Primary and pollen-associated hazelnut allergy in school-aged children in Germany: A birth cohort study.

BACKGROUND: Primary hazelnut allergy is a common cause of anaphylaxis in children, as compared to birch-pollen associated hazelnut allergy. Population-based data on hazelnut and concomitant birch-pollen allergy in children are lacking. We aimed to investigate the prevalence of primary and pollen-associated hazelnut allergy and sensitization profiles in school-aged children in Berlin, Germany. METHODS: 1570 newborn children were recruited in Berlin in 2005-2009. The school-age follow-up (2014-2017) was based on a standardized web-based parental questionnaire and clinical evaluation by a physician including skin prick tests, allergen specific immunoglobulin E serum tests and placebo-controlled double-blind oral food challenges, if indicated. RESULTS: 1004 children (63.9% response) participated in the school-age follow-up assessment (52.1% male). For 1.9% (n = 19, 95%-confidence interval 1.1%-2.9%) of children their parents reported hazelnut-allergic symptoms, for half of these to roasted hazelnut indicating primary hazelnut allergy. Symptoms of birch-pollen allergy were reported for 11.6% (n = 116 95%-CI 9.7%-13.7%) of the children. Both birch-pollen allergy and hazelnut allergy associated symptoms affected 0.6% (n = 6, 95%-CI 0.2%-1.3%) of children. Assessment of allergic sensitization was performed in 261 participants and showed that almost 20% of these children were sensitized to hazelnut, being the most frequent of all assessed food allergens, or birch-pollen, the majority to both. CONCLUSIONS: Based on parental reports hazelnut-allergic symptoms were far less common than sensitization to hazelnut. This needs to be considered by physicians to avoid unnecessary changes in diet due to sensitization profiles only, especially when there is a co-sensitization to hazelnut and birch-pollen.

Electrochemical and optical biosensing platforms for the immunorecognition of hazelnut Cor a 14 allergen.

Two immunosensors were advanced to target hazelnut Cor a 14 based on electrochemical and optical transduction. Both approaches were developed with two types of custom-made antibodies, namely anti-Cor a 14 IgG (rabbit) and anti-Cor a 14 IgY (hen's egg) targeting the Cor a 14 allergen. Antibody immobilisation was performed via EDC/NHS onto disposable screen-printed electrodes. The detection limit (LOD) of the electrochemical immunoassay for Cor a 14 was 5-times lower than the optical, being down to 0.05 fg mL-1 with a dynamic range of 0.1 fg mL-1 to 0.01 ng mL-1. Antibody selectivity was verified against non-target 2S albumins (potential cross-reactive plant species). Anti-Cor a 14 IgY exhibited the best specificity, presenting minor cross-reactivity with peanut/walnut. Preliminary results of the application of anti-Cor a 14 IgY electrochemical immunosensor to incurred foods established a LOD of 1 mg kg-1 of hazelnut in wheat (0.16 mg kg-1 hazelnut protein).

Component-Resolved Diagnosis of Hazelnut Allergy in Children.

Hazelnuts commonly elicit allergic reactions starting from childhood and adolescence, with a rare resolution over time. The definite diagnosis of a hazelnut allergy relies on an oral food challenge. The role of component resolved diagnostics in reducing the need for oral food challenges in the diagnosis of hazelnut allergies is still debated. Therefore, three electronic databases were systematically searched for studies on the diagnostic accuracy of specific-IgE (sIgE) on hazelnut proteins for identifying children with a hazelnut allergy. Studies regarding IgE testing on at least one hazelnut allergen component in children whose final diagnosis was determined by oral food challenges or a suggestive history of serious symptoms due to a hazelnut allergy were included. Study quality was assessed by the Quality Assessment of Diagnostic Accuracy Studies-2 tool. Eight studies enrolling 757 children, were identified. Overall, sensitivity, specificity, area under the curve and diagnostic odd ratio of Cor a 1 sIgE were lower than those of Cor a 9 and Cor a 14 sIge. When the test results were positive, the post-test probability of a hazelnut allergy was 34% for Cor a 1 sIgE, 60% for Cor a9 sIgE and 73% for Cor a 14 sIgE. When the test results were negative, the post-test probability of a hazelnut allergy was 55% for Cor a 1 sIgE, 16% for Cor a9 sIgE and 14% for Cor a 14 sIgE. Measurement of IgE levels to Cor a 9 and Cor a 14 might have the potential to improve specificity in detecting clinically tolerant children among hazelnut-sensitized ones, reducing the need to perform oral food challenges.

