牛病毒性下痢（BVD）是牛隻疾病中重要的接觸傳染疾病之一，造成呼吸道、消化道與生殖道疾病。臨床症狀包括腸道下痢及出血。懷孕牛會導致不孕、流產、胎兒缺損、虛弱仔牛及小牛持續性感染。牛病毒性下痢廣佈於世界各地，台灣從1977年首發至今，造成國內經濟損失。在目前診斷方法須時間進行檢測、且耗費較大，在現場運用困難。本研究目的將研製出牛病毒性下痢免疫層析檢驗試條，檢測牛隻抗體為目的，其效果較為有效及快速，並評估此方法之敏感性和特異性。實驗結果，製備標記物溶液適當的pH值為8-9及抗體結合膠體金的最穩定濃度為100 μg/mL 。此試條與ELISA比較結果，敏感性85%、特異性75%、精確性84%。期望此研發將有助於臨床獸醫師快速診斷本病及有效的控制，未來可用於清除計畫的快速診斷方法。

Bovine viral diarrhea（BVD）, one of the most important cattle contact diseases, causing respiratory tract, enteric, and reproductive problems. The major clinical signs included enteric diarrhea, and hemorrhage. Pregnate cow during gestation could lead to infertile, abortion, early embryonic deformity, weak calf and remain persistently infection in calf. The spread of BVD is worldwide, it had been causing economic lost. Since 1977,the first case of BVD was reported in Taiwan. In current, the diagnostic methods are waste of time and money, which makes the manipulation more difficult in the field. The purpose of this study is to develop immunochromatographic test for detection the antibody of BVD, which is more effective and fast. Also, the study will detect the sensitivity and specificity. Preliminary results of experiments：the colloidal gold optimum pH value is 8-9 and the
best range of colloidal gold conjugate to antibody is very stability in 100 μg/mL. The diagnostic strip and ELISA compare of the resulting data revealed 85% in sensitivity, 75% in specificity and 84% in accuracy. The development of this diagnostic strip will be helpful for veterinarians to diagnose BVD and control this disease. It can be used to rapid diagnosis of clear plans in the future.

目 錄
中文摘要………………………………………………………………Ⅰ
英文摘要………………………………………………………………Ⅱ
誌謝……………………………………………………………………Ⅳ
目錄……………………………………………………………………Ⅴ
圖表目錄…………………………………………………………..…Ⅷ
第1章 前言…………………………………………………………...1
第2章 文獻回顧…………………………………………….........3
2.1牛病毒性下痢的流行病學……........….…………………....3
2.1.1致病因子………………………………..………….…………...3
2.1.2病毒的結構………………….………….……………....…3
2.1.3傳播途徑........……………………………………….….4
2.1.4 臨床症狀……………..………………………….........4
2.1.5臺灣地區牛病毒性下痢爆發情形……………………......5
2.2目前常用之診斷方法及比較…………………………….….……..5
2.2.1病毒分離及螢光抗體檢測…………………………………..5
2.2.2 ELISA檢測………..…………………………..…….....6
2.2.3 電子顯微鏡檢查………………………………………....6
2.2.4 聚合酶連鎖反應……………………………….………....6
2.3檢測診斷技術產品市場概況……………………………….…....7
2.3.1醫用之檢測診斷試劑產品尚需要具備以下特性…….…...7
2.4 免疫層析檢測之簡介………………………………………….…..8
2.4.1 免疫層析試紙之檢測原理…………………………….……8
2.4.2 免疫層析試紙之組成………………………………….……8
2.4.2.1檢體墊 (sample pad)………………………………….....10
2.4.2.2結合墊 (conjugate pad)……………………………………10
2.4.2.3硝化纖維薄膜………………………………………………..10
2.4.2.4吸收墊……………………..…..…………….…………...11
2.4.3 膠金標誌………………………...……...…….…....……11
2.4.4蛋白質吸附於膠體金和溶液pH…………………………..….12
2.5免疫層析試驗……………………………….…………....…..12
第3章 材料與方法………………………………………………….…14
3.1 牛病毒性下痢抗原製備………………………..……….….14
3.1.1 牛病毒性下痢病毒株………………………………...14
3.1.2 病毒繁殖和細胞培養………………………………...14
3.1.3 蔗糖梯度法之病毒純化……………………………...14
3.1.4螢光抗體檢測…………………………….…………...15
3.2 病毒RNA鑑定…..……………………..………………....…15
3.2.1 病毒RNA萃取……………………………………….…15
3.2.2 cDNA…………………………………………………..16
3.2.3 RT-PCR………………………………………….......16
3.2.4 電泳分析……………………………………………...16
3.2.5病毒電顯分析……………………………............17
3.3 陽性血清抗體製備…………………………………………..17
3.4 ELISA檢測…………………………………………………...17
3.4.1 抗原抗體定量………………………………………...17
3.4.2 陽性抗體ELISA Titer 判定……………………....19
3.4.3 刪除值計算…………………………………………...19
3.4.4酵素免疫吸附法之評估分析………………….……………...20
3.5免疫層析試紙檢測……………………………….……………...21
3.5.1 免疫層析試紙的製作………………………………...21
3.5.2 膠體金的製備…………………………………….…..22
3.5.3抗體及抗原濃度檢測…………....………………....22
3.5.4 膠體金吸附抗體的最適合之pH值……………………23
3.5.5膠體金吸附抗體的最適合濃度…………………………23
3.5.6 Goat-Anti-Bovine IgG抗體結合膠金………………24
3.5.7 BVDV之抗原敏感性比較………………………………24
3.5.8 BVDV抗體免疫層析之特異性……………………..…24
第4章 結果……………………………………………………….....25
4.1蔗糖梯度法離心病毒之位置……………………….………….25
4.2病毒螢光染色………………………………………..…………26
4.3 BVD病毒之穿透式電子顯微鏡(TEM)………………………….28
4.4病毒RNA萃取………………………………………………..….29
4.5 Box titration 抗原抗體定量…...……………….………30
4.6血清ELISA titer 判定…………………………………………32
4.7 刪除值計算…………………………………………………...33
4.8檢體墊裁切對顯色結果的影響………………………………..34
4.9體金吸附抗體之最適合pH值…………………………………..35
4.10膠體金吸附抗體的最佳蛋白濃度………………....……….37
4.11免疫層析試紙裁切大小……………………………………39
4.12穩定劑濃度對免疫層析試紙檢測之影響……………………40
4.13 BVDV 抗原免疫層析之敏感性檢測…………………………….40
4.14比較ELISA與免疫層析法之待測陽性血清敏感性……………….41
4.15 BVDV抗體免疫層析之特異性測試....………………...……..42
4.16 BVDV免疫層析試紙與ELISA特異性抗體之比較……………...43
第5章 討論………………..………………..……………..........44
參考文獻…………………..……………………………………..….48
附錄一……………………………………………………………………55
1. 細胞培養材料…………..……..………………….…..…..55
2. 膠體金配置及抗體...………………………………………….55
3. 免疫層析試紙………………………………………………….55
4. 螢光抗體…………………………………………………………56
5. ELISA酵素免疫結合吸附法………………………………….…56
6. 蛋白質分析Protein assay……..………..…………………….58
7. 穿透式電子顯微鏡…………………………………………...…58
8. PCR………………………………………………………………….59
9. 福氏佐劑…………………………………………………………..59
10. 儀器…………………………………………………...……..…59
附錄二................................................….61
1. 抗原濃度以Protein assay分析結果………………………………61
2.Salmonella typhimurium抗原濃度，以Protein assay分析結果.61
作者簡介……………………………………………………………....62

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