Detection by real time PCR of walnut allergen coding sequences in processed foods.
A quantitative real-time PCR (RT-PCR) method, employing novel primer sets designed on Jug r 1, Jug r 3, and Jug r 4 allergen-coding sequences, was set up and validated. Its specificity, sensitivity, and applicability were evaluated. The DNA extraction method based on CTAB-phenol-chloroform was best for walnut. RT-PCR allowed a specific and accurate amplification of allergen sequence, and the limit of detection was 2.5pg of walnut DNA. The method sensitivity and robustness were confirmed with spiked samples, and Jug r 3 primers detected up to 100mg/kg of raw walnut (LOD 0.01%, LOQ 0.05%). Thermal treatment combined with pressure (autoclaving) reduced yield and amplification (integrity and quality) of walnut DNA. High hydrostatic pressure (HHP) did not produce any effect on the walnut DNA amplification. This RT-PCR method showed greater sensitivity and reliability in the detection of walnut traces in commercial foodstuffs compared with ELISA assays.
Prospective investigation on the transfer of Ara h 2, the most potent peanut allergen, in human breast milk.
BACKGROUND: Peanut allergy is one of the most severe food allergies. Whether breast feeding induces tolerance to peanuts or on the contrary, predisposes at risk-babies to occult allergic sensitization to peanuts is still a matter of discussion. We sought to investigate the transfer of the most potent peanut allergen Ara h 2 into human breast milk in a German breast milk study and to shed light on the time kinetics of Ara h 2 appearance. METHODS: We recruited 32 lactating, non-peanut allergic women, and collected breast milk samples at different time points after consumption of 100 g dry roasted peanuts. Breast milk samples were investigated for Ara h 2 with different immunological methods: by 2D immunoblotting with a patient's serum, by affinity enrichment using a monoclonal antibody against Ara h 2 followed by LC-MS/MS based detection and by a competitive inhibition ELISA for the detection of Ara h 2 and its digestion resistant peptides (DRP-Ara h 2). RESULTS: In a qualitative analysis Ara h 2 could be identified in a breast milk sample by 2D immunoblot by means of a patient's serum and furthermore by immunoaffinity enrichment followed by LC-MS/MS analysis. In a semi-quantitative analysis Ara h 2 and its digestion-resistant peptides were detected in the breast milk of 9 out of 32 subjects. Evidence suggests that Ara h 2 is excreted individually either rapidly (after 1 h, 2 h, 3 h or 4 h) or delayed (after 8 h or 12 h) and in different concentrations. CONCLUSIONS: Time and concentration of secreted Ara h 2 in breast milk appears to be individually regulated. The identification of Ara h 2 in breast milk is the prerequisite for the investigation of its sensitizing or tolerogenic properties. This article is protected by copyright. All rights reserved.
Is Microarray Analysis Really Useful and Sufficient to Diagnose Nut Allergy in the Mediterranean Area?
BACKGROUND: Component-based diagnosis on multiplex platforms is widely used in food allergy but its clinical performance has not been evaluated in nut allergy. OBJECTIVE: To assess the diagnostic performance of a commercial protein microarray in the determination of specific IgE (sIgE) in peanut, hazelnut, and walnut allergy. METHODS: sIgE was measured in 36 peanut-allergic, 36 hazelnut-allergic, and 44 walnut-allergic patients by ISAC 112, and subsequently, sIgE against available components was determined by ImmunoCAP in patients with negative ISAC results. ImmunoCAP was also used to measure sIgE to Ara h 9, Cora 8, and Jug r 3 in a subgroup of lipid transfer protein (LTP)-sensitized nut-allergic patients (positive skin prick test to LTP-enriched extract). sIgE levels by ImmunoCAP were compared with ISAC ranges. RESULTS: Most peanut-, hazelnut-, and walnut-allergic patients were sensitized to the corresponding nut LTP (Ara h 9, 66.7%; Cor a 8, 80.5%; Jug r 3, 84% respectively). However, ISAC did not detect sIgE in 33.3% of peanut-allergic patients, 13.9% of hazelnut-allergic patients, or 13.6% of walnut-allergic patients. sIgE determination by ImmunoCAP detected sensitization to Ara h 9, Cor a 8, and Jug r 3 in, respectively, 61.5% of peanut-allergic patients, 60% of hazelnut-allergic patients, and 88.3% of walnut-allergic patients with negative ISAC results. In the subgroup of peach LTP-sensitized patients, Ara h 9 sIgE was detected in more cases by ImmunoCAP than by ISAC (94.4% vs 72.2%, P < .05). Similar rates of Cora 8 and Jug r 3 sensitization were detected by both techniques. CONCLUSIONS: The diagnostic performance of ISAC was adequate for hazelnut and walnut allergy but not for peanut allergy. sIgE sensitivity against Ara h 9 in ISAC needs to be improved.
