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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 鄭益謙 | |
| dc.contributor.author | Jiun-Chi Gan | en |
| dc.contributor.author | 甘昀騏 | zh_TW |
| dc.date.accessioned | 2021-05-17T09:15:32Z | - |
| dc.date.available | 2017-08-28 | |
| dc.date.available | 2021-05-17T09:15:32Z | - |
| dc.date.copyright | 2012-08-28 | |
| dc.date.issued | 2012 | |
| dc.date.submitted | 2012-08-09 | |
| dc.identifier.citation | Abu Elzein, E.M.E. and Crowther, J.R. 1978. Enzyme-labelled immunosorbent assay techniques in foot-and-mouth disease virus research. J Hyg Camb 80: 391-399.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/6633 | - |
| dc.description.abstract | 口蹄疫疫苗免疫後，主要是透過血清中和試驗 (serum neutralization test, SN test) 來了解免疫動物獲得中和抗體保護力的情形，但SN test必須使用口蹄疫的活病毒，因而衍生許多的不便及限制。本實驗室使用口蹄疫病毒O/TW/97所製備的單源抗體Q10E-3，已被證明能夠精準辨認口蹄疫病毒涵蓋RGD的中和抗原決定位Site 1序列；利用此單源抗體為tracer Ab，搭配原核系統所表現的口蹄疫重組結構蛋白VP1，建構一個重組結構蛋白VP1阻斷型酵素連結免疫吸附試驗 (recombinant VP1 blocking enzyme-linked immunosorbent assays, rVP1- bELISA)，來檢測免疫動物體內的抗體，並以SN test作為黃金標準 (gold standard)。試驗樣本包含不同物種及不同SN力價的血清，分別為豬血清674個、牛血清100個及羊血清100個。結果顯示，以樣本PI值 (percentage inhibition value) 與SN力價所繪製的標準曲線圖，可看出兩個試驗在三種種別動物樣品皆有極顯著的相關性 ( p < 0.001)。而試驗血清所得之cut-off值、敏感性及特異性，在豬血清樣本為20%、58.8% (173/294)、92.9% (353/380)，牛血清樣本為10%、66.7% (40/60)、67.5% (27/40)，羊血清樣本則為30%、73.3% (44/60)、67.5% (27/40)。推測同一組SN力價血清樣本經過rVP1-bELISA檢測後PI值的差異，可能是因為血清樣本中含有抗不同中和抗原決定位的抗體，或是血清樣本影響了tracer Ab與rVP1的結合所致。本實驗的結果證明了rVP1-bELISA與SN test的正相關性，或許未來可以嘗試加入口蹄疫病毒的其他結構蛋白，再篩選出抗各個中和抗原決定位的單源抗體去搭配組合作用，以獲得更佳的敏感性及特異性，而更能精準地呈現出SN test的檢測結果。 | zh_TW |
| dc.description.abstract | To know the protection of foot-and-mouth disease virus (FMDV) vaccine, intensive serological surveillance has been implemented. And serum neutralization test (SN test) is a conventional serological assay of FMD. The test operates live virus desperately, so it should be handled in P3 class laboratory. To overcome the restriction, we try to establish a recombinant structural protein VP1 blocking enzyme-linked immunosorbent assays (rVP1-bELISA) to replace the SN test in the future hopefully. The rVP1 was expressed by prokaryotic system. The monoclonal antibody (MAb) Q10E-3 recognizing neutralization antigenic site 1 (RGD motif) on VP1 was raised by the immunization against FMDV O/Taiwan/1997. The rVP1-bELISA was checked with swine, cattle and goat sera samples with different