- 學位論文
薄膜上免疫聚合鍊環反應之開發以促進側流動偵測極限
Development of a Membrane-Based Immuno-PCR Assay for Enhancing the Sensitivity of Lateral Flow Detection
摘要
中文摘要 本研究在硝化纖維膜上發展免疫PCR技術，以增加橫向流免疫層析法的靈敏度。此測試遵循傳統橫向流免疫層析法，但是以DNA 共軛抗體取代金奈米粒子共軛抗體的顯色方式，並令DNA 報告分子藉由PCR擴增程序，以達到提高偵測靈敏度的目的。 本研究測試DNA 對streptavidin 和抗體對DNA-Streptavidin 複合物的最佳混合比例，找尋DNA-Streptavidin 複合物的最佳化反應方式。研究發現，抗體對DNA-Streptavidin複合物以1：1的比例測試會得到最佳實驗結果，本實驗因此以此比例做為偵測基準。抗體和DNA 反應時間5 分鐘與捕獲抗體佈放濃度0.5 mg/ml，經測試結果可得最佳訊號。該條件應用於偵測human IL-6，其偵測靈敏度為 <0.001 pg/ml。進一步改善試劑和分析方法，以減少本研究存在高背景值的問題是，未來必須努力的方向。
並列摘要
Abstract Membrane-based immuno-PCR procedure was developed by applying the immuno-PCR technique on membrane nitrocellulose to increase the sensitivity of the lateral flow immunochromatography assay. The test procedure followed the traditional lateral flow immunochromatography assay. But instead the gold nanoparticles-conjugated antibody, this study employed DNA-conjugated antibody as the signal reporter. The DNA reporter was amplified by PCR to enhance the detection sensitivity. The detector antibody was optimized its conjugation method for the DNA-streptavidin complex. Two methodologies were tested the effect of the ratio of DNA to streptavidin and the ratio antibody to DNA-streptavidin complex. The 1:1 ratio of antibody to DNA-streptavidin complex was found to give the best result and therefore selected for further test. Premixing of the detector antibody and DNA, 5 minutes waiting time, and 0.5 mg/ml spraying concentration of capture antibody were concluded the best condition to give the best signal. This condition was demonstrated on the membrane-based immuno-PCR procedure to detected human IL-6 and reported the detection sensitivity is <0.001 pg/ml of human IL-6. Further improvement of reagents and assay formats is necessary to reduce the high background problem in this study.