Immuno-PCR 於鼻咽癌早期診斷應用之研究

Abstract

鼻咽癌為華人罹患率以及死亡率較高的疾病之一，根據衛生署的統計，近幾年來罹患鼻咽癌所造成的死亡人數至少有800個人。以現有的診斷方式，病人在診斷出鼻咽癌時，往往是癌症末期，癌細胞已遠處轉移。本研究以免疫聚合酶連鎖反應作為診斷工具，利用抗原-抗體與酵素-受質間的作用，架構一套具高度靈敏且具有專一性之檢驗方法：免疫聚合酶連鎖反應(immuno-PCR)，以期能早期診斷出鼻咽癌病灶。 首先以玻璃珠為基材，利用帶有環氧基的矽氧烷進行表面接枝改質，使原本帶OH之基材帶有環氧基，並以水接觸角、原子力顯微鏡與X射線光電子能譜儀(XPS)來評估分析。接著在利用鼻咽癌抗原(EBNA1)上之胺基與接枝於基材之環氧基做鍵結，此抗原會捕獲病人血清中的相對應的anti-EBNA1 IgA抗體，再與生物素標定的二次抗體(biotinylated anti-human IgA)結合。再以鏈黴親合素(streptavidin)架橋將生物素標定之二抗和生物素標定DNA連接在一起，最後以引子(primer)將生物素標定DNA 大量擴增，產物利用洋菜膠電泳法來分析判讀。 由實驗結果得知，在選定生物素標定DNA之濃度為0.1ng/ml、鏈黴親合素濃度為100ng/ml與生物素標定二次抗體濃度為0.1μg/ml的反應條件下，可有效增加系統的靈敏度與降低背景值；在此的實驗參數下，免疫聚合酶連鎖反應之偵測靈敏度至少較傳統檢測用之酵素連結免疫吸附法(ELISA)超出10倍。因此，未來在疾病早期測定上，免疫聚合酶連鎖反應可望成為主要臨床檢測的工具，扮演著非常重要的角色。

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.