SN titers with sample size 674, 100 and 100 respectively. SN test is the gold standard in this experiment. The current result shows the significant correlation between rVP1-bELISA and SN test (p < 0.001) among three species samples. The cut-off value, sensitivity and specificity of swine, cattle and goat samples is 20%, 58.8% (173/294), 92.9% (353/380); 10%, 66.7% (40/60), 67.5% (27/40) and 30%, 73.3% (44/60), 67.5% (27/40), respectively. The reason of the variance of results between same SN titer sample, might be the effect of antibody that induced against different antigenic site. In the future, we can try to add other FMDV structural proteins and MAb that against different antigenic sites in our ELISA kit. It may be able to obtain better sensitivity, specificity and consistency between test results of rVP1-bELISA and SN test. | en |
| dc.description.provenance | Made available in DSpace on 2021-05-17T09:15:32Z (GMT). No. of bitstreams: 1 ntu-101-R99629024-1.pdf: 3176779 bytes, checksum: f915ffc90ddc861e98084d211c7d74b3 (MD5) Previous issue date: 2012 | en |
| dc.description.tableofcontents | 口試委員審定書 i
致謝 ii 摘要 iii Abstract iv 目錄 v 圖目錄 xi 表目錄 xiii 第一章 序言 1 第二章 文獻回顧 3 2.1 歷史背景與簡介 3 2.2 病毒特性 4 2.2.1 病毒分類與結構 4 2.2.2 病毒基因與蛋白 5 2.2.3 病毒抗原構造 5 2.2.4 病毒物理化學特性 6 2.2.5 病毒生長特性 6 2.3 病毒流行病學 7 2.3.1 傳播途徑 7 2.3.2 宿主動物 8 2.3.3 致病機轉 8 2.3.4 臨床症狀 9 2.3.5 組織病理變化 10 2.3.6 宿主免疫反應 10 2.4 診斷方法 11 2.4.1 補體結合試驗 (Complement Fixation Test, CFT) 11 2.4.2 病毒分離 (Virus Isolation) 11 2.4.3 酵素連結免疫吸附試驗 (ELISA) 12 2.4.4 聚合酶鏈鎖反應 (Polymerase Chain Reaction, PCR) 12 2.4.5 血清中和試驗 (Serum Neutralization Test, SN test) 13 2.5 預防與控制 13 2.5.1 移動管制 13 2.5.2 撲殺 13 2.5.3 疫苗免疫 14 2.5.4 血清學監控 14 2.6 酵素連結免疫吸附試驗 15 2.6.1 ELISA原理 15 2.6.2 ELISA分類 16 2.6.2.1 直接型ELISA (Direct ELISA) 16 2.6.2.2 間接型ELISA (Indirect ELISA) 16 2.6.2.3 三明治ELISA (Sandwich ELISA) 16 2.6.2.4 競爭型ELISA (Competitive ELISA) 17 2.6.2.5 阻斷型ELISA (Blocking ELISA, bELISA) 17 2.6.3 ELISA應用 18 2.7 單源抗體 18 2.7.1 歷史背景 18 2.7.2 單源抗體原理 18 2.7.3 單源抗體應用 19 第三章 材料與方法 20 3.1 病毒製備 20 3.1.1 口蹄疫病毒株 20 3.1.2 細胞培養與繼代 20 3.1.3 口蹄疫病毒的增殖 21 3.1.4 口蹄疫病毒力價測定 21 3.1.5 口蹄疫病毒濃縮 21 3.2 單源抗體製備 22 3.2.1 單源抗體來源 22 3.2.2 單源抗體生產 22 3.2.3 單源抗體純化 23 3.2.4 單源抗體透析 23 3.2.5 單源抗體定量 24 3.2.6 單源抗體標示過氧化氫酶 (horseradish peroxidase, HRP) 24 3.3 重組結構蛋白質 (rVP1) 表現與確認 25 3.3.1 重組蛋白質表現 25 3.3.2 重組蛋白質之電泳分析與確認 26 3.3.2.1 聚丙烯醯胺膠片電泳 (SDS-PAGE) 26 3.3.2.2 西方墨點法 (Western blot, WB) 27 3.3.3 重組蛋白質純化 