Peach extract induces systemic and local immune responses in an experimental food allergy model.

Peach allergy is among the most frequent food allergies in the Mediterranean area, often eliciting severe anaphylactic reactions in patients. Due to the risk of severe symptoms, studies in humans are limited, leading to a lack of therapeutic options. This study aimed to develop a peach allergy mouse model as a tool to better understand the pathomechanism and to allow preclinical investigations on the development of optimized strategies for immunotherapy. CBA/J mice were sensitized intraperitoneally with peach extract or PBS, using alum as adjuvant. Afterwards, extract was administered intragastrically to involve the intestinal tract. Allergen provocation was performed via intraperitoneal injection of extract, measuring drop of body temperature as main read out of anaphylaxis. The model induced allergy-related symptoms in mice, including decrease of body temperature. Antibody levels in serum and intestinal homogenates revealed a Th2 response with increased levels of mMCPT-1, peach- and Pru p 3-specific IgE, IgG1 and IgG2a as well as increased levels of IL-4 and IL-13. FACS analysis of small intestine lamina propria revealed increased amounts of T cells, neutrophils and DCs in peach allergic mice. These data suggest the successful establishment of a peach allergy mouse model, inducing systemic as well as local gastrointestinal reactions.

Validation of an ELISA method for quantification of the major Timothy grass pollen allergen Phl p 5a (BSP090).

Progress towards standardisation of allergen products has been made in recent years. Nevertheless, no standardised test method to quantify the allergen content of grass pollen allergen products is available at present. One aim of the BSP090 project was to validate a quantitative assay for a major Timothy grass (Phleum pratense) pollen allergen, Phl p 5. Qualification of a candidate ELISA system was performed with regard to range, robustness and cross-reactivity in preliminary studies. The assay specifically detected Phl p 5 with a quantification range from 3.9 ng/mL to 62.5 ng/mL. Suitability to quantify recombinant and natural Phl p 5 was further assessed in a collaborative study including 14 laboratories in Europe and the USA. Precision and accuracy of the assay was satisfactory with 93% of calculated Phl p 5 concentrations and 100% of total recoveries being within the ± 30% acceptance range. Similar results were obtained for spike recoveries, with exclusion of the lowest concentration spike, showing spike recoveries exceeding the acceptance range for six laboratories. Inter-assay (repeatability) and inter-laboratory (reproducibility) variability were satisfactory, in the format used in the present study. Robustness towards different statistical methods for data analysis was demonstrated. In conclusion, the assay can easily be established in routine testing and results of the preliminary testing and collaborative study support the proposal of the assessed Phl p 5-specific ELISA as a European Pharmacopoeia general method.

Distinct epitope structures of defensin-like proteins linked to proline-rich regions give rise to differences in their allergenic activity.

BACKGROUND: Art v 1, Amb a 4, and Par h 1 are allergenic defensin-polyproline-linked proteins present in mugwort, ragweed, and feverfew pollen, respectively. We aimed to investigate the physicochemical and immunological features underlying the different allergenic capacities of those allergens. METHODS: Recombinant defensin-polyproline-linked proteins were expressed in E. coli and physicochemically characterized in detail regarding identity, secondary structure, and aggregation status. Allergenic activity was assessed by mediator releases assay, serum IgE reactivity, and IgE inhibition ELISA using sera of patients from Austria, Canada, and Korea. Endolysosomal protein degradation and T-cell cross-reactivity were studied in vitro. RESULTS: Despite variations in the proline-rich region, similar secondary structure elements were observed in the defensin-like domains. Seventy-four percent and 52% of the Austrian and Canadian patients reacted to all three allergens, while Korean patients were almost exclusively sensitized to Art v 1. This was reflected by IgE inhibition assays demonstrating high cross-reactivity for Austrian, medium for Canadian, and low for Korean sera. In a subgroup of patients, IgE reactivity toward structurally altered Amb a 4 and Par h 1 was not changed suggesting involvement of linear epitopes. Immunologically relevant endolysosomal stability of the defensin-like domain was limited to Art v 1 and no T-cell cross-reactivity with Art v 125-36 was observed. CONCLUSIONS: Despite structural similarity, different IgE-binding profiles and proteolytic processing impacted the allergenic capacity of defensin-polyproline-linked molecules. Based on the fact that Amb a 4 demonstrated distinct IgE-binding epitopes, we suggest inclusion in molecule-based allergy diagnosis.

