臺灣博碩士論文加值系統

本論文以酵素顯色法在靈敏度及多重分析上的發展為研究目的。酵素顯色法利用酵素與特定受質反應產生色素沉澱而達到偵測效果。論文中分別針對酵素顯色法與活化標記佈放(CARD)顯色增益法之動態範圍與偵測極限作探討，同時比較兩種方法在分析上訊號的一致性，並以實際篩選基因驗證結果。在CARD顯色增益法的實例應用上，則研究人類鼻咽癌細胞在抗癌藥物Camptothecin(CPT)處理前後基因表現的情形。根據實驗結果，利用活化標記佈放(CARD)顯色增益法，可將酵素顯色法的偵測靈敏度提高至原來的六十倍，並得到與酵素顯色法一致的結果。
在多色酵素顯色系統的發展方面，利用在標記探針的過程中加入不同比例混合的半抗原，理論上可標記出無限多種顯色後呈現不同呈色的探針。論文中比較以聚合脢連鎖反應及隨機引子法標記多色探針的效率，及顯色結果與加入之半抗原比例的相對應關係﹔實驗最後並以隨機引子法成功的標記出二十一種顯色結果經數值化分析後可解析之探針，若運用在實際分析上，將可達到同時分析多種樣品的目的，使分析的效率大大提昇。

The goals of the research project are on the development of novel methods for enhancing the sensitivity of enzyme colorimetric detection and multiplex detection based on multicolor enzyme colorimetry. Enzyme colorimetric detection is achieved by the enzymatic reaction to create a colour precipitate on the immediate area. The thesis focuses on the dynamic range and limit of detection for both the enzyme colorimetric detection and the signal amplification method by Catalyzed Reporter Deposition(CARD). With the amplification of CARD, studies have been done on the effect of camptothecin(CPT) to the expression profile of human nasopharyngeal cell line(KB cell). The experimental results show that CARD amplification can raise the sensitivity of the technique by sixty folds comparing with enzyme colorimetric detection. Data consistency is achieved at the same time.
On the development of multicolor enzyme colorimetric detection, combining different ratios of different haptens in probe labeling can create many probes with different colors. Two different labeling methods, polymerase chain reaction and random-primed labeling, were compared on the labeling efficiency and the correlation of hapten ratios with color . Using random-primed method, 21 probes with distinct colors were successfully created. They can be used to analyze up to 21 samples simultaneously, which greatly increases the efficiency of analysis.