Inverse relation between structural flexibility and IgE reactivity of Cor a 1 hazelnut allergens.

A major proportion of allergic reactions to hazelnuts (Corylus avellana) are caused by immunologic cross-reactivity of IgE antibodies to pathogenesis-related class 10 (PR-10) proteins. Intriguingly, the four known isoforms of the hazelnut PR-10 allergen Cor a 1, denoted as Cor a 1.0401-Cor a 1.0404, share sequence identities exceeding 97% but possess different immunologic properties. In this work we describe the NMR solution structures of these proteins and provide an in-depth study of their biophysical properties. Despite sharing highly similar three-dimensional structures, the four isoforms exhibit remarkable differences regarding structural flexibility, hydrogen bonding and thermal stability. Our experimental data reveal an inverse relation between structural flexibility and IgE-binding in ELISA experiments, with the most flexible isoform having the lowest IgE-binding potential, while the isoform with the most rigid backbone scaffold displays the highest immunologic reactivity. These results point towards a significant entropic contribution to the process of antibody binding.

A Food, a Bite, a Sip: How Much Allergen Is in That?

Detailed information about the amount of allergenic protein ingested by the patient prior to an allergic reaction yields valuable information for the diagnosis, guidance and management of food allergy. However, the exact amount of ingredients is often not declared on the label. In this study the feasibility was studied for estimating the amount of allergenic protein from milk, eggs, peanuts and hazelnuts in frequently consumed composite and non-composite foods and per bite or sip size in different age groups in the Netherlands. Foods containing milk, egg, peanut or hazelnut most frequently consumed were selected for the age groups 2-3, 4-6 and 19-30 years. If the label did not yield clear information, the amount of allergenic protein was estimated based on food labels. Bite or sip sizes were determined in these age groups in 30 different foods. The amount of allergenic protein could be estimated in 47/70 (67%) of composite foods, which was complex. Estimated protein content of milk, egg, peanut and hazelnut was 2-3 g for most foods but varied greatly from 3 to 8610 mg and may be below threshold levels of the patient. In contrast, a single bite or sip can contain a sufficient amount of allergenic protein to elicit an allergic reaction. Bite and sip sizes increased with age. In every day practice it is hard to obtain detailed and reliable information about the amount of allergenic protein incorporated in composite foods. We encourage companies to disclose the amount of common allergenic foods on their labels.

Simplified AIT for allergy to several tree pollens-Arguments from the immune outcome analyses following treatment with SQ tree SLIT-tablet.

BACKGROUND: The SQ tree SLIT-tablet (containing birch extract) proved clinically significant effects during the pollen season for birch as well as alder/hazel. Immune outcomes of this treatment for allergens from multiple birch homologous trees need further investigation. We hypothesize that birch pollen extract AIT modulates a highly cross-reactive immune response and that this may be the basis for the observed clinical cross-protection. METHODS: Blood samples were collected from 397 birch allergic patients during SQ tree SLIT-tablet or placebo treatment (1:1) for up to 40 weeks. Serum IgE and IgG4 specific to birch, and birch homologous tree pollens from alder, hazel, hornbeam, beech and chestnut were measured by ImmunoCAP. IgE-Blocking Factor (IgE-BF) for alder, birch and hazel during treatment was measured by Advia Centaur and blocking effects for birch and all these birch homologous tree pollens were further investigated by basophil activation (BAT). Antibody readouts were investigated in patient subsets. T-cell responses (proliferation) to allergen extracts and peptide pools (group 1 allergens) were investigated in T-cell lines from 29 untreated birch pollen-allergic individuals. RESULTS: Significant Pearson correlations between serum IgE towards birch, alder, hazel, hornbeam and beech were observed (r-values > .86). T-cell reactivity was observed throughout the birch homologous group. Almost identical kinetics for changes in IgE towards birch, alder and hazel were observed during treatment and similar species-specific changes were seen for serum-IgG4 . IgG4 reactivity towards birch and alder, hazel, hornbeam and beech correlated significantly at end-of-treatment (r-values > .72). Treatment resulted in similar IgE-BF kinetics for alder, birch, and hazel and blocking of BAT for multiple trees in most actively treated patients investigated. CONCLUSIONS: Systematic analyses of T-cell and antibody cross-reactivities before and during birch pollen extract AIT provide the immunological basis for the observed clinical effect of SQ tree SLIT-tablet treatment of tree pollen allergy induced by multiple trees in the birch homologous group.

