臺灣博碩士論文加值系統

花生是一種重要的食物過敏原，僅微量的花生及會產生嚴重的免疫反應，其中以Ara h 1為花生主要過敏原。目前沒有有效的方法可以治療食物過敏，最好的方法就是避免食用含有過敏原的食物，因此建立偵測過敏原的方法是必要的。皆由磁珠從樣品中捕捉與分離目標物，可以增加檢測系統的靈敏度。首先，從花生中以硫酸銨沉澱法(70-100% )與陰離子交換管柱純化得到花生過敏原Ara h 1。另外以Ara h 1全蛋白製備抗體，以領導胜肽(leader peptide) 的Epitope-L (KSSPYQKKTENPCAQR) 及核心區域胜肽(core region)的Epitope-M (SNREVRRYTARLKEG)製備胜肽抗體。抗Ara h 1與抗Epitope-M抗體對其他堅果與種子無非專一性結合，然而抗Epitope-L抗體對杏仁、夏威夷豆、亞麻、腰果、油菜籽有非專一性結合。因此選用抗Ara h 1抗體修飾在磁珠上形成免疫磁珠，作為捕捉抗體。選擇抗Epitope-M抗體作為偵測抗體。以100 µg/mL抗Ara h 1抗體製備免疫磁珠，此條件下抗體接合率達96.3%，而偵測抗體的最佳稀釋倍數為1/10,000。結果顯示本實驗所建立的免疫磁珠檢測系統具有良好的專一性，，其線性範圍在25-200 µg/ml，檢測極限為14 µg/ml。當用來偵測6種食用油發現有三種油品含有Ara h 1，故此建立的免疫磁珠檢測系統可以應用於油中花生過敏原Ara h 1的檢測。

Peanut plays an important role in food allergy because only small amounts of peanut proteins can induce severe allergic reactions, and the Ara h 1 is a major allergen. There is no effective treatment for food allergy and the only way is to avoid specific allergens in food. Therefore, a method to detect peanut allergen is necessary. The magnetic beads are an efficient tool to enhance sensitivity by capturing and isolating target molecules from samples. Firstly, crude peanut proteins were obtained from defatted peanut and then precipitated at higher concentrations of ammonium sulfate (70-100%). Subsequently, Ara h 1 was purified in a HiTrap Q HP with a linear salt gradient (0 - 1 M NaCl). Furthermore, three antibodies against Ara h 1 were produced, by immunizing rabbits with 2 immune-dominant epitopes of Ara h 1 and pure Ara h 1 whole protein. Epitope-L (KSSPYQKKTENPCAQR) was from the leader peptide region of Ara h 1, while Epitope-M (SNREVRRYTARLKEG) was from the core region of Ara h 1. Both anti-Ara h 1 antibodies and anti-Epitope-M antibodies have no cross-reactivity with other peanut proteins or proteins from 10 nuts and seeds; while anti-Epitope-L antibodies displayed the reactivity with proteins from almond, macadamia, cashew, flax, and rapeseed. Therefore, anti-Ara h 1 antibody was conjugated to the surface of immunomagnetic beads (IMBs) to capture the Ara h 1 in samples, and anti-Epitope-M Ab was used as the detection antibody to react with protein A-HRP conjugate. Because of 96.3 % of antibody conjugation, 100 µg/mL anti-Ara h 1 Ab was selected to be coated on the surface magnetic beads. The dilution of detection antibody was 1/10,000 due to the high optical density in the presence of the allergen and low blank signals. Furthermore, the specificity of the developed assay was testified by analyzing various nuts and seeds. The specificity of immunomagnetic beads assay was highly specific for Ara h 1 with no cross-reactivity. The developed assay allowed the quantification of Ara h 1 between 25 and 200 µg/ml with a limit of detection of 14 µg/ml. For the detection of Ara h 1 in commercial oil products, three oil among six specimens contained Ara h 1. Therefore, this developed assay would be a rapid method for detecting the major peanut allergen- Ara h 1 in oil samples.

摘要 i
Abstract ii
Contents iii
Figure contents v
Table contents vi
Chapter 1: Introduction 1
1.1 Peanut allergy 1
1.1.1. Prevalence 1
1.1.2. Symptoms 2
1.1.3. Diagnosis 4
1.2. Peanut allergen 6
1.2.1. Vicilin, Ara h 1 6
1.2.2. Glycinin, Ara h 3 and Ara h 4 7
1.2.3. Conglutins, Ara h 2, Ara h 6 and Ara h 7 8
1.2.4. Profilins, Ara h 5 8
1.2.5. Pathogenesis-Related Proteins (PRs), Ara h 8 9
1.2.6. The non-specific Lipid Transfer Protein, Ara h 9, Ara h 16 and Ara h 17 9
1.2.7. Oleosins 9
1.2.8. The Peanut Defensins Ara h 12 and Ara h 13 10
1.3. Detection methods 14
1.3.1. DNA-based methods 14
1.3.2. Immunoanalytical methods 15
1.4. Immunomagnetic beads 20
Chapter 2: Materials and Methods 23
2.1. Materials 23
2.2. RNA extraction protocol 23
2.3. Expression of recombinantAra h 1 24
2.4. Purification of Ara h 1 25
2.5. Protein extraction from oil 26
2.6. Production of antibodies against Ara h 1 27
2.7. Purification of antibody 27
2.8. The sensitivity and specificity of antibodies against Ara h 1 in Western blotting 27
2.9. The sensitivity and specificity of antibodies against Ara h 1 in magnetic bead assay…… 28
2.9.1. Preparation of immunomagnetic beads 28
2.9.2. Immunomagnetic beads assay 28
2.10. SDS-PAGE and Western Blot 29
3.1. Quantity and quality of isolated RNA extraction 32
3.2. PCR and DNA sequencing analysis 32
3.3. Cloning of Ara h 1 gene and the expression of recombinantAra h 1 33
3.4. Purification of peanut allergen Ara h 1 34
3.5. Protein extraction from peanut oil 35
3.6. Purification of antibody 35
3.7. The specificity of antibodies 36
3.8. Optimized the concentration of antibody coated on the surface immunomagnetic beads 37
3.9. Optimization of detection antibody in the immunomagnetic beads assay 38
3.10. Sensitivity and specificity of immunomagnetic beads assay 38
3.11. Application to the analysis of oil samples 39
Chapter 4: Conclusion and Future Work 41
References 58

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