Quality requirements for allergen extracts and allergoids for allergen immunotherapy.

All allergen products for allergen immunotherapy currently marketed in the European Union are pharmaceutical preparations derived from allergen-containing source materials like pollens, mites and moulds. Especially this natural origin results in particular demands for the regulatory requirements governing allergen products. Furthermore, the development of regulatory requirements is complicated by the so far missing universal link between certain quality parameters, in particular biological potency, on the one hand and clinical efficacy on the other hand. As a consequence, each allergen product for specific immunotherapy has to be assessed individually for its quality, safety and efficacy. At the same time, biological potency of allergen products is most commonly determined using IgE inhibition assays based on human sera relative to product-specific in house references, ruling out full comparability of products from different manufacturers. This review article aims to summarize the current quality requirements for allergen products including the special requirements implemented for control of chemically modified allergen extracts (allergoids).

Minor allergen patterns in birch pollen allergen products-A question of pollen?

BACKGROUND: Contrary to the scientific differentiation between major and minor allergens, the regulatory framework controlling allergen products in the EU distinguishes relevant and non-relevant allergens. Given the lack of knowledge on their clinical relevance, minor allergens are usually not controlled by allergen product specifications. Especially, in birch pollen (BP) allergen products, minor allergens are commonly disregarded. OBJECTIVES: To quantify three minor allergens in BP allergen products from different manufacturers and to assess the influence of the utilized BP on minor allergen patterns. METHODS: Apart from common quality parameters such as Bet v 1 content, Bet v 4, Bet v 6 and Bet v 7 were quantified in 70 BP allergen product batches from six manufacturers, using ELISA systems developed in-house. Batch-to-batch variability was checked for agreement with a variability margin of 50%-200% from mean of the given batches for individual allergen content. Subsequently, minor allergen patterns were generated via multidimensional scaling and related to information on the pollen lots used in production of the respective product batches. RESULTS: Like the already established Bet v 4 ELISA, the ELISA systems for quantification of Bet v 6 and Bet v 7 were successfully validated. Differences in minor allergen content between products and batch-to-batch consistency were observed. Correlations between minor and major allergen content were low to moderate. About 20% of batches exceeded the variability margin for at least one minor allergen. Interestingly, these fluctuations could not in all cases be linked to the use of certain BP lots. CONCLUSIONS AND CLINICAL RELEVANCE: The impact of the observed minor allergen variability on safety and efficacy of BP allergen products can currently not be estimated. As the described differences could only in few cases be related to the used pollen lots, it is evident that additional factors influence minor allergens in BP allergen products.

The conformational IgE epitope profile of soya bean allergen Gly m 4.

BACKGROUND: Birch pollen-related soya allergy is mediated by Gly m 4. Conformational IgE epitopes of Gly m 4 are unknown. OBJECTIVE: To identify the IgE epitope profile of Gly m 4 in subjects with birch pollen-related soya allergy utilizing an epitope library presented by Gly m 4-type model proteins. METHODS: Sera from patients with (n = 26) and without (n = 19) allergy to soya as determined by oral provocation tests were studied. Specific IgE (Bet v 1/Gly m 4) was determined by ImmunoCAP. A library of 59 non-allergenic Gly m 4-type model proteins harbouring individual and multiple putative epitopes for IgE was tested in IgE binding assays. Primary, secondary and tertiary protein structures were assessed by mass spectrometry, circular dichroism and nuclear magnetic resonance spectroscopy. RESULTS: All subjects were sensitized to Gly m 4 and Bet v 1. Allergen-specific serum IgE levels ranged from 0.94 to > 100 kUA /L. The avidities of serum IgE were 5.06 ng (allergic) and 1.8 ng (tolerant) as determined by EC50 for IgE binding to Gly m 4. 96% (46/48) of the protein variants bound IgE. Model proteins had Gly m 4-type conformation and individual IgE binding clustered in six major surface areas. Gly m 4-specific IgE binding could be inhibited to up to 80% by model proteins harbouring individual IgE binding sites in an epitope-wise equimolar fashion. Receiver operating curve analysis revealed an area under fitted curve of up to 0.88 for model proteins and 0.66 for Gly m 4. CONCLUSION AND CLINICAL RELEVANCE: Serum levels and avidity of Gly m 4-specific IgE do not correlate with clinical reactivity to soya. Six IgE-binding areas, represented by 23 amino acids, account for more than 80% of total IgE binding capacity of Gly m 4. Model proteins may be used for epitope-resolved diagnosis to differentiate birch-soya allergy from clinical tolerance.

