Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/98003
標題: 建立奈米磁珠側層流免疫分析法以檢測食品中花生主要過敏原Ara h 1
Develop a magnetic nanoparticle labeled lateral flow assay for the detection of a major peanut allergen Ara h 1 in food.
作者: 李亦婷
Yi-Ting Li
關鍵字: 花生過敏
Ara h 1
免疫磁珠
側層流免疫分析法
peanut allergy
Ara h 1
magnetic nanoparticle
lateral flow assay
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摘要: 花生過敏可能導致高危險的急性症狀發生,僅微量的花生過敏原就能引起嚴重的過敏反應,且可能藉由加工過程交互汙染,因此需要建立一個快速偵測花生過敏原的系統去預防以及產品品質管控。側層流免疫分析法做為一個便利且肉眼可視的檢測系統已廣泛應用在各種領域上,結合免疫磁珠分離法可以有效增加靈敏度。Ara h 1為花生的主要過敏原,且在熱處理的過程中可能增加致敏性,因此以Ara h 1做為系統的偵測對象。選擇Anti-Ara h 1全蛋白抗體及一株核心區域胜肽抗體配對後,針對試紙與磁珠進行條件最適化。試紙與磁珠的抗體皆為Anti-Ara h 1全蛋白抗體,試紙AE99以glycine做為blocker、檢量線抗體密度為2.5 μg/cm;磁珠150 nm以750 μg/ml抗體修飾,並以2.5%脫脂牛奶blocking;磁珠樣品比例為20:300。以此模式在溶液中的肉眼可視極限為0.01 μg/ml,標準方程式y = 30.967ln(x) + 166.6,R² = 0.9914。針對進行巧克力、餅乾及牛奶三種基質進行測試,餅乾與牛奶檢測極限為0.5 μg/ml而巧克力為1 μg/ml,並在食品樣品中可正確檢測到含有花生的產品。
Peanut can induce severe allergic reaction by very small amount. In the food processing, peanut allergen residues may contaminate other products which do not contain peanut, it will be an important problem to peanut-allergic consumers. In order to prevent peanut allergen from contamination and improve the quality control, an analytical method for rapid detecting peanut allergen is necessary. Lateral flow assay (LFA) is known for its simple procedure to detect target rapidly. Magnetic nanoparticles can against matrix effect and improve detection result. As a peanut major allergen, Ara h 1, may increase allergenicity after heat processing. Therefore, in this study, a LFA model based on magnetic nanoparticle for the rapid detection of Ara h 1 was developed. Firstly, anti-Ara h 1 polyclonal antibody was selected to be tagged on magnetic nanoparticles and immobilized on the test line of strip. Following was the optimal conditions: Nitrocellulose membrane type was AE99; antibody density on the test line is 2.5 μg/cm; blocker on membrane was glycine; magnetic nanoparticle size was 150 nm modified antibody concentration is 750 μg/ml; magnetic beads blocker was non-fat dried milk; ratio of magnetic beads and sample is 20:300 (v:v). The visual limit of detection is 0.01 μg/ml Ara h 1 in PBS, and the formula was y = 30.967ln(x) + 166.6 with R square of 0.9914. This assay was used to detect peanut allergen-Ara h 1 in cookie, milk and chocolate product, and the detection limit was 0.5 μg/ml (cookie, milk) and 1μg/ml (chocolate). Moreover, Ara h 1 in various food smples were detected by the developed assay.
URI: http://hdl.handle.net/11455/98003
文章公開時間: 10000-01-01
Appears in Collections:食品暨應用生物科技學系

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