- 學位論文
孔雀石綠與嘔吐毒素多株抗體的生產並將其應用於酵素連結免疫分析法及奈米金粒子免疫層析試紙分析法的開發
Production of Polyclonal Antibodies and Development of ELISA and Gold Nanoparticle Immunochromatographic Strip Assay for Malachite Green and Deoxynivalenol
摘要
小分子毒素在食物中分佈十分廣泛，容易因誤食而導致動物及人類的健康受到威脅。本研究以孔雀石綠 (Malachite green, MG) 與脱氧雪腐鐮刀菌烯醇 (Deoxynivalenol, DON) 為主題，希望分別建立一套能有效且快速的方法來檢測這兩種小分子毒素。 MG為一良好的抗菌劑，廣泛應用於水產養殖漁業; DON又稱嘔吐毒素（vomitoxin），是一種常見的黴菌毒素，尤其是養殖場的豬隻對 DON 特別敏感，嚴重影響養殖豬場上的經濟效益，為了能快速的在食品中檢測出MG及DON的含量，本研究利用抗原-抗體之間專一性鍵結的特性製備了MG及DON的多株抗體，並建立非直接競爭型酵素連結免疫吸附分析法 (Competitive indirect enzyme-linked immunosorbent assay, ciELISA) 和直接競爭型酵素連結免疫吸附分析法 (Competitive direct enzyme-linked immunosorbent assay, cdELISA) 。利用MG抗體在ciELISA和cdELISA檢測毒素中，其抑制一半所需要的濃度 (IC50) 分別為5、5.5 ng/mL，結果顯示有很好的一致性。利用先前實驗室已生產的DON多株抗體，來建立一套 ELISA 的分析方法，在ciELISA和cdELISA的檢測DON毒素中，其抑制一半所需要的濃度 (IC50) 分別為200、5.4 ng/mL。 本研究成功生產出 MG 之多株抗體並且利用已知DON抗體分別建立於ELISA 能有效的檢測出毒素，以避免受到這些毒素的危害。由於 ELISA只適用於實驗室方便操作，無法適用於一般大眾。因此本研究將奈米金粒子與多株抗體相結合，形成抗體奈米金探針，開發免疫層析試紙分析法(Immunochromatographic strip,immunostrip) ，但是結果顯示游離毒素無法競爭掉測試區上的毒素-蛋白接合物而呈現陰性反應。
並列摘要
Small molecule toxins are widely spread in daily life. It’s easily to induce health threat to humans and animals. This study focused on malachite green (MG) and deoxynivalenol (DON), hoping to establish an effective detection method to detect these small molecule toxins. MG is a great antiseptic widely used in aquaculture; DON also called vomitoxin, is a common mycotoxins, pigs are especially more sensitive to DON and will seriously affect the economic benefits in pig industry. In order to detect the content of MG and DON, respectively in food rapidly.We have produced polyclonal antibody of MG and established the competitive indirect enzyme-linked immunosorbent assay, ciELISA and competitive direct enzyme-linked immunosorbent assay, cdELISA. In the ciELISA and cdELISA, the concentration causing 50% inhibition (IC50) of MG was 5 ng/ml and 5.5 ng/mL, respectively. The result shows that both have a good consistence by ELISA. Additionally, Using polyclonal antibody of DON which was generated in own laboratory to establish the ELISA. In the ciELISA and cdELISA, the concentration causing 50% inhibition (IC50) of DON was 200 ng/mL and 5.4 ng/mL, respectively. The ELISA method is feasible only in the laboratory and can not be used on site test for the general public. Therefore, gold nanoparticles were conjugated with polyclonal antibodies to form antibody nano-gold probe to develop immunochromatographic strip for on-site test for MG and DON,respectively which is more simple, fast and applicable to the general public. However the result showed that the free toxin displace the test line toxin-protein conjugate which showed negative results.