- 學位論文
T-2毒素與萊克多巴胺抗體之製備並將其應用於酵素免疫吸附分析法及免疫層析試紙分析法之開發
Production of Antibody and Development of Enzyme-Linked Immunosorbent Assay and Immunochromatographic Strip for T-2 toxin and Ractopamine
摘要
T-2 毒素為一黴菌毒素，是由鐮刀菌真菌所產生的次級代謝物，常存在於糧食穀物或未保存良好之加工品中，人們容易誤食被污染之食物而導致腹瀉等急性症狀，有研究證明 T-2 具有細胞毒性、抑制免疫反應、DNA 和 RNA 合成等。萊克多巴胺 (Ractopamine, Rac) 是一種人工合成苯乙醇胺類的乙型受體 (β-receptor) 興奮劑，添加於飼料中，可降低動物的脂肪合成並且增加肌肉生長，亦稱瘦肉精，研究指出 Rac 導致心臟相關症狀，包括心跳過速、心律不整或心悸等。因此，本研究藉由抗體與抗原間具有專一的特性，希望分別建立一套快速且靈敏檢測 T-2 與 Rac的方法，用於檢測食品。由於 T-2 與 Rac 皆屬於小分子化合物，因此需與載體蛋白質接合進而對實驗動物進行免疫，分別產生對 T-2 與 Rac 具有專一性之抗體，並以此抗體建立免疫檢測方法。於 T-2 部分，在專一性抗體開發上，我們嘗試許多接合載體蛋白質的方法，然而進行實驗動物免疫後並無具專一性抗體的產生，利用市售的 T-2 多株抗體我們成功建立了直接與非直接競爭型酵素連結免疫吸附法，其 IC50 分別為 4.33 與 19.6 ng/mL，而在免疫層析試紙分析法的開發上，將抗體接合奈米金粒子作為探針，其顯色的效果並不佳。至於 Rac 部分，以 Rac-SH-BTG 作為免疫抗原免疫小鼠，進一步與小鼠骨髓瘤細胞融合後，篩選出可分泌抗 Rac 單株抗體之融合瘤細胞株 (6G8F4F2)，建立直接競爭型酵素連結免疫吸附法，其 IC50 為 19.7 ng/mL。本研究已成功建立 T-2 與 Rac 的免疫檢測方法，但需改善 T-2 與 Rac 接合載體蛋白質，增加檢測方法的靈敏度，以更容易篩檢食品中殘留之毒素。
並列摘要
T-2 toxin (T-2), a mycotoxin, is a secondary metabolite produced by the Fusarium fungus. It is often found in a variety of grain and the processed food which preserved poorly. If people consumed the T-2 contaminated food, it may cause the acute toxic effects including diarrhea and skin blistering. Previous studies have shown that T-2 led to the cytotoxicity, immunosuppression, and inhibition of DNA or RNA synthesis. Ractopamine (Rac) is a synthetic beta-receptor agonist. As an animal feed additive, Rac can reduce fat synthesis and increase muscle growth. Rac has been reported to cause in heart-related symptoms like tachycardia, arrhythmia and palpitations. Therefore, to detect the residues of T-2 and Rac in the food, the development of high sensitive and rapid detection methods for T-2 and Rac are nessessary. Both T-2 and Rac, belong to small molecule weight compounds, need to conjugate with carrier proteins to render their immunogenicity. Toxin-proetin conjugates as antigens were immunized the animal to produce the antibody against T-2 and Rac, respectively. Based on these antibodies, the immunoassay methods were established. In the T-2 section, although several methods for antigen conjugation were attempted, there was no specific antibody production after immunizing animals. Based on the commercial anti-T-2 antibody, the direct and indirect competitive enzyme-linked immunosorbent assay were successful developed with the IC50 of 4.33 and 19.6 ng/mL, respectively. However, immunochromatographic strip was still needed to improve due to the effect of the color reaction by antibody-gold nanoparticle conjugate. In the Rac section, the spleen cell of the mouse which immunized Rac-SH-BTG was fused with mouse myeloma Sp2/0-Ag14 cell. The anti-Rac monoclonal antibody was produced from a hybridoma, 6G8F4F2, and the competitive direct enzyme-linked immunosorbent assay was successful developed with the IC50 of 19.7 ng/mL. In this study, we have successfully established the ELISA methods for detecting T-2 and Rac, respectively. It is still necessary to improve both T-2-protein and Rac-protein conjugation effectiveness and enhance the sensitivity of the methods for screening the residues of toxins in the food.
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