Molecular cloning of plane pollen allergen Pla a 3 and its utility as diagnostic marker for peach associated plane pollen allergy.

BACKGROUND: Non-specific lipid transfer proteins (nsLTP) are considered to provoke allergic symptoms to plane tree pollen, which are frequently associated with peach allergy. OBJECTIVE: The objective was to clone the cDNA of plane pollen nsLTP Pla a 3, to characterize IgE-binding and allergenic potency of recombinant Pla a 3 in comparison to its natural counterpart and peach nsLTP Pru p 3. METHODS: Natural Pla a 3 was purified from plane pollen and analysed by mass spectrometry (MS). Recombinant Pla a 3 was characterized by SDS-PAGE and CD spectroscopy. Specific IgE to extract, components of plane pollen and Pru p 3 was measured by ImmunoCAP in sera of patients allergic to either plane pollen (n = 10), peach (n = 15) or both (n = 15). Biological potency of the proteins was investigated by in vitro mediator release assays and IgE cross-reactivity by competitive ELISA. RESULTS: Two Pla a 3 isoforms were identified. Recombinant Pla a 3 showed high purity, structural integrity, IgE-binding capacity comparable to nPla a 3 and biological potency. Sensitization to plane pollen extract was confirmed in 24/25 plane pollen allergics. The frequency of sensitization to Pla a 3 was 53% among patients allergic to both plane pollen and peach and 10% among plane pollen allergics tolerating peach where most patients were sensitized to Pla a 1. Pla a 3 and Pru p 3 showed strong bi-directional IgE cross-reactivity in patients allergic to peach and plane pollen, but not in peach allergics tolerating plane pollen. Levels of IgE-binding were generally higher to Pru p 3 than to Pla a 3. CONCLUSION: Sensitization to Pla a 3 is relevant in a subgroup of plane pollen allergics with concomitant peach allergy. IgE testing with Pla a 3 may serve as a marker to identify plane pollen allergic patients at risk of LTP-mediated food reactions and thereby improve in vitro diagnostic procedures.

IgE recognition patterns in peanut allergy are age dependent: perspectives of the EuroPrevall study.

BACKGROUND: We tested the hypothesis that specific molecular sensitization patterns correlate with the clinical data/manifestation in a European peanut-allergic population characterized under a common protocol. METHODS: Sixty-eight peanut-allergic subjects and 82 tolerant controls from 11 European countries were included. Allergy to peanut and lowest symptom-eliciting dose was established by double-blind placebo-controlled food challenge in all but anaphylactic subjects. Information of early or late (before or after 14 years of age) onset of peanut allergy was obtained from standardized questionnaires. IgE to peanut allergens rAra h 1-3, 6, 8-9, profilin and CCD was determined using ImmunoCAP. RESULTS: Seventy-eight percent of peanut allergics were sensitized to peanut extract and 90% to at least one peanut component. rAra h 2 was the sole major allergen for the peanut-allergic population. Geographical differences were observed for rAra h 8 and rAra h 9, which were major allergens for central/western and southern Europeans, respectively. Sensitization to rAra h 1 and 2 was exclusively observed in early-onset peanut allergy. Peanut-tolerant subjects were frequently sensitized to rAra h 8 or 9 but not to storage proteins. Sensitization to Ara h 2 ≥ 1.0 kUA /l conferred a 97% probability for a systemic reaction (P = 0.0002). Logistic regression revealed a significant influence of peanut extract sensitization and region on the occurrence of systemic reactions (P = 0.0185 and P = 0.0436, respectively). CONCLUSION: Sensitization to Ara h 1, 2 and 3 is usually acquired in childhood. IgE to Ara h 2 ≥ 1.0 kUA /l is significantly associated with the development of systemic reactions to peanut.

