鍊格孢酸與鍊格孢醇多株抗體之生產並將其應用於酵素連結免疫吸附分析法及免疫試紙之開發及應用

鍊格孢酸與鍊格孢醇皆屬於鍊孢格屬黴菌產生的次級代謝物，鍊孢格屬黴菌廣泛存在於土壤、空氣及植物表面，其中部分種類為常見過敏原，可引發呼吸道疾病及免疫力低下者的皮膚與指甲感染，對人類健康構成潛在威脅。鍊格孢酸及鍊格孢醇已分別被證實具有細胞毒性及基因毒性。鍊格孢屬的黴菌可以引起植物葉斑病、果實腐爛與莖腐病。因此，本研究旨在分別製備出鍊格孢酸與鍊格孢醇專一性抗體，並用來開發準確的檢測方法，保護人類免於鍊格孢酸及鍊格孢醇的毒性傷害。首先，分別將鍊格孢酸與鍊格孢醇與載體蛋白質接合，使其具有免疫原性，並免疫紐西蘭大白兔與小鼠以誘導鍊格孢酸與鍊格孢醇專一性抗體產生。接著，利用這些專一性抗體進行酵素連結免疫吸附分析法的開發。結果顯示，本研究分別透過免疫 BALB/c小鼠及紐西蘭大白兔產生鍊格孢酸專一性的多株抗體，並成功建立直接競爭型酵素連結免疫吸附分析法，其抑制 50% 抗體與鍊格孢酸-酵素接合物結合時，所需的鍊格孢酸標準品濃度 (IC50) 分別為 36.4 ng/mL 與 120 ng/mL。另一方面，本研究也分別透過免疫 BALB/c 小鼠及紐西蘭大白兔產生對鍊格孢醇具有專一性多株抗體，且成功建立直接競爭型酵素連結免疫吸附分析法，而其抑制 50% 抗體與鍊格孢醇-酵素接合物結合時，所需的鍊格孢醇標準品濃度分別為 94 ng/mL 及 49.4 ng/mL。此外，本研究利用鍊格孢酸多株抗體進行免疫層析試紙製備，並成功探索了鍊格孢酸免疫層析試紙製備條件，但其靈敏度尚不足以應用於實際樣品檢測。雖然鍊格孢酸與鍊格孢醇的檢測方法尚未完全優化，研究中所採用的策略及對挑戰的探討為未來相關研究提供了重要參考與方向。

Tenuazonic Acid (TeA) and Alternariol (AOH) are secondary metabolites produced by Alternaria, which are widely distributed in soil, air, and on plant surfaces. Some Alternaria species are common allergens, capable of inducing respiratory issues in humans and animals, as well as skin and nail infections in immunocompromised individuals. TeA has been reported to exhibit cytotoxic potential, while AOH has been demonstrated to possess genotoxic properties, underscoring the significant health risks these metabolites pose to humans. Additionally, Alternaria cause significant agricultural losses by inducing diseases such as leaf spot, fruit rot, and stem blight in plants. This study aims to produce specific antibodies against alternariol and tenuazonic acid, respectively, and develop accurate detection methods for these mycotoxins to protect humans from the toxic effects of TeA and AOH. Initially, TeA and AOH were respective conjugated to carrier proteins, respectively, to enhance immunogenicity and subsequently used to immunize BALB/c mice and New Zealand White rabbits, inducing the production of specific polyclonal antibodies. These antibodies were then utilized to develop enzyme-linked immunosorbent assay (ELISA) for TeA or AOH detection. For TeA, specific polyclonal antibodies were successfully produced from mice and rabbit, and the competitive direct ELISA (cdELISA) was developed, achieving the concentration causing 50% inhibition (IC50) of TeA-HRP binding by free TeA were 36.4 and 120 ng/mL, respectively. Similarly, for AOH, polyclonal antibodies were produced, and the cdELISA were established, with IC50 values of 94 and 49.4 ng/mL, respectively. Additionally, this study explored the development of immunochromatographic strip (immunostrip) using TeA-specific polyclonal antibodies. Although the sensitivity of the immunuostrip was insufficient for practical application in sample detection, the research successfully identified the conditions required for their preparation. While the specific antibodies against TeA or AOH were successfully produced, the detection methods developed did not fully meet the criteria for further optimization and application. Despite these limitations, the strategies employed and the challenges addressed in this study provide valuable insights and a foundation for future research into the detection of TeA and AOH.