摘要 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 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 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 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 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 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 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 的檢測。 關鍵字: 過敏原、Ara h 1、免疫磁珠、花生 I Abstract Peanut plays an important role in food allergy because only small amounts of peanut proteins can induce severe allergic reactions, and the Ara h 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 was purified in a HiTrap Q HP with a linear salt gradient (0 - M NaCl) Furthermore, three antibodies against Ara h were produced, by immunizing rabbits with immune-dominant epitopes of Ara h and pure Ara h 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 Both anti-Ara h 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 antibody was conjugated to the surface of immunomagnetic beads (IMBs) to capture the Ara h 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 g/mL anti-Ara h 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 with no cross-reactivity The developed assay allowed the quantification of Ara h between 25 and 200 g/ml with a limit of detection of 14 g/ml For the detection of Ara h in commercial oil products, three oil among six specimens contained Ara h Therefore, this developed assay would be a rapid method for detecting the major peanut allergen- Ara h in oil samples Keywords: allergen, Ara h 1, immunomagnetic bead, peanut II Contents 摘要 Error! Bookmark not defined Abstract II Contents III Figure contents V Table contents VI Chapter 1: Introduction 1.1 Peanut allergy 1.1.1 Prevalence 1.1.2 Symptoms 1.1.3 Diagnosis 1.2 Peanut allergen 1.2.1 Vicilin, Ara h 1.2.2 Glycinin, Ara h and Ara h 1.2.3 Conglutins, Ara h 2, Ara h and Ara h 1.2.4 Profilins, Ara h 1.2.5 Pathogenesis-Related Proteins (PRs), Ara h 1.2.6 The non-specific Lipid Transfer Protein, Ara h 9, Ara h 16 and Ara h 17 1.2.7 Oleosins 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 recombinant Ara h 24 2.4 Purification of Ara h 25 2.5 Protein extraction from oil 26 2.6 Production of antibodies against Ara h 27 2.7 Purification of antibody 27 2.8 The sensitivity and specificity of antibodies against Ara h in Western blotting 27 III 2.9 The sensitivity and specificity of antibodies against Ara h in immunomagnetic 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 Chapter 3: Results and discussion 32 3.1 Quantity and quality of isolated RNA 32 3.2 PCR and DNA sequencing analysis 32 3.3 Cloning of Ara h gene and the expression of recombinant Ara h 33 3.4 Purification of peanut allergen Ara h 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 magnetic 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: Conclusions and Future Work 41 References 58 IV Figure contents Figure 2.1 Analytical procedures designed for the extraction of proteins from oil……… 30 Figure 2.2 A schematic of immunomagnetic bead assay procedure……………………… 31 Figure 3.1 Total RNA of peanut was analyzed by 1% agarose gel electrophoresis……… 43 Figure 3.2 Optimization of the annealing temperature for the amplified of Ara h gene by using various Taq polymerases ….……………………………… ……………………… 44 Figure 3.3 DNA sequencing of amplified Ara h gene was compared with other stains of peanut………………………………………………………………….…………… …… 46 Figure 3.4 Cloning of Ara h gene and the expression of recombinant Ara h1………………………………………….……………………………………………… 47 Figure 3.5 Purification of peanut allergen Ara h ……………………………………… 48 Figure 3.6 Protein extraction from oil ………….………….…………………………… 49 Figure 3.7 Purification of antibody by protein A column………………… …………… 50 Figure 3.8 Specificity of anti-Epitope-L and anti-Epitope M antibody against various peanut species.……………………………………………………………………… …….51 Figure 3.9 Specificity of anti-Ara h and anti- Epitope antibodies against proteins from nuts and seeds………………………………………………….………………………… 52 Figure 3.10 Optimizing the concentration of conjugated anti-Ara h antibody on the magnetic beads………… …………………………………………………………………53 Figure 3.11 Optimizing the dilution factor of detection antibody in the immunomagnetic bead assay…………………………………………………………………….…………….54 Figure 3.12 The sensitivity of the immunomagnetic bead assay for the detection of Ara h1 …………………………………………………………………………….