開發對原發性肝癌具有專一性之抗體及其應用

Abstract

肝癌是全球十大常見癌症中的第四位，每年約有662000人死於肝癌。尤其在台灣因為B型肝炎的流行，肝癌是十大常見癌症中的第一位。由於現行的肝癌檢測方式價格昂貴且無法有效檢測出初期的腫瘤，因此我們希望能開發出一個能檢測肝癌的系統。 近年來有研究指出在肝癌病患的血漿中有些生物標計分子會不正常的增加。在2005年的文獻中指出，將其中三種生物標計分子組合起來應用於肝癌檢測時，不論肝癌發生至哪一階段都能百分之百的檢測出肝癌，它們分別是胎兒球蛋白(Alpha-fetoprotein, AFP)、岩藻醣苷水解酶(Alpha-fucosidase, AFU)以及血管內皮增生因子(Vascular endothelial growth factor, VEGF)。因此，我們希望能發展生物辨識分子能專一的辨認這些生物標計分子。我們選定抗體來做為生物辨識分子，因為抗體具有高度的靈敏度以及辨識能力。 在本研究中我們利用分子生物學的技術將生物辨識分子利用大腸桿菌大量表達並利用管住層析純化，這些生物辨識分子被用做老鼠的免疫並且發展出多株對於這些生物辨識分子具專一性的單株抗體。

Heptocellular carcinoma (HCC) is the primary cancer of liver. It causes about 66,200 deaths per year worldwide and ranks the forth of the most common cancer. HCC is hard to be detected in its early stage by present diagnostic techniques. Recent researches indicate that the concentration of some biomolecules is abnormal in serum of HCC patients. In this study, we aim to develop biorecognition molecules specific to these target biomarkers. We chose antibody as our biorecognition molecules, based on the high sensitivity and specificity of the antibody. In this study, three proteins, alpha-fetoprotein (AFP), alpha-fucosidase (AFU), and vascular endothelial growth factor (VEGF) were chosen as target biomolecules to develop monoclonal antibodies. The cDNA of AFU and VEGF were expressed directly as fusion proteins in vector pET22a and pET28a, respectively. For AFP, two truncated regions were overexpressed. The recombinant biomarkers were used to generate monoclonal antibodies. The monoclonal antibodies, which are specific to AFU and VEGF and AFP, respectively, have been developed. To develop HCC detection system the monoclonal antibodies were conjugated to alkaline phosphatase (AP) and applied in sandwich ELISA. The detection range based on the AP conjugated antibodies was from 10 μg/ml to 1 mg/ml.