[Official experimental testing of biomedical products. Regulatory frame and importance of for quality, safety and efficacy]. / Staatliche experimentelle Produktprüfung von biomedizinischen Arzneimitteln. Regulatorischer Rahmen und Bedeutung für die Qualität, Sicherheit und Wirksamkeit.

The official experimental testing of biomedicinal products provides a very significant contribution to ensuring quality, safety and efficacy of these indispensable medicines. Already in the prelicensing phase or to elucidate clusters of increased adverse effects, official medicinal control laboratories are committed to perform experimental testing. The official batch release can be seen as external quality control of the manufacturer's release testing. For proficient performance in these tasks, scientific research is required, in particular on the development and refinement of test methods, and considering the continuous development of innovative biomedicinal products. This article is aimed at introducing the present thematic issue and in particular the regulatory basis of experimental product testing, and illustrates by means of several examples its great importance for the sake of the patients.

Kinetics, cross-reactivity, and specificity of Bet v 1-specific IgG4 antibodies induced by immunotherapy with birch pollen.

BACKGROUND: IgE antibodies specific for the major birch pollen allergen frequently cross-react with Bet v 1 homologous food proteins, for example Cor a 1 in hazelnut and Mal d 1 in apple. Specific immunotherapy with birch pollen (BP-SIT) induces IgG4 antibodies that inhibit IgE binding to Bet v 1. However, information on cross-reactivity of BP-SIT-induced Bet v 1-specific IgG4 antibodies with food allergens is limited. In this study, we investigated the kinetics of production, cross-reactivity, and IgE-blocking activity of Bet v 1-specific IgG4 antibodies emerging during conventional BP-SIT and whether IgG4-epitopes overlapped with IgE epitopes. METHODS: IgE and IgG4 levels specific for Bet v 1, Mal d 1, and Cor a 1 were determined in 42 birch pollen-allergic patients before and during BP-SIT. Inhibition of IgE binding was studied by IgE-facilitated antigen-binding assays and basophil activation tests. Furthermore, inhibition of IgE-mediated activation of food allergen-reactive Bet v 1-specific T-cell lines was assessed. Competitive immunoscreening of phage-displayed peptides was applied to select mimotopes recognized by IgE and IgG4 antibodies, respectively. The resulting mimotopes were mapped on the surface of the 3D structure of the allergens using a computer-based algorithm. RESULTS: BP-SIT significantly increased Bet v 1- and food allergen-reactive IgG4 antibodies. In parallel, allergen-specific IgE levels decreased significantly. Sera containing food allergen-reactive IgG4 antibodies inhibited IgE binding, basophil activation, and IgE-mediated food allergen-induced T-cell proliferation. Predicted IgE and IgG4 epitopes on all allergens showed high overlap. CONCLUSION: Our results indicate that BP-SIT may induce Bet v 1-specific IgG4 antibodies that cross-react with related food allergens and inhibit IgE binding by epitope competition.

Component-resolved in vitro diagnosis of carrot allergy in three different regions of Europe.

BACKGROUND: Carrot is a frequent cause of food allergy in Europe. The objective of this study was to evaluate a panel of carrot allergens for diagnosis of carrot allergy in Spain, Switzerland and Denmark. METHODS: Forty-nine carrot allergic patients, 71 pollen allergic but carrot-tolerant patients and 63 nonatopic controls were included. Serum IgE to carrot extract, recombinant carrot allergens (rDau c 1.0104; rDau c 1.0201; rDau c 4; the isoflavone reductase-like proteins rDau c IFR 1, rDau c IFR 2; the carrot cyclophilin rDau c Cyc) were analyzed by ImmunoCAP. RESULTS: The sensitivity of the carrot extract-based test was 82%. Use of the recombinant allergens increased the sensitivity to 90%. The Dau c 1 isoforms were major allergens for Swiss and Danish carrot allergic patients, the profilin rDau c 4 for the Spanish patients. The rDau c IFR 1 and rDau c IFR 2 were recognized by 6% and 20% of the carrot allergics, but did not contribute to a further increase of sensitivity. Among pollen allergic controls, 34% had IgE to carrot extract, 18% to each of rDau c 1.0104, rDau c 1.0201 and rDau c 4, 8% to rDau c IFR 1 and 7% to rDau c IFR 2. Sensitization to rDau c Cyc occurred in one carrot allergic patient and one nonatopic control. CONCLUSION: Component-resolved in vitro analyses revealed a significant difference in IgE sensitization pattern between geographical regions and in the prevalence of sensitization to carrot components between carrot allergic and carrot-tolerant but pollen sensitized patients.

