Background Diagnostic tests for fish allergy are hampered by the large number of under‐investigated fish species. Four salmon allergens are well‐characterized and registered with the WHO/IUIS while no catfish allergens have been described so far. In 2008, freshwater‐cultured catfish production surpassed that of salmon, the globally most‐cultured marine species. We aimed to identify, quantify, and compare all IgE‐binding proteins in salmon and catfish. Methods Seventy‐seven pediatric patients with clinically confirmed fish allergy underwent skin prick tests to salmon and catfish. The allergen repertoire of raw and heated protein extracts was evaluated by immunoblotting using five allergen‐specific antibodies and patients' serum followed by mass spectrometric analyses. Results Raw and heated extracts from catfish displayed a higher frequency of IgE‐binding compared to those from salmon (77% vs 70% and 64% vs 53%, respectively). The major fish allergen parvalbumin demonstrated the highest IgE‐binding capacity (10%‐49%), followed by triosephosphate isomerase (TPI; 19%‐34%) in raw and tropomyosin (6%‐32%) in heated extracts. Six previously unidentified fish allergens, including TPI, were registered with the WHO/IUIS. Creatine kinase from salmon and catfish was detected by IgE from 14% and 10% of patients, respectively. Catfish L‐lactate dehydrogenase, glyceraldehyde‐3‐phosphate dehydrogenase, pyruvate kinase, and glucose‐6‐phosphate isomerase showed IgE‐binding for 6%‐13% of patients. In salmon, these proteins could not be separated successfully. Conclusions We detail the allergen repertoire of two highly farmed fish species. IgE‐binding to fish tropomyosins and TPIs was demonstrated for the first time in a large patient cohort. Tropomyosins, in addition to parvalbumins, should be considered for urgently needed improved fish allergy diagnostics.
Understanding and predicting an individual's clinical cross-reactivity to related allergens is a key to better management, treatment and progression of novel therapeutics for food allergy. In food allergy, clinical cross-reactivity is observed in patients reacting to unexpected allergen sources containing the same allergenic protein or antibody binding patches (epitopes), often resulting in severe allergic reactions. Shellfish allergy affects up to 2% of the world population and persists for life in most patients. The diagnosis of shellfish allergy is however often challenging due to reported clinical cross-reactivity to other invertebrates including mites and cockroaches. Prediction of cross-reactivity can be achieved utilizing an in-depth analysis of a few selected IgE-antibody binding epitopes. We combined available experimentally proven IgE-binding epitopes with informatics-based cross-reactivity prediction modeling to assist in the identification of clinical cross-reactive biomarkers on shellfish allergens. This knowledge can be translated into prevention and treatment of allergic diseases. To overcome the problem of predicting IgE cross-reactivity of shellfish allergens we developed an epitope conservation model using IgE binding epitopes available in the Immune Epitope Database and Analysis Resource (http://www.iedb.org/). We applied this method to a set of four different shrimp allergens, and successfully identified several non-cross-reactive as well as cross-reactive epitopes, which have been experimentally established to cross-react. Based on these findings we suggest that this method can be used for advanced component-resolved-diagnosis to identify patients sensitized to a specific shellfish group and distinguish from patients with extensive cross-reactivity to ingested and inhaled allergens from invertebrate sources.
