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Identification, purification and characterization of allergens is crucial to the understanding of IgE-mediated disease. Immunologic and structural studies with purified allergens is essential for understanding relative immunogenicity and cross-reactivity. In this work, the complex soybean 7S vicilins (Gly m 5) with three subunits and 11S legumins (Gly m 6) with five subunits were purified and characterized along with purified peanut allergens (Ara h 1, 2, 3, and 6) by label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS). Individual subjects plasma IgE binding was tested from subjects allergic to soybeans and or peanuts by immunoblotting, ImmunoCAP™ and ISAC™ ImmunoCAP chip, comparing these soybean proteins with those of purified peanut allergens; vicilin (Ara h 1), 2S albumin (Ara h 2 and Ara h 6) and 11S globulin (Ara h 3). Results show differences between methods and subjects demonstrating the complexity of finding answers to questions of cross-reactivity., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The 2S albumin Ara h 6 is one of the most important peanut allergens. A post-translationally cleaved Ara h 6 (pAra h 6) was purified from Virginia type peanuts, and the cleavage site was mapped using high-resolution mass spectrometry. Compared to intact Ara h 6, pAra h 6 lacks a 5-amino acid stretch, resembling amino acids 43-47 (UniProt accession number Q647G9) in the nonstructured loop. Consequently, pAra h 6 consists of two chains: an N-terminal chain of approximately 5 kDa and a C-terminal chain of approximately 9 kDa, held together by disulfide bonds. Intermediate post-translationally cleaved products, in which this stretch is cleaved yet still attached to one of the subunits, are also present. The secondary structure and immunoglobulin E (IgE) binding of pAra h 6 resembles that of intact Ara h 6, indicating that the loss of the nonstructured loop is not critical for maintaining the protein structure. Commercially available monoclonal and polyclonal immunoglobulin G (IgG) antibodies directed to Ara h 6 react with both intact Ara h 6 and pAra h 6, suggesting that the involved epitopes are not located in the area that is post-translationally cleaved. No differences between intact Ara h 6 and pAra h 6 in terms of IgE binding were found, suggesting that the area that is post-translationally cleaved is not involved in IgE epitopes either. For all main cultivars Runner, Virginia, Valencia, and Spanish, intact Ara h 6 and pAra h 6 occur in peanut at similar levels, indicating that pAra h 6 is a consistent and important contributor to the allergenic potency of peanut.

Profiling allergens in complex food ingredients used in oral food challenges and immunotherapy is crucial for regulatory acceptance. Mass spectrometry based analysis employing data-independent acquisition coupled with ion mobility mass spectrometry-mass spectrometry (DIA-IM-MS) was used to investigate the allergen composition of raw peanuts and roasted peanut flour ingredients used in challenge meals. This comprehensive qualitative and quantitative analysis using label-free approaches identified and quantified 123 unique protein accessions. Semiquantitative analysis indicated that allergens Ara h 1 and Ara h 3 were the most abundant proteins and present in approximately equal amounts and were extracted in reduced amounts from roasted peanut flours. The clinically significant allergens Ara h 2 and 6 were less abundant, but relative quantification was unaffected by roasting. Ara h 5 was undetectable in any peanut sample, while the Bet v 1 homologue Ara h 8 and the lipid transfer protein allergen, Ara h 9, were detected in low abundance. The oleosin allergens, Ara h 10 and 11, were moderately abundant in the raw peanuts but were 100-fold less abundant in the defatted roasted peanut flour than the major allergens Ara h 1, 3, 2, and 6. Certain isoforms of the major allergens dominated the profile. The relative quantitation of the major peanut allergens showed little variation between different batches of roasted peanut flour. These data will support future development of targeted approaches for absolute quantification of peanut allergens which can be applied to both food ingredients used in clinical studies and extracts used for skin testing and to identify trace levels of allergens in foods.

Background: Hazelnut allergy is birch pollen-driven in Northern/Western Europe and lipid transfer protein-driven in Spain and Italy. Little is known about other regions and other allergens., Objective: Establishing a molecular map of hazelnut allergy across Europe., Methods: In 12 European cities, subjects reporting reactions to hazelnut (n = 731) were evaluated and sensitization to 24 foods, 12 respiratory allergen sources, and latex was tested by using skin prick test and ImmunoCAP. A subset (124 of 731) underwent a double-blind placebo-controlled food challenge to hazelnut. Sera of 423 of 731 subjects were analyzed for IgE against 7 hazelnut allergens and cross-reactive carbohydrate determinants by ImmunoCAP., Results: Hazelnut allergy was confirmed in 70% of those undergoing double-blind placebo-controlled food challenges. Birch pollen-driven hazelnut sensitization (Cor a 1) dominated in most cities, except in Reykjavik, Sofia, Athens, and Madrid, where reporting of hazelnut allergy was less frequent anyhow. In Athens, IgE against Cor a 8 dominated and strongly correlated with IgE against walnut, peach, and apple and against Chenopodium, plane tree, and mugwort pollen. Sensitization to seed storage proteins was observed in less than 10%, mainly in children, and correlated with IgE to nuts, seeds, and legumes. IgE to Cor a 12, observed in all cities (10% to 25%), correlated with IgE to nuts, seeds, and pollen., Conclusions: In adulthood, the importance of hazelnut sensitization to storage proteins, oleosin (Cor a 12), and Cor a 8 is diluted by the increased role of birch pollen cross-reactivity with Cor a 1. Cor a 8 sensitization in the Mediterranean is probably driven by diet in combination with pollen exposure. Hazelnut oleosin sensitization is prevalent across Europe; however, the clinical relevance remains to be established., (Copyright © 2015 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Apple (Malus domestica) is the most widely cultivated fruit crop in Europe and frequently causes allergic reactions with a variable degree of severity. So far, four apple allergens Mal d 1, Mal d 2, Mal d 3 and Mal d 4 have been identified. Mal d 1, a Bet v 1 related allergen, and Mal d 4, apple profilin, are sensitive to proteolytic degradation, whereas Mal d 2, a thaumatin-like protein and Mal d 3, a nonspecific lipid transfer protein, are rather stable to proteolytic processes. Mal d 1 and Mal d 4 were purified after expression in Escherichia coli expression system, while Mal d 2 and Mal d 3 were purified from apple fruit tissue. All purified proteins were subjected to detailed physicochemical characterisation to confirm their structural integrity and maintained IgE binding capacity. Detailed investigations of carbohydrate moieties of Mal d 2 demonstrated their involvement in the overall IgE binding capacity of this allergen. It was concluded that the folded structure and IgE binding capacity of all four allergens were preserved during purification.