Unraveling the Hook Effect: A Comprehensive Study of High Antigen Concentration Effects in Sandwich Lateral Flow Immunoassays.

Sandwich lateral flow immunoassays (LFIAs) are limited at high antigen concentrations by the hook effect, leading to a contradictory decrease in the test line (T) intensity and false-negative results. The hook effect is mainly associated with the loss of T, and research focuses on minimizing this effect. Nevertheless, the control line (C) intensity is also affected at higher analyte concentrations, undesirably influencing the T/C ratio in LFIA readers. The main aim of this work is to identify and understand these high antigen concentration effects in order to develop ubiquitous strategies to interpret and mitigate such effects. Four complementary experiments were performed: performance assessment of three different allergen LFIAs (two for hazelnut, one for peanut) over 0.075-3500 ppm, LFIAs with C only, surface plasmon resonance (SPR) binding experiments on the immobilized control antibody, and smartphone video recording of LFIAs during their development. As antigen concentrations increase, the C signal decreases before the T signal does, suggesting that distinct mechanisms underlie these intensity reductions. Reduced binding at the C occurred even in the absence of T, so the upfront T does not explain the loss of C. SPR confirmed that the C antibody favors binding with free labeled antibody compared with a labeled antibody-analyte complex, indicating that in antigen excess, binding is reduced at C before T. Finally, a smartphone-based video method was developed for dynamically monitoring the LFIA development in real time to distinguish between different concentration-dependent effects. Digitally analyzing the data allows clear differentiation of highly positive samples and false-negative samples and can indicate whether the LFIA is in the dynamic working range or at critically high concentrations. The aim of this work is to identify and understand such high antigen concentration effects in order to develop ubiquitous strategies to interpret and mitigate such effects.

Immunoglobulin E-Binding Pattern of Canadian Peanut Allergic Children and Cross-Reactivity with Almond, Hazelnut and Pistachio.

Peanut allergic individuals can be both co-sensitized and co-allergic to peanut and tree nuts. At the moment, standard diagnostic approaches do not always allow differentiation between clinically relevant sensitization and nonsignificant cross-reactions, and the responsibility of each allergen remains unclear. The objective of this study was therefore to determine a peanut sensitization profile in a cohort of Canadian peanut allergic children and assess the immunoglobulin E (IgE) molecular cross-reactivity between peanut, almond, hazelnut and pistachio. The specific IgE (sIgE) levels of each patient serum were determined by ImmunoCAP, indirect ELISA and immunoblot to examine their sIgE-binding levels and profiles to peanut proteins. Reciprocal inhibition ELISA and immunoblotting were used to study sIgE cross-reactions between peanut and the selected tree nuts using an adjusted and representative serum pool of the nine allergic patients. The results showed that the prepared peanut and tree nut protein extracts allowed for the detection of the majority of peanut and selected tree nut known allergens. The reciprocal inhibition ELISA experiments showed limited sIgE cross-reactivities between peanut and the studied tree nuts, with peanut being most likely the sensitizing allergen and tree nuts the cross-reactive ones. In the case of hazelnut and pistachio, a coexisting primary sensitization to hazelnut and pistachio was also demonstrated in the serum pool. Reciprocal inhibition immunoblotting further revealed that storage proteins (2S albumin, 7S vicilin and 11S legumin) could possibly account for the observed IgE-cross-reactions between peanut and the studied tree nuts in this cohort of allergic individuals. It also demonstrated the importance of conformational epitopes in the exhibited cross-reactions.

Natural Variation of Hazelnut Allergenicity: Is There Any Potential for Selecting Hypoallergenic Varieties?

