臺灣博碩士論文加值系統

芝麻(Sesamum indicum L.)是一種具有豐富的營養價值與發展潛力的種子作物。也是日常生活中常見的食品之一。但同時也會有芝麻過敏患者誤食造成過敏反應。常見的症狀如皮膚發炎、氣喘、呼吸困難與腹痛。因此需要一套檢測方法來檢測食品中芝麻過敏原含量。本研究的目的為建立一種三明治型酵素連結免疫吸附法 (sELISA) 以偵測食品中芝麻過敏原Ses i 6。Ses i 6為芝麻過敏蛋白中主要成分之一，其蛋白含量佔總蛋白量約 60-70%，因此選擇Ses i 6作為檢測方法的目標蛋白。sELISA經優化後條件為1 µg/mL的鹼性抗體作為捕獲抗體 (90 分鐘)、3 %甘胺酸做為阻隔蛋白、2.5 µg/mL的芝麻萃取蛋白作為抗原 (120 分鐘)、1 µg/mL的含生物素酸性抗體作為偵測抗體 (90 分鐘)、SA-HRP濃度150 ng/mL (30 分鐘)。以上述條件製作劑量反應曲線，標準曲線的公式為y = 2.0752ln(x) + 14.119 (R² = 0.9866)。以P/N = 2.1求得的上述sELISA系統之檢測極限為0.00305 μg/mL 的芝麻萃取蛋白。接著在餅乾基質中進行測試，y = 13.412x + 0.7831 (R² = 0.9967)。以P/N = 2.1求得的檢測極限分別為0.09 mg/g的芝麻萃取蛋白。本研究建立之三明治型ELISA可應用於市售食品中芝麻萃取蛋白的檢測及定量。

Sesame seed (Sesamum indicum L.) is a seed crop which rich in nutrition value and has potential. Also is the one of the common food. But it also causes allergic reactions in the same time. Including skin itching, wheezing , breathing trouble and abdominal pain. Therefore, we need an analytical method to measure the content of the major sesame allergen. The aim of the study was to develop an sandwich ELISA for the detection and quantification of sesame allergen Ses i 6 in processed foods. Ses i 6 is the one of the sesame seed major proteins, and represent 60–70% of the total seed proteins In this study, we have established a sandwich ELISA based on polyclonal antibody (pAb) to measure the content of the major sesame allergen sesame protein extracts in foods. The optimal concentrations of the capture antibody and detection antibody were 1 µg/mL of the base antibody (90 min) and 1 µg/mL of the biotin acid antibody (90 min) , respectively, and blocking with 3% glycine, samples 2.5 µg/mL of the sesame protein extracts (90 min) , and SA-HRP was used at a concentration of 150 ng/mL, which was incubated for 30 min. The limit of detection is 0.00305 μg/mL of sesame protein extracts in sELISA system, and The standard curve was y = 2.0752ln(x) + 14.119 (R² = 0.9866). This assay was used to detect sesame protein extracts in cookie products, and the detection limit were 0.09 mg/g, and the standard curve was y = 13.412x + 0.7831 (R² = 0.9967). This assay could detect sesame protein extracts in many kinds of food samples.

摘要 i
Abstract ii
目錄 iii
表目次 vi
圖目次 vii
第一章 文獻回顧 1
1.1 食品過敏原 1
1.1.1 常見的食品不良反應類型 1
1.1.2 常見食品過敏原 1
1.1.3 食品過敏蛋白質介紹 2
1.1.4 不同地區的食品過敏比例 2
1.1.5 食品過敏原間的交叉反應 2
1.1.6 台灣地區食品過敏種類 3
1.2 芝麻 5
1.2.1 芝麻成分 5
1.3 芝麻過敏原 6
1.3.1 芝麻過敏檢測方法 6
1.3.2 種子儲存蛋白類型 6
1.3.3 芝麻過敏原類型介紹 6
1.3.4 Ses i 1 7
1.3.5 Ses i 2 7
1.3.6 Ses i 3 7
1.3.7 Ses i 4 8
1.3.8 Ses i 5 8
1.3.9 Ses i 6 8
1.3.10 Ses i 7 8
1.3.11 不同處理方式對芝麻過敏原的影響 9
1.4 酵素連結免疫吸附法 12
1.4.1 ELISA類型 13
1.4.2 三明治型ELISA 13
第二章 材料與方法 18
2.1 實驗流程 18
2.2 材料與設備 19
2.3 芝麻蛋白Ses i 6萃取 19
2.4 抗體製備 19
2.4.1 捕獲抗體製備 19
2.4.2 偵測抗體製備 20
2.5 間接型ELISA(indirect ELISA) 20
2.6 三明治型酵素連結免疫吸附法(sELISA) 20
2.7 三明治型酵素連結免疫吸附法(sELISA)實驗條件優化 21
2.8 靈敏度與專一性 21
2.9 食品萃取方法 21
2.9.1 萃取方法測試 21
2.9.2 萃取方法實驗條件優化 22
2.9.3 萃取前攙入(Pre-spike) 22
2.10 市售食品樣品測試 22
第三章 結果與討論 25
3.1 芝麻蛋白萃取分離 25
3.2 芝麻抗體純化 25
3.3 以間接型ELISA(iELISA)確認抗體狀態 25
3.4 三明治型酵素連結免疫吸附法(sELISA)實驗條件優化 25
3.4.1 阻隔蛋白選擇及濃度優化 26
3.4.2 捕獲抗體與偵測抗體濃度配對 26
3.4.3 清洗緩衝液的選擇 26
3.4.4 捕獲抗體反應緩衝液的選擇 27
3.4.5 抗原加熱反應時間 27
3.4.6 抗原稀釋用緩衝液條件優化 27
3.4.7 SA HRP濃度與反應時間選擇 29
3.4.8 TMB反應時間選擇 29
3.5 劑量反應曲線 30
3.6 專一性測試 30
3.7 萃取方法實驗條件優化 30
3.7.1 萃取方法測試 30
3.7.2 萃取液 pH值選擇 31
3.7.3 萃取溫度選擇 31
3.7.4 萃取時間選擇 31
3.7.5 萃取前攙入(Pre-spike) 31
3.8 食品樣品測試 32
第四章 結論與未來發展 33
參考書目 62
附錄 74

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