Shellfish allergy affects 2% of the world’s population and persists for life in most patients. The diagnosis of shellfish allergy, in particular shrimp, is challenging due to the similarity of allergenic proteins from other invertebrates. Despite the clinical importance of immunological cross-reactivity among shellfish species and between allergenic invertebrates such as dust mites, the underlying molecular basis is not well understood. Here we mine the complete transcriptome of five frequently consumed shrimp species to identify and compare allergens with all known allergen sources. The transcriptomes were assembled de novo, using Trinity, from raw RNA-Seq data of the whiteleg shrimp (Litopenaeus vannamei), black tiger shrimp (Penaeus monodon), banana shrimp (Fenneropenaeus merguiensis), king shrimp (Melicertus latisulcatus), and endeavour shrimp (Metapenaeus endeavouri). BLAST searching using the two major allergen databases, WHO/IUIS Allergen Nomenclature and AllergenOnline, successfully identified all seven known crustacean allergens. The analyses revealed up to 39 unreported allergens in the different shrimp species, including heat shock protein (HSP), alpha-tubulin, chymotrypsin, cyclophilin, beta-enolase, aldolase A, and glyceraldehyde-3-phosphate dehydrogenase (G3PD). Multiple sequence alignment (Clustal Omega) demonstrated high homology with allergens from other invertebrates including mites and cockroaches. This first transcriptomic analyses of allergens in a major food source provides a valuable resource for investigating shellfish allergens, comparing invertebrate allergens and future development of improved diagnostics for food allergy.
The Pacific oyster is a commercially important mollusc and, in contrast to most other shellfish species, frequently consumed without prior heat treatment. Oysters are rich in many nutrients but can also cause food allergy. Knowledge of their allergens and cross-reactivity remains very limited. These limitations make an optimal diagnosis of oyster allergy difficult, in particular to the Pacific oyster (Crassostrea gigas), the most cultivated and consumed oyster species worldwide. This study aimed to characterise IgE sensitisation profiles of 21 oyster-sensitised patients to raw and heated Pacific oyster extract using immunoblotting and advanced mass spectrometry, and to assess the relevance of recombinant oyster allergen for improved diagnosis. Tropomyosin was identified as the major allergen recognised by IgE from 18 of 21 oyster-sensitised patients and has been registered with the WHO/IUIS as the first oyster allergen (Cra g 1). The IgE-binding capacity of oyster-sensitised patients’ IgE to purified natural and recombinant tropomyosin from oyster, prawn, and dust mite was compared using enzyme-linked immunosorbent assay. The degree of IgE binding varied between patients, indicating partial cross-sensitisation and/or co-sensitisation. Amino acid sequence alignment of tropomyosin from these three species revealed five regions that contain predicted IgE-binding epitopes, which are most likely responsible for this cross-reactivity. This study fully biochemically characterises the first and major oyster allergen Cra g 1 and demonstrates that the corresponding recombinant tropomyosin should be implemented in improved component-resolved diagnostics and guide future immunotherapy.
Background Clinical cross‐reactivity between bony fish, cartilaginous fish, frog, and chicken muscle has previously been demonstrated in fish‐allergic patients. In indicative studies, two reports of anaphylaxis following the consumption of crocodile meat and IgE‐cross‐binding were linked to the major fish allergen parvalbumin (PV). This study investigates IgE‐binding proteins in crocodile meat with a focus on PV and their clinical relevance. Methods Proteins were extracted from muscle tissue of crocodile, three bony fish, and two cartilaginous fish. A cohort of fish‐allergic pediatric patients (n = 77) underwent allergen skin prick testing (SPT) to three fish preparations (n = 77) and crocodile (n = 12). IgE‐binding proteins were identified and quantified by SDS‐PAGE, mass spectrometric analyses, and immunoblotting using commercial and in‐house antibodies, as well as individual and pooled patients’ serum. PV isoforms were purified or recombinantly expressed before immunological analyses, including human mast cell degranulation assay. Results Of the tissues analyzed, PV was most abundant in heated crocodile preparation, triggering an SPT of ≥3 mm in 8 of 12 (67%) fish‐allergic patients. Seventy percent (31 of 44) of fish PV‐sensitized patients demonstrated IgE‐binding to crocodile PV. Crocodile β‐PV was the major IgE‐binding protein but 20‐fold less abundant than α‐PV. Cellular reactivity was demonstrated for β‐PV and epitopes predicted, explaining frequent IgE‐cross‐binding of β‐PVs. Both PV isoforms are now registered as the first reptile allergens with the WHO/IUIS (β‐PV as Cro p 1 and α‐PV as Cro p 2). Conclusion Fish‐allergic individuals may be at risk of an allergy to crocodile and should seek specialist advice before consuming crocodilian meat.
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