Establishment of recombinant major allergens Bet v 1 and Phl p 5a as Ph. Eur. reference standards and validation of ELISA methods for their measurement. Results from feasibility studies.

The potency of allergen extracts is determined as total allergenic activity without consideration of their composition and the units differ from one manufacturer to another, making it very difficult to compare the different products. Recently, purified major allergens have been obtained by recombinant DNA technology and produced under Good Manufacturing Practice (GMP) conditions. In principle, such recombinant allergens could be established as reference standards and could help for the standardisation of the major allergen content of allergen extracts. Two recombinant major allergens, one from birch pollen, rBet v 1, and one from Timothy grass pollen, Phl p 5a, have been selected at the end of the CREATE programme as a potential starting point for the establishment as European Pharmacopoeia (Ph. Eur.) Reference Standards through a project run by the Biological Standardisation Programme (BSP) of the European Directorate for the Quality of Medicines & HealthCare (EDQM). To this end, bulk candidate recombinant materials, produced under GMP conditions, were procured from two European manufacturers and subsequently formulated and lyophilised. Four ELISA systems from three different manufacturers were included in the project, two for Bet v 1 and two for Phl p 5a with the aim of establishing reference methods for determination of the respective major antigens both in natural allergen extracts as well as in recombinant allergen products. The project was run in 3 phases: a preparatory and preliminary testing phase (feasibility phase or Phase 1), an extended feasibility phase carried out in 3 laboratories (Phase 2) to confirm the transferability of the methods and an international collaborative study with a large number of participating laboratories (Phase 3). This article describes the work done in Phase 1 and Phase 2, i.e. the physico-chemical and biological characterisation of the recombinant candidate reference standards, the assessment of their suitability for the intended purpose as well as the evaluation of the candidate ELISA systems. The results show that both candidate reference standards are suitable for the intended purpose. In addition, three out of the four ELISA systems that were included in the preliminary phase were found to be appropriate for further evaluation in the collaborative study which was organised in 2011. The results of the collaborative study will be published separately.

Impact of native, heat-processed and encapsulated hazelnuts on the allergic response in hazelnut-allergic patients.

BACKGROUND: Pollen-associated food allergy is common. However, systemic reactions or even life-threatening anaphylaxis are rare. OBJECTIVE: The aim of this study was to investigate the clinical impact of native, heat-processed and encapsulated hazelnuts (HN) in patients with proven HN allergy. METHODS: One hundred and thirty-two patients with a positive history of HN allergy were recruited. Sensitization was confirmed by a skin prick test (SPT) and sIgE against HN. After an HN-free diet, double-blind placebo-controlled challenges were performed with increasing amounts of native and roasted HN. A subset of patients were given HN capsules to circumvent the oral mucosa. Basophil activation was measured by flow cytometry before and after provocation but also ex vivo using native and roasted HN extracts. RESULTS: Three groups of HN-allergic patients were identified depending on their clinical reaction pattern. The dosages by which allergic reactions were elicitated varied for native HN from 0.01 to 2.0 g, with a median of 0.1 g, for roasted HN from 0.01 to 10.0 g, with a median of 0.23 g, and for encapsulated HN from 0.1 to 3.0 g, with a median of 0.3 g. Accordingly, the SPT was more frequently positive and resulted in greater weal reactions if native HN was used. This finding was confirmed by ex vivo basophil activation showing that significantly higher allergen extract concentrations (roasted>native) were necessary to induce 50% basophil activation. CONCLUSION: Our data show that heat processing of HN reduces its allergenicity. SPT but also the basophil activation test can be used to determine the reactivity of an allergen extract.

