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標題: 建立三種快速檢測食物中小麥過敏原的方法藉由高靈敏度的石英晶體微天平、高專一性的微脂球-免疫磁珠及快速的條碼式免疫層析試紙
Development of three rapid assays for the detection of wheat allergens in foods by the use of highly sensitive quartz crystal microbalance assay, highly specific liposomal nanovesicles-immunomagnetic bead assay, and rapid barcode style dipstick strip
作者: 朱霈慈
Pei-Tzu Chu
關鍵字: Gliadin
Quartz crystal microbalance
Immunomagnetic bead
Barcode style dipstick strip
引用: 8098043004-b
摘要: Gluten causes both wheat allergy and coeliac disease. It is the dough-forming proteins and is formed by gliadins and glutenins. An in vitro study found out that all gliadins can trigger allergic reaction. Therefore, the objective of this project is to develop three rapid immunoassays for detecting wheat allergens in the foods. We immunized the 40-week-old white leghorn chicken with the gliadin from wheat and purified the polyclonal antibody (IgY) from egg yolk. To evaluate the specificity of IgY antibody, eight cereals were tested and prolamins of oats, rice, corn, foxtail millet, buckwheat and soybean did not have cross-reaction in the assay. First, a quartz crystal microbalance (QCM) immunosensor that the IgY was immobilized on Au surface has been developed for analysis of gliadin. Successfully, the sensitivity of this QCM detection system could reach 20 ppb of gliadin and linear range was 2×101 to 2×104 ppb gliadin. In real food samples, the detection platform could reach 1 ppm of gliadin and could be done within 40 minutes. Then, we had developed a high throughput IMB/IMLN fluorescent assay. This detection platform exhibited good sensitivity and specificity for gliadin with a detection limit as low as 0.6 ppm of gliadin. The consistency of this detection method with AOAC ELISA kit was assessed by Bland-Altman plot, and the correlation was 95%. Finally, the barcode style dipstick strip based on AuNPs had been developed. This assay could be finish within 40 minutes, and had advantages such as convenience, disposability and visual results. Furthermore, the detection limit of these three methods was below to the international standards of gluten-free food, and these methods could be widely used to monitor the food safety in the future.
Gluten是由醇溶蛋白gliadin和 glutenins所組成,是造成小麥過敏和腹腔疾病的主要過敏原。在體外研究發現,所有醇溶蛋白都具有引起過敏反應的特性。因此,本研究的目的是建立三種快速偵測食物中小麥過敏原的免疫檢測平台。以小麥的醇溶蛋白注射40週齡的白色來亨雞,並從蛋黃中純化出多株抗體IgY,再以八種穀物進行專一性測試,發現本研究所製備的抗醇溶蛋白抗體對燕麥、稻米、玉米、小米、蕎麥和大豆所萃取出的醇溶蛋白並無交叉反應產生,故此抗體具有良好之專一性。首先,將抗醇溶蛋白抗體固定在石英晶體微天平的金電極表面來建立一個檢測平台。在偵測 60%酒精中小麥之醇溶蛋白時,其最低偵測極限為20 ppb,其線性範圍在 2 × 101到2 × 104 ppb之間;而在食物樣本的檢測中,最低偵測極限則為1 ppm,整個檢測過程可於40分鐘內完成。接下來,我們完成免疫微脂體結合免疫磁珠及微?滴定盤的螢光分析方法。此醇溶蛋白檢測平台具有良好的靈敏度和特異性,偵測極限為0.6 ppm 的醇溶蛋白。以Bland-Altman plot 方式評估此檢測平台與AOAC ELISA kit的結果,可獲得95%的一致性。最後,?用奈米金作為檢測試劑發展出條碼式的免疫層析試紙,此檢驗可在40分鐘內完成並具有簡?、可拋棄式和?用肉眼即可辨?結果的優點。另外,此三套檢驗方法的最低偵測極限皆低於國際無麩質食品檢測標準,因此未來可廣泛應用於食品安全的監控上。
文章公開時間: 2017-08-31
Appears in Collections:食品暨應用生物科技學系



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