Prevalence of Peanut, Tree Nut, Sesame, and Seafood Allergy in Mexican Adults.
OBJECTIVE: To identify the prevalence of perceived and probable allergic reactions to peanuts, tree nuts, sesame seed, or seafood and its association with the personal history of allergic disease. METHODS: A cross-sectional study was performed in four cities of the metropolitan area of Guadalajara, located in western Mexico. Through sampling by gender and age, 1,126 subjects were included. Using a structured questionnaire, we investigated: (i) history of atopic disease, (ii) perception of allergic reaction after food intake, and (iii) probable allergic reaction to peanuts, tree nuts, sesame, or seafood. Prevalence and 95% confidence intervals were calculated. A multivariate analysis of factors associated to perceived and probable allergic reactions to food was performed by logistic regression. RESULTS: Men were 49.8%; mean age was 28.1 years; personal history of atopic disease included: allergic rhinitis (6.9%), asthma (6.8%), and atopic dermatitis (3.8%). Prevalence of perceived and probable food allergy was, respectively: pecan 0.4 and 0.3%; peanut 0.6 and 0.6%; sesame 0.1 and 0.1%; shellfish 4.2 and 4.0% (mainly shrimp); mollusk 0.8 and 0.7%; fish 1.4 and 1.2%. Asthma was significantly associated with perceived and probable allergy to pecans, peanuts, fish, or seafood. Atopic dermatitis was associated with perceived and probable allergic reaction to sea snail, seafood, shellfish, or mollusks. Finally, allergic rhinitis was associated with allergy to shrimp and crustaceans. CONCLUSIONS: This study shows that the prevalence of peanut, tree nut, sesame seed, and seafood allergy in this Mexican population is similar to that reported in developed countries.
Purification and Characterization of Anacardium occidentale (Cashew) Allergens Ana o 1, Ana o 2, and Ana o 3.
In this study a fast and simple purification procedure for the three known allergens from cashew (7S globulin Ana o 1, 11S globulin Ana o 2, and 2S albumin Ana o 3) is described. The purified allergens are characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blot, glycoprotein stain, and protein identification. The purified proteins still bind IgE, and this IgE binding varied between different pools of patient serum. Ana o 1 was found to be a glycoprotein. Ana o 3 has been studied more in detail to identify both the small and large subunits, both displaying microheterogeneity, and epitope mapping of Ana o 3 has been performed.
Allergic Reactions to Pine Nut: A Review
Pine nut is a nutrient-rich food with a beneficial impact on human health. The many bioactive constituents of pine nut interact synergistically to affect human physiology in a favorable way. However, pine nut can trigger dangerous allergic reactions. Severe anaphylactic reactions to pine nut accounted for most of the 45 cases reported in the scientific literature. Pine nut allergy seems to be characterized by low IgE cross-reactivity with other commonly consumed nuts and a high monosensitization rate. The present review provides updated information on allergic reactions to pine nut, molecular characterization of its allergens, and potential homologies with other nut allergens.
Development and inter-laboratory transfer of a decaplex polymerase chain reaction assay combined with capillary electrophoresis for the simultaneous detection of ten food allergens.
