Application of chromatographic strip on genetic, enzymaticand immunological assays
| Autor: | Wei-Feng Chang, 張瑋峰 |
|---|---|
| Rok vydání: | 2014 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 102 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. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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