Hazelnuts (Corylus avellana L.) have an important role in human nutrition and health. However, they are a common cause of food allergy. Due to hazelnut varietal diversity, variety-dependent differences in the IgE-binding properties may be suspected, which could allow therapeutic strategies based on the use of hypoallergenic varieties to induce desensitization. In a proteogenomic approach, we aimed to evaluate the allergenic potential of a genetically diverse set of hazelnuts (n = 13 varieties). Minor differences were found at the level of genes encoding important allergens, namely Cor a 8, Cor a 9, and Cor a 14. Nevertheless, IgE-reactivity was similar for all varieties using sera from seven allergic individuals. The predominant IgE-reactive proteins were Cor a 9 (100%) and Cor a 1.04 (60%), with the former being the most frequently identified by a two-dimensional gel electrophoresis (2-DE)-based proteomic approach. Therefore, it seems that the conventional exclusion diet will hold its ground for the time being.

Allergen components in diagnosing childhood hazelnut allergy: Systematic literature review and meta-analysis.

BACKGROUND: Hazelnut-specific IgE antibodies (sIgEs) in serum support the diagnosis of hazelnut allergy, but extract-based tests have low diagnostic specificity, commonly leading to over-diagnosis. Measuring sensitization to individual allergen components may enhance the diagnosis of hazelnut allergy. We systematically examined data on diagnostic accuracy of sIgE to commercially available hazelnut components to compare their individual contributions in diagnosing hazelnut allergy. METHODS: Seven databases were searched for diagnostic studies on patients suspected of having hazelnut allergy. Studies employing component-specific IgE testing on patients whose final diagnosis was determined by oral food challenges were included in the meta-analysis. Study quality was assessed as recommended by Cochrane. RESULTS: Seven cross-sectional studies and one case-control study were identified, seven presenting data on children (N = 635), and one on a mixed age population. Overall, the diagnostic accuracies of sIgE to both Cor a 9 and Cor a 14 were significantly higher than for Cor a 1-sIgE (P < .05). In children, the specificity of Cor a 14-sIgE at 0.35 kUA /L cutoff was 81.7% (95% CI 77.1, 85.6), and 67.3% (60.3, 73.6) for Cor a 9-sIgE. The specificities for Cor a 1-sIgE and hazelnut-sIgE were 22.5% (7.4, 51.2) and 10.8% (3.4, 29.8), respectively. The sensitivity of Cor a 1-sIgE (60.2% [46.9, 72.2]) was lower than for hazelnut extract-sIgE (95.7% [88.7, 98.5]), while their specificities did not differ significantly. CONCLUSION: sIgE to Cor a 14 and Cor a 9 hazelnut storage proteins increases diagnostic specificity in assessing hazelnut allergy in children. The combined use of hazelnut extract and hazelnut storage proteins may improve diagnostic value.

Component-resolved diagnostics can be useful for identifying hazelnut allergy in Japanese children.

BACKGROUND: Cor a 9 and Cor a 14 are effective markers for predicting hazelnut allergy. However, there have been no reports on the component-resolved diagnostics (CRD) of hazelnut allergy using an oral food challenge (OFC) for diagnosis in Asia. We hypothesized that CRD would improve the accuracy of diagnosing hazelnut allergies in Japanese children. METHODS: We recruited 91 subjects (median age: 7.3 years) who were sensitized to hazelnuts and had performed a hazelnut OFC at the National Hospital Organization Sagamihara National Hospital between 2006 and 2017. All subjects were classified as allergic or asymptomatic to 3 g of hazelnuts. The sIgE levels (hazelnut/Cor a 1/Cor a 8/Cor a 9/Cor a 14/alder pollen) were measured using ImmunoCAP. We aimed to determine the predictive factors of hazelnut allergy. RESULTS: Nine subjects (10%) were allergic to ≤3 g of hazelnuts. Levels of sIgE for Cor a 9 in hazelnut-allergic subjects were significantly higher than those in asymptomatic subjects (4.47 vs. 0.76 kUA/L, p = 0.039). Levels of sIgE to alder pollen and Cor a 1 in hazelnut-allergic subjects were significantly lower than those in asymptomatic subjects (<0.10 vs 13.0 kUA/L, p = 0.004; <0.10 vs 5.03 kUA/L, p = 0.025). The area under the receiver operating characteristics curve for hazelnut/alder/Cor a 1/Cor a 9 was 0.55/0.78/0.72/0.71, respectively, with p = 0.651/0.006/0.029/0.040, respectively. CONCLUSIONS: The findings of a high sIgE level for Cor a 9 and a low sIgE level for Cor a 1 can improve the diagnostic accuracy to better identify Japanese children sensitized to hazelnuts.