Food allergies cause health risks to susceptible consumers and regulations on labeling of food allergen contents have been implemented in many countries and regions. To achieve timely and accurate food allergen labeling, the development of fast and effective allergen detection methods is very important. Herein, a decaplex polymerase chain reaction (PCR) assay combined with capillary electrophoresis was developed to detect simultaneously 10 common food allergens from hazelnut, pistachio, oat, sesame, peanut, cashew, barley, wheat, soybean and pecan. The absolute limit of detection (LODa) of this system is between 2 and 20 copies of haploid genome, and the relative LOD (LODr) is as low as 0.005% (w/w) in simulated food mixtures. The developed assay was subsequently applied to 20 commercial food products and verified the allergen ingredients stated on the labels. Furthermore, results using this decaplex PCR assay was successfully replicated in three other laboratories, demonstrating the repeatability and applicability of this assay in routine analysis of the 10 food allergens.
Allergenicity Attributes of Different Peanut Market Types.
Four different market classes of peanut (Runner, Virginia Spanish, and Valencia) are commonly consumed in Western countries, but for some consumers peanuts are a main cause of food-induced anaphylaxis. Limited information is available on the comparative allergenicity of these distinct market classes. The aim of this study was to compare allergenicity attributes of different peanut cultivars. The protein content and protein profiles were highly comparable for all tested cultivars. All cultivar samples contained the major allergens Ara h 1, Ara h 2, Ara h 3 and Ara h 6, as assessed by SDS-PAGE and RP-HPLC, although some minor differences in major allergen content were found between samples. All samples were reactive in commercial ELISAs for detection and quantification of peanut protein.. IgE-binding potency differed between samples with a maximum factor of 2, indicating a highly comparable allergenicity. Based on our observations, we conclude that peanuts from the main market types consumed in Western countries are highly comparable in their allergenicity attributes, indicating that safety considerations with regard to peanut allergy are not dependent on the peanut cultivar in question.
COR a 14- Specific IgE Predicts symptomatic hazelnut allergy in children.
The self-reported prevalence of tree nut allergy in children has been increasing in the past decade (1). Hazelnuts are in particularly widespread use and this makes them difficult to avoid completely. It is therefore important to correctly diagnose hazelnut allergy to avoid any unjustified diet. The aim of the present study was to assess the utility of currently-available hazelnut molecular components (Cor a 1-, Cor a 8-, Cor a 9-, and Cor a 14-specific IgE) in identifying cases of symptomatic hazelnut allergy among Italian children sensitized to hazelnut and referred to a tertiary medical center. This article is protected by copyright. All rights reserved.
A review of oral food challenges in children presenting to a single tertiary centre with perceived or true food allergies.
INTRODUCTION: The prevalence of perceived food allergies exceeds that of true food allergies. Unnecessary food avoidance may increase parental and patient anxiety, reduce quality of life and increase the risk of nutritional deficiency. An oral food challenge (OFC) can provide an objective measure regarding the presence or absence of food allergies in a child. This study reviews the indications for and outcomes of OFCs performed on children. METHODS: A retrospective review was performed on all children who underwent OFCs at the Allergy Unit of the National University Hospital, Singapore, over a three-year period. RESULTS: A total of 197 OFCs were performed among 58 patients (34 male, 24 female). Most of the tests were for allergies to tree nuts (n = 107). Among the OFCs, 43.1% were for foods that were avoided and never eaten due to perceived food allergies, 25.9% were for foods that had previously resulted in positive skin prick tests (SPTs) and/or immunoassay results, 16.2% were for foods thought to worsen eczema and 14.7% were for foods thought to have caused a previous reaction. Of all the OFCs, 5% were positive, although adverse reactions were mostly cutaneous. Challenge-positive patients had either positive SPTs (wheal > 3 mm) or raised serum immunoglobulin E levels to specific foods that they reacted to during the challenges. No episodes of anaphylaxis were reported after the challenge. Most of the patients were able to safely introduce the avoided foods into their diets. CONCLUSION: OFCs provide an objective assessment for suspected food allergies.