The principle of homologous groups in regulatory affairs of allergen products--a proposal.

Among other legal regulations, the Note for Guidance on Allergen Products CPMP/BWP/243/96 released by the European Medicines Agency provides regulatory instructions regarding the quality of allergen extracts for diagnostic or therapeutic purposes. The current revision of this guideline intends to transform the so-called 'principle of taxonomic families' to the 'principle of homologous groups'. According to this concept, the data of one allergen extract demonstrating stability, efficacy and safety can, to a limited extent, be extrapolated to other allergen extracts belonging to the same homologous groups. The present work proposes the formation of homologous groups for pollen species and animal-derived materials on the basis of similar biochemical composition and homology/cross-reactivity of allergens or allergen sources. Some tree pollen species could be assigned to three different homologous groups, some weed pollen species to one homologous group and numerous grass pollen species to one homologous group on condition that data rely on single defined representative species. A homologous group for mites is limited to the Dermatophagoides species and the grouping of vertebrate-derived materials such as dander could be possible under restrictions. The criteria for the formation of the proposed homologous groups are illustrated in detail to provide an opportunity for extending existing homologous groups by further species in case of new insights in allergens and cross-reactivity of allergen sources. In this way, the concept of homologous groups could serve as a dynamic tool in the regulation of allergen products.

Assessment of three human FcepsilonRI-transfected RBL cell-lines for identifying IgE induced degranulation utilizing peanut-allergic patient sera and peanut protein extract.

Specific IgE sera screening studies are employed to investigate protein cross-reactivity. Such nonfunctional immunochemical methods cannot measure the biological activity of proteins. Therefore, an assay using RBL cells transfected with human FcepsilonRI was developed. Our objective was to evaluate the degranulation of three cell-lines expressing either the alpha-(RBL-hEI(a)-2B12 and RBL-30/25cells) or alpha-, beta-, and gamma-subunits (RBL SX-38) of the human FcepsilonRI by beta-hexosaminidase release. Purified human IgE and serum-derived polyclonal IgE from peanut-allergic subjects following challenge with anti-IgE or peanut protein extract, respectively, were utilized. Robust degranulation was induced in all three: RBL-30/25 (84%), -hEI(a)-2B12 (54%), SX-38 (94%), respectively, using purified IgE+anti-human IgE. Good release (18%, 40-45%, and 65%, respectively) occurred for one peanut-allergic subject+peanut extract with all cell-lines. With serum from three other peanut-allergic subjects, no beta-hexosaminidase release occurred with RBL-hEI(a)-2B12 cells+peanut extract, while only serum from one subject induced good degranulation, 30% and 60%, respectively, with RBL-30/25 and RBL SX 38 cells. Consistent degranulation with a potent food allergen (peanuts) was not observed. The assay's utility in safety assessment, predictive value and reproducibility for evaluating the cross-reactivity of proteins with allergens needs further investigation with additional proteins and well-characterized sera.

Strategy for allergenicity assessment of 'natural novel foods': clinical and molecular investigation of exotic vegetables (water spinach, hyacinth bean and Ethiopian eggplant).

BACKGROUND: Foods not commonly consumed in the European Union must be proven safe before being brought to market, including an assessment of allergenicity. We present a three-stepwise strategy for allergenicity assessment of natural novel foods using three novel vegetables, namely, water spinach, hyacinth bean, Ethiopian eggplant. METHODS: First, vegetable extracts were analyzed for the presence of pan-allergens [Bet v 1 homologous proteins, profilins, nonspecific lipid transfer proteins (LTP)] by immunoblot analysis with specific animal antibodies. Secondly, the IgE-binding of the food extracts was investigated by EAST (Enzyme-allergosorbent test) and immunoblot analysis using sera with IgE-reactivity to known pan-allergens or to phylogenetically related foods from subjects (i) allergic to birch, grass and mugwort pollen, (ii) with food allergy to soy, peanut, tomato, multiple pollen-related foods and (iii) sensitized to LTP. Thirdly, the clinical relevance of IgE-binding was assessed in vivo by skin prick testing (SPT) and open oral food challenges (OFC). RESULTS: Profilin and LTP were detected by animal antibodies in all vegetables, a Bet v 1 homologue selectively in hyacinth bean. IgE-binding to LTP, profilin and a Bet v 1 homologue was proven by immunoblot analysis and EAST. Positive SPT and OFC results were observed for all vegetables in pollen-allergic patients. CONCLUSIONS: Our stepwise procedure confirmed the presence and IgE-binding capacity of novel vegetable proteins homologous to known allergens in endemic vegetable foods. In vivo testing proved the potential of the novel vegetables to elicit clinical allergy. Hence, our described algorithm seems to be applicable for allergenicity testing of natural novel foods.

