鐮孢菌酸多株抗體及適體之製備並將其運用於酵素連結免疫分析法及奈米金粒子免疫層析試紙

鐮孢菌酸 (Fusaric acid, FA) 是鐮孢菌屬 (Fusarium) 產生的次級代謝產物，主要構造為芳香環和羧酸。鐮孢菌酸常見於土壤中，被視為引起植物病害的因素，對植物細胞可能產生毒性影響，若是穀物保存不善，寵物穀物飼料中曾被檢測到高達 35 ppm 的 FA 汙染。此外，FA 對人體也可能會造成危害，如破壞 DNA 合成、抑制人體細胞生成及抑制多巴胺 β-羥化酶酶活性，長期食用汙染的穀物可能導致人類或動物罹患疾病。然而，傳統的 FA 檢測法費時而且昂貴。因此，本研究主旨在利用抗體-抗原的專一結合的特性來開發快速免疫檢測法;同時，也利用核酸適體-標的物親和結合的特性，開發快速之適體分析法。首先，本研究將 FA 接合至載體蛋白質使其具有免疫原性，並用來免疫紐西蘭大白兔及 Balb/c 小鼠，來產生 FA 高度專一性多株抗體。本研究已經成功利用紐西蘭大白兔製備出高專一性之 FA 多株抗體，並利用此 FA 多株抗體建立直接競爭型酵素連結免疫吸附分析法 (Competitive direct enzyme-linked immunosorbent assay, cdELISA)，其抑制 50% 抗體與 FA-酵素接合物結合需的 FA 標準品濃度 (IC50) 為 40 ng/mL；檢測區間 (IC20 – IC80) 為 3.32 – 大於 5000 ng/mL；檢測極限 (IC10) 為 1.08 ng/mL。利用此 cdELISA 於外添加毒素 (Spiked) 陰性穀物飼料樣品檢測，結果顯示其回收率介於 86 – 125% 之間；且 CV (Coefficient of Variation) 值介於 6.1 – 8.8% 之間。接著於市面抽樣 15 種穀物飼料，經由 cdELISA 檢測結果顯示，FA 汙染量介於 441 – 1733 ng/g 之間；汙染發生率為 27%。最後，利用 HPLC (High performance liquid chromatography) 來確認 cdELISA 分析樣品之結果，顯示 cdELISA 與 HPLC 檢測結果有高度一致性。因此本研究所開發之 cdELISA 可實際應用於穀類樣品檢測。此外，cdELISA 結果需要儀器判讀，因此本研究擬利用 FA 多株抗體來開發免疫層析試紙，結果顯示此一免疫層析試紙視覺檢測極限 (Visual detection limit, vLOD) 大於 500 ng/mL。其檢測靈敏度還不足以應用於實際樣品檢測上，但可提供未來改進的方向。另一方面，本研究利用 SELEX (Systematic evolution of ligands by exponential enrichment) 篩選出一對 FA 親和性之核酸適體，將此 FA 核酸適體應用於 FA 適體分析法，其 IC50 大於 5000 ng/mL，其檢測敏感度尚不足以用於實際樣品檢測。綜合以上所述，本研究以成功製備 FA 多株抗體，並利用此一多株抗體開發出可應用於實際穀類飼料樣品檢測之 cdELISA，儘管 FA 免疫層析試紙及核酸適體分析法檢測靈敏度較差，但提供了未來開發 FA 免疫檢測法有價值的參考。最終希望本研究成果有助於監測食品中的 FA 汙染。

Fusaric acid (FA) is a secondary metabolite of the Fusarium genus, characterized by a primary structure consisting of an aromatic ring and a carboxylic acid group.Fusaric acid is commonly found in soil and is considered a factor contributing to plant diseases. It may exert toxic effects on plant cells. In cases of inappropriate storage of grains, Fusaric acid contamination has been detected in pet grain feed at levels as high as 35 ppm. Furthermore, Fusaric acid (FA) may pose risks to human health, such as disrupting DNA synthesis, inhibiting cell proliferation in the human body, and suppressing the activity of dopamine β-hydroxylase. Long-term consumption of contaminated grains may lead to the development of diseases in humans or animals. However, traditional Fusaric acid (FA) detection methods are time-consuming and expensive. Therefore, this study aims to develop a rapid immunochemical method by utilizing the specific binding properties of antibodies to antigens. Simultaneously, the study also aims to develop a rapid aptamer analysis method by leveraging the specific binding properties of nucleic acid aptamers to their targets. Firstly, in this study, Fusaric acid (FA) was conjugated to a carrier protein to render it immunogenic and was used to immunize New Zealand White rabbits and Balb/c mice, aiming to generate highly specific polyclonal antibodies against FA. The study successfully produced highly specific polyclonal antibodies against FA using New Zealand White rabbits. These antibodies were then employed to establish a competitive direct enzyme-linked immunosorbent assay (cdELISA), with an half maximal inhibitory concentration (IC50) of FA standard required for 50% antibody binding to the FA-enzyme conjugate being 40 ng/mL. The detection range (IC20 – IC80) was 3.32 – greater than 5000 ng/mL, and the detection limit (IC10) was 1.08 ng/mL. Using this cdELISA for the detection of spiked toxin in negative grain feed samples, the results indicated recovery rates ranging between 86% and 125%. The coefficient of variation (CV) values fell within the range of 6.1% to 8.8%. Subsequently, in the analysis of 15 commercially available grain feed samples using cdELISA, Fusaric acid (FA) contamination levels were found to range from 441 to 1733 ng/g, with an incidence rate of contamination at 27%. Finally, the results obtained from the cdELISA analysis were confirmed using High Performance Liquid Chromatography (HPLC), demonstrating a high degree of consistency between the cdELISA and HPLC detection methods. Therefore, the developed cdELISA in this study can be practically applied to the detection of grain samples. Additionally, as the interpretation of cdELISA results requires instrumentation, this study aims to utilize the Fusaric acid (FA) polyclonal antibodies to develop an immunochromatographic test strip. The results indicate that the visual detection limit (vLOD) of this immunochromatographic test strip is greater than 500 ng/mL. While its detection sensitivity is currently insufficient for practical sample testing, it serves as a basis for future improvements. On another note, this study utilized Systematic Evolution of Ligands by Exponential Enrichment (SELEX) to screen FA nucleic aptamers. Applying these FA nucleic aptamers to the FA aptamer analysis method resulted in an IC50 exceeding 5000 ng/mL. The detection sensitivity is currently inadequate for practical sample testing, suggesting potential areas for enhancement in the future. In summary, this study successfully generated Fusaric acid (FA) polyclonal antibodies and utilized them to develop a competitive direct enzyme-linked immunosorbent assay (cdELISA) suitable for practical grain feed sample testing. Although the sensitivity of FA immunochromatographic test strips and nucleic aptamer analysis methods is currently lower, they provide valuable references for future developments in FA detection methods. Ultimately, it is hoped that the outcomes of this research contribute to monitoring FA contamination in food.