Identification of a natural ligand of the hazel allergen Cor a 1.

Hazelnut is one of the most frequent causes of food allergy. The major hazel allergen in Northern Europe is Cor a 1, which is homologous to the major birch pollen allergen Bet v 1. Both allergens belong to the pathogenesis related class PR-10. We determined the solution structure of Cor a 1.0401 from hazelnut and identified a natural ligand of the protein. The structure reveals the protein fold characteristic for PR-10 family members, which consists of a seven-stranded antiparallel ß-sheet, two short α-helices arranged in V-shape and a long C-terminal α-helix encompassing a hydrophobic pocket. However, despite the structural similarities between Cor a 1 and Bet v 1, they bind different ligands. We have shown previously that Bet v 1 binds to quercetin-3-O-sophoroside. Here, we isolated Cor a 1 from hazel pollen and identified the bound ligand, quercetin-3-O-(2"-O-ß-D-glucopyranosyl)-ß-D-galactopyranoside, by mass spectrometry and nuclear magnetic resonance spectroscopy (NMR). NMR experiments were performed to confirm binding. Remarkably, although it has been shown that PR-10 allergens show promiscuous binding behaviour in vitro, we can demonstrate that Cor a 1.0401 and Bet v 1.0101 exhibit highly selective binding for their specific ligand but not for the respective ligand of the other allergen.

Antibody Cross-Reactivity between Proteins of Chia Seed ( Salvia hispanica L.) and Other Food Allergens.

Chia seeds are becoming increasingly common in Europe because of their functional and nutritional properties. Despite this, few studies have focused on the allergic potential and antibody cross-reactivity among storage proteins in chia seed and other plants. The aim of this study was to identify chia seed's immunoglobulin G (IgG) and immunoglobulin E (IgE) binding proteins ( Salvia hispanica L.) and to investigate the antibody cross-reactivity among its storage proteins and those of other seeds. Extracted chia seed proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Immunodetection was performed with commercial antibodies against sesame seed, hazelnut, and peanut and sera from 33 patients with a hazelnut allergy and five with a sesame allergy. Cross-reactivity of certain antibodies with storage proteins of chia seed, sesame seed, and hazelnut was assessed using an enzyme-linked immunosorbent assay (ELISA) inhibition, blot inhibition, and surface plasmon resonance (SPR) spectroscopy. IgG binding proteins were identified at molecular weight (MW) 70, 49, 34, 23, and 20 kDa by applying commercial antibodies. Furthermore, the interaction of chia proteins with sera from sesame-allergic patients led to identify IgE binding proteins at MW 49, 45, 31, 20, and 12 kDa, while IgEs in sera from hazelnut-allergic patients reacted with proteins at MW 300, 140, 49, 45, 31, 20, and 6 kDa. The results of ELISA inhibition and blot inhibition indicated chia seed proteins are similar to sesame seed and hazelnut proteins in the primary structure. The antisesame antibodies' binding to sesame proteins was more strongly inhibited by the chia globulin fraction (GLO) than the antihazelnut antibodies' binding to hazelnut proteins. SPR results confirmed the presence of IgG binding proteins in GLO and the high similarity of epitopes on globulins of chia seed and sesame seed. Thus, chia seed consumption might lead to cross-sensitization in patients with a sesame allergy.

Retrospective definition of reaction risk in Italian children with peanut, hazelnut and walnut allergy through component-resolved diagnosis.