Enhancement of hazelnut extract for IgE testing by recombinant allergen spiking.

BACKGROUND: Hazelnuts are a common cause of food allergic reactions. Most hazelnut allergic individuals in central and northern Europe are sensitized to Cor a 1, a member of the PR-10 protein family, while the lipid transfer protein Cor a 8 acts as a major allergen in the south of Europe. Other allergens, including profilin and seed storage proteins, may be important in subgroups of patients. Reliable detection of specific IgE in the clinical diagnosis of food allergy requires allergen reagents with a sufficient representation of all relevant allergen components. Some reported observations suggest that natural hazelnut extract may not be fully adequate in this respect. METHODS: The capacity of immobilized natural hazelnut extract to bind Cor a 1-, Cor a 2- and Cor a 8-specific IgE and IgG antibodies was investigated by serum adsorption and extract dilution experiments and by the use of allergen specific rabbit antisera. All measurements were performed with the ImmunoCAP assay platform. RESULTS: The experimental results revealed an incomplete capacity of immobilized hazelnut extract to capture IgE antibodies directed to the major allergen Cor a 1. Spiking of hazelnut extract with recombinant Cor a 1.04 prior to solid phase coupling gave rise to significantly enhanced IgE antibody binding from Cor a 1 reactive sera. The spiking did not negatively affect the measurement of IgE to extract components other than Cor a 1. CONCLUSION: A hazelnut allergen reagent with enhanced IgE detection capacity can be generated by supplementing the natural food extract with recombinant Cor a 1.04.

Characterization of the T cell response to the major hazelnut allergen, Cor a 1.04: evidence for a relevant T cell epitope not cross-reactive with homologous pollen allergens.

BACKGROUND: IgE antibodies specific for the major birch-pollen allergen, Bet v 1, cross-react with homologous allergens in particular foods, e.g. apples, carrots and hazelnuts. In a high number of tree pollen-allergic individuals, this cross-reactivity causes clinical symptoms, commonly known as the 'birch-fruit-syndrome'. OBJECTIVE: To characterize the T cell response to the Bet v 1-related major allergen in hazelnuts, Cor a 1.04, and its cellular cross-reactivity with Bet v 1 and the homologous hazel pollen allergen, Cor a 1. METHODS: Using recombinant Cor a 1.04, T cell lines (TCL) and T cell clones (TCC) were established from peripheral blood mononuclear cells of tree pollen-allergic patients with associated food allergy. T cell epitopes were determined using overlapping synthetic peptides in Cor a 1.04-reactive TCL and TCC. In parallel, reactivity to Bet v 1 and Cor a 1 was tested. RESULTS: In total, 20 distinct T cell epitopes on the hazelnut allergen were identified. Several Cor a 1.04-specific TCL and TCC reacted with pollen allergens albeit less pronounced than with the hazelnut allergen. Several Cor a 1.04-specific TCC did not react with pollen allergens. Interestingly, these clones were found to react with the Bet v 1-related major allergen in carrots, Dau c 1. The cellular cross-reactivity between both food allergens could be associated with the most frequently recognized T cell epitope of Cor a 1.04, Cor a 1.04(142-153). CONCLUSIONS: The major hazelnut allergen cross-reacts with the major allergens of birch and hazel pollen but apparently contains a relevant T cell epitope not shared with pollen allergens. Our finding may have important implications for the specific immunotherapy of tree pollen-allergic patients suffering from concomitant hazelnut allergy.