Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/91570
標題: 層析試紙在基因、酵素與免疫方法上之應用
Application of chromatographic strip on genetic, enzymatic and immunological assays
作者: Wei-Feng Chang
張瑋峰
關鍵字: chromatography strip
lactate
Mycobacterium tuberculosis diagnosis
fragment
ESAT-6/CFP-10
Rabbit anti-ESAT-6/CFP-10
colloidal gold
Dengue Virus type II
Streptavidin
FITC
anti-FITC
Biotin
PCR
層析試紙
乳酸
結核桿菌診斷
蛋白質片段
ESAT-6/CFP-10
Rabbit anti-ESAT-6/CFP-10
膠體金
登革熱
Streptavidin
FITC
anti-FITC
Biotin
PCR
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摘要: 免疫層析檢測(immunochromatographic test, ICT),其優點 為?用簡單步驟即可完成檢測,可以廣泛的應用於?床上的診斷、汙 染檢測、農業與獸醫診斷。層析試紙本身具有成本低?、操作容?、 原?簡單,使用者?需要長期的訓?,可以即時檢驗的優點,?用肉 眼就可以判斷免疫試紙檢測結果,?進一步結合各?型光學式的判? 儀器,用以定?的分析。 第一部份研究中,在乳酸檢測試紙方面,人體中乳酸以L-乳酸與 D-乳酸?種型態存在,而血清主要以L-乳酸為主成分,可以用?鑑 別組織敏感的指標。在本研究中開發專一性酵素分析?同型態之乳酸, ?用四氮唑鹽的特性,以黃遞?做為NADH 和四氮唑鹽之間反應的 橋?並結合脫氫酵素,在免疫層析試紙上呈色檢測L-乳酸濃?。所 得到的最適化條件為硝化纖維薄膜上固定黃遞?濃?為8x10-3 U、移 動相磷酸緩衝液pH 為9.0、tetrazolium salt 之NBT 濃?為12mM、乳 酸脫氫?濃?為0.25U 及反應時間為10 分鐘,可檢測乳酸的線性範 圍在0.039-5mM 及偵測極限為0.053 mM。研究中所開發的層析試紙, 檢體可以直接分析?必經過稀釋,試紙檢涵蓋?床上檢測範圍,可以 達到操作方?與即時檢測的目的。 研究的第二部份,著重於半定?層析檢測試紙偵測結核分枝桿菌 II 群,實驗室希望針對結核病患在醫院的送檢的檢體中,經過BACTEC MGIT(mycobacteria growth indicator tube) 960 system培養後,初步判 斷為分枝桿菌的病患檢體,?用試紙進?快篩分辨是否為結核桿菌群, 所得到對於anti- ESAT-6/CFP-10結合膠體?的最適化條件如下:兔 anti-ESAT-6/CFP-10與膠體?結合最適化濃?為(稀釋倍?25倍)、膠體 ?結合pH值為pH 7,結合之反應溫?為37℃與結合反應時間為30分 鐘,半定?層析檢測試紙可以檢驗ESAT-6/CFP-10濃?介於60-976 μg/ml。 第三部份的研究,開發PCR產物結合層析試紙,?用層析試紙上 固定Streptavidin,將膠體?結合小白鼠 anti-FITC作為標誌物,可以 做為一個規格化的檢驗試紙?取代電泳法,減少照膠系統這一步驟, 增加?實驗的??性,所得到的層析試紙最適化結果如下:小白鼠 anti-FITC與膠體?結合最適化濃?為6.25x10-2mg/ml(序?稀釋倍? 24倍)、膠體?結合pH值為pH 8,結合之反應溫?為28℃與結合反應 時間為30分鐘,檢測?用登革熱2型專一性primer之PCR產物,於層析 試紙可以有效且快速的鑑別出結果。
The immunochromatographic test (ICT) is an advanced technique widely used in clinical diagnosis, pollution monitoring, agricultural and veterinary inspections. There are many advantages for this technique, such as low cost, easy to operate, simple and convenient. Long term training is not needed for the users and the tests results can be easily and immediately obtained. The results from ICT can be interpreted by visual observation in a short time and the quantitative analysis can also be carried out by using appropriate optical devices. In this thesis, the studies are divided into three parts. First of the studies is the development of ICT systems, for the clinical applications. The ICT displays a good profit on its advantages in the diagnosis, such as the lactate strips. In this study, the analysis of L-lactate with specific L-lactate dehydrogenase enzymes is applied via the addition of the tetrazolium salt. The detection of L-lactate on the strips is carried out by using the diaphorase redox reaction between the NADH and tetrazolium salts. The optimal conditions of the reactions are 8x10-3 U of immobilized diaphorase on nitrocellulose membrane, pH 9 PBS buffer, 12mM NBT tetrazolium salt, 0.25 U lactate dehydrogenase and reaction time of 10 min. The linear detection range of lactate is 0.039-5.0 mM, while the optimal detection limit of the reaction is 0.053 mM. In the lactate ICT detection, the samples can be analyzed directly without any dilutions because the detection range is within the clinical applications. In the second part of this study, it is focused on the detection of Mycobacterium tuberculosis complex on the semi-quantitative strips. The IV samples of the patients are incubated in BACTEC MGIT (Mycobacteria growth indicator tube) 960 systems to obtain the initial diagnosis data, then a rapid screen of the strips is designated to distinguish the Mycobacterium tuberculosis complex from the samples. The optimal antibody conjugate CG conditions are: 6.25x10-2mg/ml anti-ESAT-6/CFP-10 pH 7.0, 37 ℃ and reaction time of 30 min. The semi-quantitative detection range of ESAT-6/CFP-10 is in the range of 60-976 μg/ml. In the third part of this study, the development of PCR combined chromatographic strips is established. The Streptavidin is immobilized on the strips. The Mouse anti-FITC with colloidal gold served as a biomarker. The strips are used to replace the electrophoresis with the ease of operation. The optimal conjugate conditions are 6.25x10-2mg/ml mouse anti-FITC conjugate colloidal gold, reaction under pH 8.0, reaction temperature of 28℃ and reaction time of 30 min. The Dengue Virus Type II detection was performed and the PCR products from its specific primers are detected effectively and rapidly by using the strips.
URI: http://hdl.handle.net/11455/91570
其他識別: U0005-2811201416183092
文章公開時間: 10000-01-01
Appears in Collections:化學工程學系所

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