目 錄
一、 緒論 1
二、 酵素顯色法 4
2.1 緒言 4
2.2 材料與方法 5
2.2.1 酵素耦合抗體的定量5
2.2.2 酵素顯色法之動態範圍與偵測極限 7
2.3 結果 11
2.3.1 酵素耦合抗體的定量11
2.3.2 酵素顯色法之動態範圍與偵測極限 12
2.4 討論 13
三、 活化標記佈放(CARD)顯色增益法的發展與性質分析16
3.1 緒言 16
3.2 材料與方法 18
3.2.1 Biotinyl-tyramide的合成18
3.2.2 CARD顯色增益法之動態範圍與偵測極限20
3.3 結果22
3.3.1 Biotinyl-Tyramide的合成22
3.3.2 CARD顯色增益法之動態範圍與偵測極限22
3.4 討論23
四、 CARD顯色增益法與酵素顯色法之比較及其在生醫科學上的應用 27
4.1 緒言27
4.2 材料與方法 28
4.2.1 酵素顯色法與CARD顯色增益法訊號一致性研究28
4.2.2 篩選表現受CPT影響的基因31
4.2.3 CARD顯色增益法之實例應用31
4.3 結果 32
4.3.1 酵素顯色法與CARD顯色增益法訊號一致性研究32
4.3.2 篩選表現受CPT影響的基因33
4.3.3 CARD顯色增益法之實例應用33
4.4 討論34
五、 多色酵素顯色偵測系統之發展37
5.1 緒言37
5.2 材料與方法 40
5.2.1 聚合脢連鎖反應標記探針40
5.2.2 以隨機引子法標記探針 42
5.2.3 酵素聯結多色偵測法顯色反應的進行43
5.3 結果45
5.3.1 聚合脢連鎖反應標記探針45
5.3.2 隨機引子法標記探針46
5.4 討論47
六、 總結51
七、 參考文獻92
表 目 錄
表1 可應用於酵素顯色反應之半抗原及酵素偶合抗體組合.53
表2 常用於產生不溶性有色沉澱的酵素與其特定受質.53
表3 常見用於免疫分析領域中結合反應的反應基團.54
表4 擷取影像所用之照相機及掃描器.55
表5 利用聚合連鎖反應增殖人類基因序列所使用之載體及引子.56
表6 同時經由酵素顯色法及CARD顯色增益法偵測後篩選出之受CPT 增
強之人類基因片段.57
表7 九千六百個人類基因片段中篩選出表現受CPT抑制或加強的基因.58
表8 聚合連鎖反應標記探針之半抗原比例.59
表9 作為探針標記模板之二十一個基因.60
表10 利用聚合連鎖反應以不同基因為模板標記探針所使用之引子.61
表11 應用於多色顯色反應之半抗原及酵素偶合抗體.62
表12 二十一種以隨機引子法標記之探針經三次顯色結果.63
圖 目 錄
圖1 酵素顯色法偵測流程圖.64
圖2 雜交反應裝置及流程圖.65
圖3 SA-b-Gal 定量圖.66
圖4 不同SA- b -Gal稀釋倍率下酵素顯色法之線性圖.67
圖5 Anti-Dig-AP 定量圖.68
圖6 不同Anti-Dig-AP稀釋倍率下酵素顯色法之線性圖.69
圖7 酵素顯色法-探針分子數與訊號線性關係圖.70
圖8 酵素級聯反應流程圖.71
圖9 ABC法訊號放大流程圖.72
圖10 酵素放大法流程圖.73
圖11 CARD顯色增益法流程圖.74
圖12 Biotinyl-Tyramide 結構圖.75
圖13 Biotinyl-Tyramide 合成反應圖.75
圖14 CARD顯色增益法-探針分子數與訊號線性關係圖.76
圖15 經KB cell cDNA探針雜交並分別以酵素顯色法及CARD顯色增益法偵
測訊號之薄膜.77
圖16 CARD顯色增益法之訊號線性圖.77
圖17 薄膜分別經過KB cell及KB cell+CPT之cDNA探針雜交與經酵素顯色
法及CARD顯色增益法偵測後結果.78
圖18 酵素顯色法偵測帶正電尼龍薄膜上之人類基因圖.79
圖19 CARD顯色增益法偵測帶正電尼龍薄膜上之人類基因圖.79
圖20 佈放於帶正電尼龍薄膜上之九千六百個人類基因片段分別經KB cell
及KB cell+CPT之cDNA探針雜交後以CARD顯色增益法偵測結果.80
圖21 圖20所得結果以KB cell雜交所得訊號為橫軸，KB cell+CPT雜交所
得結果為縱軸做圖.81
圖22 隨機引子標記法流程圖.82
圖23 缺斷轉譯標記法流程圖.83
圖24 PCR法標記探針流程圖.84
圖25 核醣核酸反轉錄標記法流程圖.85
圖26 尾端標記法流程圖.86
圖27 利用PCR法標記相同模板所得探針顯色結果與探針電泳圖.87
圖28 利用PCR及隨機引子法標記二十一種不同模板探針顯色結果及電泳
圖.88
圖29 以隨機引子法標記之探針三次顯色結果在C-M、C-Y及M-Y座標圖上分
布情形 .89
圖30 半抗原比例與顯色結果對應關係圖.90
圖31 實驗中常用之耦合酵素-Gal 、AP與HRP與相對應受質X-Gal、
Fast Red TR/AS-MX及DAB之顯色反應示意圖….91

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