檢測花生過敏原之三明治型ELISA之建立

花生 (Arachis hypogaea) 為一營養豐富且大眾化的食品，但據估計全世界約有1%至2%的人口對花生過敏，患者若不慎誤食花生可能會引發嚴重的過敏反應，而花生中主要的過敏原為Ara h 1、Ara h 2及Ara h 3。現今常見用來檢測過敏原的方式為酵素連結免疫吸附分析法 (Enzyme-linked immunosorbent assay, ELISA)，然而目前市面上相關檢測套組數量少，且價格相當昂貴，因此本研究目標為開發可用於檢測花生過敏原之三明治型ELISA。本研究首先從花生中萃取花生蛋白，以硫酸銨鹽析初步純化，並使用膠體過濾層析提高純化蛋白質之純度，接著以紐西蘭白兔進行免疫生產多株抗體，再利用免疫親和層析法純化血清中的抗體，以山葵過氧化酶 (Horseradish peroxidase, HRP) 標記後作為偵測抗體，並以此建立檢測花生過敏原之三明治型ELISA，最後進行一系列確效分析測試。實驗結果顯示，Ara h 1、Ara h 2及Ara h 3透過SDS-PAGE得知具有良好的純度，將它們作為免疫原個別以紐西蘭白兔製備抗體，並利用免疫擴散法進行測試，可以發現抗血清具有良好的效價。接著將純化後之Ara h 3抗體以HRP標記，透過直接法ELISA測試，結果顯示其可以作為偵測抗體使用，且稀釋之比例以1:20000為最佳。最後將Ara h 3抗體作為捕獲抗體，純化之Ara h 3為標準品，HRP標記之Ara h 3抗體作為偵測抗體，建立三明治型ELISA，捕獲抗體濃度為1 μg/mL，標準品濃度範圍為0.024-25 μg/mL，R2 = 0.999。確效分析方面，intra-day及inter-day之變異係數分別為0.84-5.21%及0.87-6.12%，兩者皆低於10%。LOD及LOQ分別為0.023 μg/mL及0.033 μg/mL。而本方法之回收率介於95.5%-107.5%，表示本方法之精密度及準確度良好，且不與腰果、芝麻及黃豆產生交叉反應性。由上述結果可判定，本研究建立的三明治型ELISA可用於檢測花生中過敏原Ara h 3。未來將延伸本ELISA技術模式至Ara h 1及Ara h 2之檢測。

關鍵字

花生；過敏原； Ara h 3 ；純化；酵素連結免疫吸附分析法；抗體

並列摘要

Peanuts (Arachis hypogaea) is a nutritious and popular food, but it is estimated that about 1% to 2% of the world’s population are allergic to peanuts. If the patient accidentally eats peanut, it may cause severe allergic reactions. The major allergens of peanut are Ara h 1, Ara h 2 and Ara h 3. Nowadays, the common method used to detect allergens is Enzyme-linked immunosorbent assay (ELISA). However, the number of relevant test kits currently on the market is small and the price is quite expensive. Therefore, the aim of this study is to develop sandwich ELISA for detecting peanut allergens. Firstly, peanut protein is extracted from peanuts, then initially purified by ammonium sulfate salting out, and purified by gel filtration chromatography to improve the purity of the purified protein. Then, New Zealand white rabbits were selected for immune production of polyclonal antibodies, and the serum is purified by immunoaffinity chromatography. The antibody was labeled with horseradish peroxidase (HRP) and used as a detection antibody to develop a sandwich ELISA for the detection of peanut allergens. Finally, a series of validation analysis tests are performed. The experimental results showed that the purification of Ara h 1, Ara h 2 and Ara h 3 was found that they have good purity through SDS-PAGE. They were used as immunogens to immunize New Zealand white rabbits to prepare antibodies, and tested by immunodiffusion method, it can be found that the antiserum has good titer. Then, the purified Ara h 3 antibody was labeled to HRP and tested by direct ELISA. The experimental results showed that it can be used as a detection antibody, and the dilution ratio of 1:20000 is the best. Finally, the Ara h 3 antibody was used as the capture antibody, the purified Ara h 3 was used as the standard, and the HRP-labeled Ara h 3 antibody was used as the detection antibody to develop a sandwich ELISA. The concentration of the capture antibody was 1 μg/mL, and the concentration range of the standard was 0.024-25 μg/mL, R2 = 0.999. In terms of verification analysis, the coefficients of variation of intra-day and inter-day are 0.84-5.21% and 0.87-6.12%, respectively, both of which are lower than 10%. LOD and LOQ are 0.023 μg/mL and 0.033 μg/mL, respectively, and the recovery of this assay is between 95.5% and 107.5%, indicating that the precision and accuracy of this method is good, and it does not cross-react with cashew nuts, sesame, and soybeans. From the above results, it can be determined that the sandwich ELISA we developed in this study can be used to detect the allergen Ara h 3 in peanuts. In the future, this ELISA technology pattern will be extended to the detection of Ara h 1 and Ara h 2.

並列關鍵字

Peanut ； Allergen ； Ara h 3 ； Purification ； Enzyme-linked immunosorbent assay ； Antibody

參考文獻

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