28 3.3.3.1 鎳離子親和層析管柱純化 28 3.3.3.2 切膠純化 28 3.3.4 重組蛋白質定量 29 3.3.5 重組蛋白質與單源抗體結合能力測試 29 3.3.6 重組蛋白質特異性分析與確認 30 3.4 多源抗體製備 30 3.4.1 實驗動物 30 3.4.2 免疫計畫 30 3.4.3 多源抗體特性分析 31 3.4.3.1 西方墨點法 31 3.4.3.2 血清中和試驗 31 3.5 血清樣本 31 3.5.1 標準血清 32 3.5.2 樣本血清 32 3.5.2.1 豬血清樣本 32 3.5.2.2 牛血清樣本 32 3.5.2.3 羊血清樣本 32 3.6 發展檢測血清中和抗體之阻斷型ELISA 33 3.6.1 塗鍍重組結構蛋白VP1阻斷型ELISA (rVP1-bELISA) 33 3.6.1.1 最佳化rVP1-bELISA條件 33 3.6.1.2 rVP1-bELISA操作流程 33 3.6.1.3 rVP1-bELISA標準血清分析與確認 34 3.6.1.4 rVP1-bELISA樣本血清檢測 34 3.6.1.5 A450讀值轉換成抑制百分比 (PI value) 34 3.6.1.6 rVP1-bELISA與SN test相關性之分析 34 3.6.1.7 rVP1-bELISA之cut-off值、敏感性、特異性與一致性分析 35 3.6.2 塗鍍口蹄疫全病毒阻斷型ELISA (virus-bELISA) 36 3.6.2.1 最佳化virus-bELISA條件 36 3.6.2.2 virus-bELISA操作流程 36 3.6.2.3 virus-bELISA標準血清分析與確認 37 第四章 結果 38 4.1 病毒製備 38 4.1.1 口蹄疫病毒力價測定 38 4.2 單源抗體製備 38 4.2.1 單源抗體純化、透析與定量 38 4.2.2 單源抗體標示過氧化氫酶 38 4.3 重組結構蛋白質 (rVP1) 表現與確認 39 4.3.1 重組蛋白質表現 39 4.3.2 重組蛋白質純化與定量 39 4.3.3 重組蛋白質與單源抗體結合能力測試 39 4.3.4 重組蛋白質特異性分析與確認 40 4.4 多源抗體製備 40 4.4.1 多源抗體特性分析 40 4.4.1.1 西方墨點法 40 4.4.1.2血清中和試驗 40 4.5 發展檢測血清中和抗體之阻斷型ELISA 40 4.5.1 塗鍍重組結構蛋白VP1阻斷型ELISA (rVP1-bELISA) 40 4.5.1.1 最佳化rVP1-bELISA條件 40 4.5.1.2 rVP1-bELISA標準血清分析與確認 41 4.5.1.3 rVP1-bELISA樣本血清檢測結果 41 4.5.1.3.1 豬血清樣本 41 4.5.1.3.2 牛血清樣本 41 4.5.1.3.3 羊血清樣本 42 4.5.1.4 rVP1-bELISA與SN test相關性之分析 42 4.5.1.4.1 豬血清樣本 42 4.5.1.4.2 牛血清樣本 42 4.5.1.4.3 羊血清樣本 43 4.5.1.5 rVP1-bELISA之cut-off值、敏感性、特異性與一致性分析 43 4.5.1.5.1 豬血清樣本 43 4.5.1.5.2 牛血清樣本 43 4.5.1.5.3 羊血清樣本 43 4.5.2 塗鍍口蹄疫全病毒阻斷型ELISA (virus-bELISA) 44 4.5.2.1 最佳化virus-bELISA條件 44 4.5.2.2 virus-bELISA標準血清分析與確認 44 第五章 討論 45 參考文獻 51 附錄 95 | |
| dc.language.iso | zh-TW | |
| dc.title | 抗口蹄疫病毒結構蛋白VP1單源抗體應用於酵素連結免疫吸附試驗 | zh_TW |
| dc.title | Establishment of ELISA with Monoclonal Antibody against Structural Protein VP1 of Foot-and-Mouth Disease Virus | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 100-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 王金和,林有良,陳啟銘,蔡向榮 | |
| dc.subject.keyword | 口蹄疫,單源抗體,結構蛋白VP1,阻斷型酵素連結免疫吸附試驗,血清中和試驗, | zh_TW |
| dc.subject.keyword | foot-and-mouth disease,monoclonal antibody,structural protein VP1,blocking enzyme-linked immunosorbent assay,serum neutralization test, | en |
| dc.relation.page | 95 | |
| dc.rights.note | 同意授權(全球公開) | |
| dc.date.accepted | 2012-08-09 | |
| dc.contributor.author-college | 獸醫專業學院 | zh_TW |
| dc.contributor.author-dept | 獸醫學研究所 | zh_TW |
| 顯示於系所單位： | 獸醫學系 | |
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