Immuno-PCR Application on the Early Nasopharyngeal Carcinoma Cancer Detection

Autor: Hsiang-Yin Lu, 盧香吟
Rok vydání: 2006
Druh dokumentu: 學位論文 ; thesis
Popis: 94
Nasopharyngeal carcinoma (NPC) is a common disease with high mortality. According to the survey of Department of Health, there are more than 800 people died in the cause of NPC. NPC commonly is diagnosed late because of its deep location and vague symptoms, and this late diagnosis leads to decreased survival. We had developed a sensitive method which is very powerful in detecting NPC in early phase. In this study, we utilized immuno-PCR as a tool for diagnostics. Our purpose was to develop a sensitive method to evaluate the anti-Epstein-Barr nuclear antigen 1 IgA (anti-EBNA1 IgA) from patient serum. First, glass beads were coated with an epoxy-terminated silane and analyzed by contact angle measurements, atomic force microscope, and X-ray photoelectron spectroscopy (XPS). Then we conjugated the NH2 group of EBNA1 with epoxy group on glass beads. The EBNA1 conjugated on glass beads was used to detect human anti-EBNA1 IgA, then the anti-EBNA1 IgA were recognized by biotinylated goat anti-human IgA. After the biotinylated goat anti-human IgA bound to the human anti-EBNA1 IgA, free streptavidin was used to link a biotinylated DNA to the biotinylated goat anti-human IgA. The biotinylated DNA was amplified by PCR, and analyzed by gel electrophoresis. In order to increase the sensitivity and reduce background noise, the amount of reporter DNA (0.1ng/ml), streptavidin (100ng/ml) and biotinylated second mAb(0.1μg/ml) were optimized. The optimized concentration of reporter DNA, streptavidin and biotinylated second Ab could improve the sensitivity and reduce background noise. Comparing with conventional ELISA, the sensitivity of immuno-PCR was 10 folds higher than ELISA and proved that immuno-PCR would be an important tool for diagnosis in the future.
Databáze: Networked Digital Library of Theses & Dissertations
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