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以Immuno-PCR 方法結合磁性奈米碳管於鼻咽癌早期篩檢之研究
Fabrication of Magnetite Multiwalled Carbon Nanotubes for the Early Screening of Nasopharyngeal Carcinoma by Immuno-PCR
摘要
鼻咽癌為於鼻咽腔或上咽喉部的癌症，好發於中國東南部及台灣，根據衛生署的統計，近幾年於台灣因罹患鼻咽癌所造成的死亡人數至少有1000 個人。由於現行檢測方式，病人常檢測出癌症時，已為末期或癌症已遠處轉移，因此若能發展出早期診斷鼻咽癌之病灶，可及早治療及降低此癌症之致死率。因此本研究以免疫聚合酶連鎖反應(immuno-PCR)作為診斷方式，利用抗原-抗體及酵素-受質之間相互作用，建構出一套具有高靈敏度且專一性之檢測平台，期能進行早期檢測鼻咽癌之病灶。 本研究以透過化學沉積方式製備之磁性奈米碳管(CNTs/Fe3O4)為基材，並進行表面改質，使基材表面帶有胺基。透過交聯劑(EDC)進行化學鍵結將鼻咽癌抗原(EBNA1)固定於基材表面，EBNA1 抗原會捕獲病人血清中的anti-EBNA1 IgA抗體，再與生物素標定的二次抗體(biotinylated anti-human IgA)進行結合。再利用鏈黴親合素(streptavidin)架橋將生物素標定之二抗和生物素標定DNA 連接在一起，最後透過用real-timePCR 方式將生物素標定DNA 進行擴增，並進行結果分析。根據實驗結果得知，於生物素標定DNA 之濃度為5ng/ml、鏈黴親合素濃度為100ng/ml 及生物素標定二次抗體濃度為0.1μg/ml 之反應條件下，可有效增加系統的靈敏度與降低背景值。且於此試驗參數下，可得知免疫聚合酶連鎖反應之偵測靈敏度比傳統檢測用之酵素連結免疫吸附法(ELISA)至少高出100 倍。因此，若未來於癌病早期檢測方面，期望免疫聚合酶連鎖反應可成為臨床主要檢測之工具。
並列摘要
Nasopharyngeal carcinoma (NPC) is the most common cancer originating in the nasopharynx, the uppermost region of the pharynx, and it is a highly prevalent malignancy in southern China and Taiwan. According to the survey of Department of Health, there are more than 1000 people died in the cause of NPC per year. NPC commonly is diagnosed late because of its deep location and vague symptoms, and this late diagnosis leads to decreased survival. We developed the sensitive method which is powerful of detecting NPC in early phase. In this study, we utilized immuno-PCR as a tool for diagnostics. The purpose of study was to develop a sensitive method to evaluate the anti-Epstein-Barr nuclear antigen 1 IgA antibody (anti-EBNA1 IgA) from patient serum. First, we used synthesis CNTs/Fe3O4 nanocomposites by chemical co-precipitation to be substrate. And surface modification by formatting amide bond by conjugated with poly(ethylene glycol) bis(amine). Then we conjugated the carboxyl group of EBNA-1 antigen via EDC coupling with CNTs/Fe3O4 nanocomposites. The EBNA1 antigen conjugated on CNTs/Fe3O4-PEG nanocomposites were used to detect human anti-EBNA1 IgA antibody, then the anti-EBNA1 IgA antibody were recognized by biotinylated goat anti-human IgA secondary antibody. And the streptavidin was used to link the biotinylated DNA with the biotinylated goat anti-human IgA. The biotinylated DNA was amplified and analyzed by real-time PCR. The optimized parameter was the amount of reporter DNA (5ng/ml), streptavidin (100ng/ml) and biotinylated secondary antibody(0.1μg/ml). The optimized concentration of reporter DNA, streptavidin and biotinylated secondary antibody could improve the sensitivity and specificity. Comparing with conventional ELISA, the sensitivity of immuno-PCR was 100 folds higher than ELISA and proved that immuno-PCR would be a powerful tool for diagnosis in the future.