橘黴素(citrinin, CTN)與嘔吐毒素(deoxynivalenol, DON)主要分別由Penicillium與Fusarium 屬所產生的黴菌毒素，此類毒素經常污染食品與穀物，長期食用可能會導致人類或動物疾 病，甚至包括細胞癌化的發生。由於近年氣候異常，高熱潮溼更令此類毒素對於人類健康的 威脅性大幅升高，因此開發各式高靈敏度抗體，並且建立快速免疫檢測分析法來分析調查食 品，穀物或飼料中橘黴素CTN與嘔吐毒素DON的污染是一項食品安全的重要課題，此外具 有商品化價值的抗體及相關檢測產品的開發也可以經由產學合作提升台灣生技的競爭力。因 此本計劃的第一年期將利用融合瘤技術分別生產CTN與DON的單株抗體，利用所得到的抗 體建立酵素免疫分析法(ELISA)來分析市售樣品的毒素分布和含量。此外由於近年來利用噬 菌體表達技術(Phage display antibody system)來表達生產單鏈抗體已逐漸成熟，因此本計畫第 二年期將以大腸桿菌重組表達CTN與DON的單鏈抗體(Single chain variable fragment, ScFv)，力求抗體來源多元化，降低動物使用率及抗體生產保存的成本，並且進一步建立其 專屬之高靈敏酵素免疫分析系統。最後為了建立簡易，快速而且符合政府法規毒素管制限量 的快速篩檢法，本計畫第三年期除了將第一年期和第二年期的傳統單株抗體和單鏈抗體ScFv 應用在CTN與DON奈米金粒子及奈米量子點的免疫檢測試紙開發，並且由於作物中常見的 黴菌毒素汙染包括黃麴毒素B1 (AFB1)和赭麴毒素(ochratoxin A, OTA)，因此希望將4種毒素 (CTN/DON/ AFB1/OTA)的抗體同時合併於免疫奈米試紙的裝置中，因而能夠在十分鐘內以 目視判讀多重毒素的檢測結果，適合衛生機關及一般大眾推廣使用。 Citrinin (CTN) and deoxynivalenol (DON) are mycotoxins which are produced by Penicillium and Fusarium and. These two mycotoxins are commonly found in wheat, cereals, and food/feed products and are known to cause various toxic effects on human and livestock. The recent high humidity and temperature greatly potentiate the exposure risk of human/animal to various mycotoxins. Therefore, production of specific antibodies and development of rapid detection methods for these mycotoxins are an important issue in food safety. The first year objective of this proposal is to generate monoclonal antibodies specific to CTN and DON, and also establish enzyme-linked immunosorbent assays (ELISA) for the toxins. Recently, because the phage display antibody system has been widely used to express promising ScFv (single chain variable fragment) antibody, the objective of the second year is to produce ScFv antibodies against CTN and DON through E. coli by applying phage display antibody system. The ScFv antibody will also be used to establish ELISA for further analysis of commercialized food/feed samples. The application of nanotechnology in biological detecting is on the cutting edge of trends. Thus, in the third- year plan, we will develop gold/quantum dot nanoparticle associated immunochromatographic strips (immunostrip) assay for CTN and DON. The whole process of immunostrip can be completed within 10 min and the results can be determined using naked eye without any instrument assistance. Finally, we will try to establish a single immunostrip which can simultaneously detect CTN, DON, aflatoxin B1 (AFB1), and ochratoxin A (OTA). The data and products from this proposal will be able to provide an alternative tool for the widely use of government and the publics.