…………… 55 Figure 3.13 The specificity of immunomagnetic bead assay against various nuts and seeds……………………………………………………………………………… ……… 56 Figure 3.14 Detection of Ara h from various oil specimens by immunomagnetic bead assay……… ………………………………………………………………………………57 V Table contents Table 1.1 Manifestation of peanut allergy on different organ compartments and its symptoms with increasing severity………………………………………………………… Table 1.2 Frequency of target system involvement in first presentation of peanut allergy Table 1.3 Performance characteristics of diagnostic tests for peanut allergy……… … … Table 1.4 Protein family, allergens, isoallergens and variants, molecular weight (MW), isoelectric point, prevalence, and biological function…………………………………… 11 Table 1.5 Characteristics and major results of selected methods for peanut allergen quantification by real time PCR…………………………………………………………… 15 Table 1.6 The major results of immunochemical methods to detect peanut allergen….… 17 Table 1.7 Determination of the Ara h concentration (in mg g−1) in different food extracts by a commercial ELISA kit……………………………………………………………… 20 Table 1.8 The major results of magnetic beads for detecting allergens and pathogens… 22 Table 3.1 Alignment of Epitope L & M with other nuts and seeds proteins.………………………………………………………………………………….…42 VI Chapter 1: Introduction 1.1 Peanut allergy Peanut is acknowledged not only as one of the major allergenic foods, but also as the source of one of the most severe food allergies because of the prevalence, persistence, and the potential severity of the allergic reaction While other food allergies occurring in childhood usually resolve themselves spontaneously with age, peanut sensitivity appears early in life and often persists indefinitely [1] 1.1.1 Prevalence With increasing news coverage on peanut allergy in the past few years, the percentage of peanut allergy increased from 0.6% in 1997 to 1.2% in 2002 and 2.1% in 2007 for children less than 18 years old in the U.S 1% of individuals were reported to have peanut allergies in a similar survey in Canada [2] The prevalence of allergies increased about 6-8% of children under years old, more than that of children over 10 years old in the USA with 4% [3] The proportion of peanut allergy is estimated to be lower for the 20 years old, there is a 4-fold increase in hospitalization rates for peanut-related anaphylaxis in New York State from 1990 to 2006 [4] In France, 1% of the population has peanut allergies out of 3.24% of food allergy population [5] Peanut allergies were present in 1.34% of a group of children surveyed at the primary school in Canada According to Skolnick’s study, it was demonstrated that people on Isle of Wight having peanut allergy had increased by 1.5% [6] In a study of 512 infants with a known milk or egg allergy or moderate-to-severe atopic dermatitis and a positive skin test to milk or egg, 69% were sensitized to peanut (a particularly high rate given that infants with known peanut sensitization were excluded [7] In 50,000 cases of sensitive food allergy, there are approximately 100 deaths per year, and especially, for allergy to peanut, which affects around % of children and 0.6% of adults in the U.S [8] The Food Allergy Research and Education (FARE) reported that it is about 25-40% of individuals who have either tree nut and/or peanut allergy In summary, peanut allergy usually starts early in life Its prevalence is different among countries, and the highest rates was in the US, Canada and the UK (1-3%) and the lowest was in France, Denmark and Israel (0.2-0.7%) In general, the world’s population is effected at an average of 1-2% [9] However, the prevalence of peanut allergy is a growing problem worldwide, especially in developed countries [10] 1.1.2 Symptoms In the last few years, there has been an escalation in the identification and sequencing of food allergens including peanut allergens The popular symptoms of peanut allergic patients consist of mild urticaria, facial swelling, and abdominal cramp to hypertension with anaphylactic shock [11] In some cases, severe anaphylaxis could be lead to death, which is considered to be the most serious food-induced allergic reaction In sensitized individuals, the level of peanut allergy may induce a broad spectrum from mild to fatal anaphylactic symptoms presented in the Table 1.1 Table 1.1 Manifestation of peanut allergy on different organ compartments and its symptoms with increasing severity [12] Part of body Skin Severity of symptoms Mild Moderate Severe hives, pruritus atopic dermatitis, angioedema urticaria Respiratory tract rhino-conjunctivitis, wheezing