability to hold different subtypes of influenza A (H1N1 and N3N2) and influenza B virus, the subtype of these viruses might be differentiated by subtype-specific developing antibody for further diagnosis. In addition, the antibody conjugated bead preloaded with virus can be used to detect anti-viral antibody. Both IgM and IgG anti-dengue or anti-EV 71 antibodies were detected simultaneously. The ratio of IgG and IgM analyzed by flow cytometry, it could be able to distinguish primary or secondary infection, and provide information about the infection caused by different serotypes of dengue virus. The immunodetection system of using flow cytometry analysis combined with sialic acid or antibody-conjugated beads can promise a specific and sensitive viral infection diagnosis with the detection of either viron or anti-virus IgM/IgG antibody.

中文摘要...........................................I
英文摘要...........................................II
誌謝...............................................III
總目錄.............................................V
表目錄.............................................VII
圖目錄.............................................VIII

主文
緒論...............................................1
材料與方法.........................................11
A. 材料
1. 病毒株..........................................11
2. 病人之血清......................................11
3. 抗體............................................12
4. 細胞株..........................................14
5. 藥品............................................14
6. 實驗耗材........................................17
7. 儀器............................................16
B. 方法
1. 病毒株的備製....................................19
2. 抗體的備製......................................20
3. 以西方墨點法確認抗體之特性......................20
4. 接合上抗體的磁珠備製............................21
5. 接合上唾液酸的塑膠珠備製........................21
6. 專一性抗體磁珠/塑膠珠抓取不同病毒...............22
7. 以流式細胞儀針測病毒顆粒........................22
8. 以流式細胞儀偵測血清中抗特定病毒的IgG 及........23
結果...............................................24
1. 利用磁珠偵測登革病毒顆粒........................24
2. 利用磁珠偵測腸病毒七十一型病毒顆粒..............24
3. 利用磁珠偵測流行性感冒病毒顆粒..................24
4. 利用塑膠珠偵測流行生感冒病毒顆粒................25
5. 利用磁珠偵測血清中具登革病毒專一性之抗體........25
6. 利用磁珠偵測血清中具腸病毒七十一型專一性之抗體..26

討論...............................................28
使用流式細胞儀偵測病毒顆粒.........................28
使用流式細胞儀偵測血清裡的抗體.....................29
使用流式細胞儀作為病毒感染分析的優點...............31
參考文獻...........................................35
圖表...............................................39
附圖...............................................53
作者簡歷...........................................55

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