BACKGROUND: Serum IgE evaluation of peanut, hazelnut and walnut allergens through the use of component-resolved diagnosis (CRD) can be more accurate than IgE against whole food to associate with severe or mild reactions. OBJECTIVES: The aim of the study was to retrospectively define the level of reaction risk in children with peanut, hazelnut and walnut sensitization through the use of CRD. METHODS: 34 patients [n=22 males, 65%; median age eight years, interquartile range (IQR) 5.0-11.0 years] with a reported history of reactions to peanut and/or hazelnut and/or walnut had their serum analyzed for specific IgE (s-IgE) by ImmunoCAP® and ISAC® microarray technique. RESULTS: In children with previous reactions to peanut, the positivity of Arah1 and Arah2 s-IgE was associated with a history of anaphylaxis to such food, while the positivity of Arah8 s-IgE were associated with mild reactions. Regarding hazelnut, the presence of positive Cora9 and, particularly, Cora14 s-IgE was associated with a history of anaphylaxis, while positive Cora1.0401 s-IgE were associated with mild reactions. Concerning walnut, the presence of positive Jug r 1, Jug r 2, Jug r 3 s-IgE was associated with a history of anaphylaxis to such food. ImmmunoCAP® proved to be more useful in retrospectively defining the risk of hazelnut anaphylaxis, because of the possibility of measuring Cor a14 s-IgE. CONCLUSIONS: Our data show that the use of CRD in patients with allergy to peanut, hazelnut and walnut could allow for greater accuracy in retrospectively defining the risk of anaphylactic reaction to such foods.

Sensitization profiles to hazelnut allergens across the United States.

BACKGROUND: Measurement of IgE antibody to hazelnut components can aid in the prediction of allergic responses to the food. OBJECTIVE: To investigate the association between patient demographics (age, location) and patterns of allergic sensitization to hazelnut components across the United States and to investigate the degree of correlation between hazelnut sensitization with sensitization to other tree nuts, peanuts, and their components. METHODS: Serum samples from 10,503 individuals with hazelnut extract specific IgE (sIgE) levels of 0.35 kUA/L or higher were analyzed for IgE antibodies to Cor a 1, 8, 9, and 14 by ImmunoCAP. A subset of these patients were analyzed for IgE antibodies to peanut, walnut, and cashew nut IgE along with associated components. RESULTS: Among hazelnut sensitized individuals, children (<3 years old) were predominantly sensitized to Cor a 9 and Cor a 14. Conversely, Cor a 1 sIgE sensitization was much higher in adults than children, especially in the Northeastern United States. Cor a 8 sensitization was relatively constant (near 10%) across all ages. Cosensitization of hazelnut with other tree nuts and peanuts was related to correlation of IgE concentrations of individual component families. CONCLUSION: We conclude that sensitization to individual hazelnut components is highly dependent on age and/or geographic location. Component correlations suggest that cosensitization to hazelnut and walnut may be caused by their pathogenesis-related protein 10 allergens, nonspecific lipid transfer proteins, or seed storage proteins, whereas hazelnut and peanut cosensitization is more often caused by cross-reactivity of pathogenesis-related protein 10 (Cor a 1 and Ara h 8) and nonspecific lipid transfer proteins (Cor a 8 and Ara h 9).

Rapid Antibody Selection Using Surface Plasmon Resonance for High-Speed and Sensitive Hazelnut Lateral Flow Prototypes.

Lateral Flow Immunoassays (LFIAs) allow for rapid, low-cost, screening of many biomolecules such as food allergens. Despite being classified as rapid tests, many LFIAs take 10⁻20 min to complete. For a really high-speed LFIA, it is necessary to assess antibody association kinetics. By using a label-free optical technique such as Surface Plasmon Resonance (SPR), it is possible to screen crude monoclonal antibody (mAb) preparations for their association rates against a target. Herein, we describe an SPR-based method for screening and selecting crude anti-hazelnut antibodies based on their relative association rates, cross reactivity and sandwich pairing capabilities, for subsequent application in a rapid ligand binding assay. Thanks to the SPR selection process, only the fast mAb (F-50-6B12) and the slow (S-50-5H9) mAb needed purification for labelling with carbon nanoparticles to exploit high-speed LFIA prototypes. The kinetics observed in SPR were reflected in LFIA, with the test line appearing within 30 s, almost two times faster when F-50-6B12 was used, compared with S-50-5H9. Additionally, the LFIAs have demonstrated their future applicability to real life samples by detecting hazelnut in the sub-ppm range in a cookie matrix. Finally, these LFIAs not only provide a qualitative result when read visually, but also generate semi-quantitative data when exploiting freely downloadable smartphone apps.