shortness of breath, asthma acute severe asthma Gastrointestinal tract diarrhea, nausea and vomiting, OAS abdominal pain laryngeal edema Systemic reaction anaphylactic shock It has to be considered that all symptoms may occur singularly as well as in combination Allergic symptoms following the ingestion of peanuts occur in display around the oral pruritus, nausea, vomiting, urticaria and angioedema to bronchospasm from within minutes to a few hours [13] The proportion of patients manifesting oral symptoms to peanut was the lowest in the USA (10%), as compared to that in Sweden (14.3%) and Spain (19.3%) In serious cases, the number of American patients frequently suffering from the cause of anaphylaxis by peanut is higher (21.4%) than the number of Spanish patients (41.7%), and so is the number of visits to the emergency department after peanut ingestion (64.3% of American patients and 48.6% Spanish patients) [4] Peanuts account for the majority of food-related anaphylaxis in children, adolescents and adults [14] A characteristic of peanut allergy is its tendency to persist through to adulthood, with only 21.5% of peanut-allergic individuals experiencing resolution of this type of food allergy with increasing age [15] Thus, lifelong vigilance is essential for the majority of bearers of peanut allergy In the other study, analysis includes infants (mean age, 7.8 months) There was 17% peanut-specific IgE sensitization (≥0.35 kU/L) in groups of patients with severe eczema, egg allergy, or both but 0-mm peanut skin prick test (SPT) wheal responses (n = 542), and was 56% of group patients with severe eczema, egg allergy, or both and 1- to 4-mm peanut wheal responses (n = 98) Moreover, 91% of those in group of patients with greater than 4-mm peanut wheal responses (n = 76) had peanut-specific IgE (PN-IgE) sensitization [16] The systems affected and symptoms of peanut allergies are summarized in Table 1.2 [17] More than 70% of children are allergic to peanut allergens [11] Indications of peanut allergy start to happen during their first known exposure Since the allergic reactions are mediated through the IgE antibody, an initial exposure to an allergen induces immunologic sensitization It is obvious that occult exposure occurs Possible routes of occult sensitization include fetal exposure to allergens from which mothers take from peanuts during pregnancy and/or infants ingest from breast milk It strongly recommend that mothers at high-risk of developing allergies should avoid potential allergic food sources They should not consume products from peanuts An infant is considered at risk for atopy if both parents, or one parent and a sibling, have atopic features Table 1.2 Frequency of target system involvement in first presentation of peanut allergy Target system % of patients 89 Cutaneous Urticaria, erythema, angioedema 42 Respiratory Wheezing, stridor, cough, dyspnea, throat-tightness, nasal congestion 26 Gastrointestinal Vomiting, diarrhea, abdominal pain Cardiovascular Hypotension, arrhythmia, cardiac arrest 1.1.3 Diagnosis There are different approaches used to diagnose food allergy Skin prick tests is a rapid method to screen patients and determine which types of food that the patient is reacting to Another test is the double-blind placebo-controlled food challenge (DBPCFC), which is an oral food challenge It is also possible to measure an individual’s food-specific IgE level and from that predict the probability of reaction to a specific food The National Institute of Allergy and Infectious Diseases recommended that diagnosis should combine a detailed clinical history and physical examination, skin-prick testing (SPTs), allergen-specific serum IgE, elimination diets, and oral food challenges (OFCs) The characteristics of diagnostic tests for peanut allergy are listed in Table 1.3 [17] These are good diagnostic tools to detect peanut hypersensitivity Nevertheless, it does not give 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Ara h 16 and Ara h 17 ), oleosins (Ara h 10 , Ara h 11 , Ara h 14 and Ara h 15 ), and defensin (Ara h 12 and Ara h 13 ) [11 ] 1. 2 .1 Vicilin, Ara h Ara h is the first protein identified as a peanut allergen. .. and Immunology, 13 1 ,14 -18 45 Koppelman SJ, Hefle SL, Taylor SL & de Jong GA (2 010 ) Digestion of peanut allergens Ara h 1, Ara h 2, Ara h 3, and Ara h 6: acomparative in vitro study and partial... Ara h 9, Ara h 16 and Ara h 17 1. 2.7 Oleosins 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