Variation in Shrimp Allergens: Place of Origin Effects on Food Safety Assessment.

Due to the widespread use of shellfish ingredients in food products, accurate food labelling is urgently needed for consumers with shellfish allergies. Most crustacean allergen detection systems target the immunorecognition of the allergenic protein tropomyosin. However, this mode of detection may be affected by an origin-dependent protein composition. This study determined if the geographic location of capture, or aquaculture, influenced the allergenic protein profiles of Black Tiger Shrimp (Penaeus monodon), one of the most farmed and consumed shrimp species worldwide. Protein composition was analysed in shrimp from nine different locations in the Asia-Pacific by SDS-PAGE, immunoblotting, and mass spectrometry. Ten of the twelve known shrimp allergens were detected, but with considerable differences between locations. Sarcoplasmic calcium-binding protein, myosin light chain, and tropomyosin were the most abundant allergens in all locations. Hemocyanin-specific antibodies could identify up to six different isoforms, depending on the location of origin. Similarly, tropomyosin abundance varied by up to 13 times between locations. These findings suggest that allergen abundance may be related to shrimp origin and, thus, shrimp origin might directly impact the readout of commercial crustacean allergen detection kits, most of which target tropomyosin, and this should be considered in food safety assessments.

Thermostable allergens in canned fish: Evaluating risks for fish allergy.

BACKGROUND: Major fish allergens, including parvalbumin (PV), are heat stable and can withstand extensive cooking processes. Thus, the management of fish allergy generally relies on complete avoidance. Fish-allergic patients may be advised to consume canned fish, as some fish-allergic individuals have reported tolerance to canned fish. However, the safety of consuming canned fish has not been evaluated with comprehensive immunological and molecular analysis of canned fish products. METHODS: We characterized the in vitro immunoreactivity of serum obtained from fish-allergic subjects to canned fish. Seventeen canned fish products (salmon n = 8; tuna n = 7; sardine n = 2) were assessed for the content and integrity of PV using allergen-specific antibodies. Subsequently, the sIgE binding of five selected products was evaluated for individual fish-allergic patients (n = 53). Finally, sIgE-binding proteins were identified by mass spectrometry. RESULTS: The canned fish showed a markedly reduced PV content and binding to PV-specific antibodies compared with conventionally cooked fish. However, PV and other heat-stable fish allergens, including tropomyosin and collagen, still maintained their sIgE-binding capacity. Of 53 patients, 66% showed sIgE binding to canned fish proteins. The canned sardine contained proteins bound to sIgE from 51% of patients, followed by canned salmon (43%-45%) and tuna (8%-17%). PV was the major allergen in canned salmon and sardine. Tropomyosin and/or collagen also showed sIgE binding. CONCLUSION: We showed that canned fish products may not be safe for all fish-allergic patients. Canned fish products should only be considered into the diet of individuals with fish allergy, after detailed evaluation which may include in vitro diagnostics to various heat-stable fish allergens and food challenge conducted in suitable environments.

The first reptilian allergen and major allergen for fish-allergic patients: Crocodile ß-parvalbumin.

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 ß-PV was the major IgE-binding protein but 20-fold less abundant than α-PV. Cellular reactivity was demonstrated for ß-PV and epitopes predicted, explaining frequent IgE-cross-binding of ß-PVs. Both PV isoforms are now registered as the first reptile allergens with the WHO/IUIS (ß-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.

Expanding the allergen repertoire of salmon and catfish.

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.

Novel Allergen Discovery through Comprehensive De Novo Transcriptomic Analyses of Five Shrimp Species.

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.

Commercial fish ELISA kits have a limited capacity to detect different fish species and their products.

BACKGROUND: Fish is a major food and allergen source, requiring safety declarations on packages. Enzyme-linked immunosorbent assays (ELISAs) are often used to ensure that the product meets the required standards with regard to the presence of allergens. Over 1000 different fish species are traded and consumed worldwide, and they are increasingly provided by aquaculture. Up to 3% of the general population is at risk of sometimes fatal allergic reactions to fish, requiring strict avoidance of this commodity. The aim of this study is to evaluate the capacity of three commercially available ELISA tests to detect a wide variety of bony and cartilaginous fish and their products, which is essential to ensure reliable and safe food labeling. RESULTS: The detection rates for 57 bony fish ranged from 26% to 61%. Common European and North American species, including carp, cod, and salmon species, demonstrated a higher detection rate than those from the Asia-Pacific region, including pangasius and several mackerel and tuna species. Among the 17 canned bony fish products, only 65% to 86% were detected, with tuna showing the lowest rate. None of the cartilaginous fish (n = 9), other vertebrates (n = 8), or shellfish (n = 5) were detected. CONCLUSIONS: We demonstrated that three commercial fish ELISA kits had a limited capacity to detect fish and their products. The complexity of fish as a protein source that is increasingly utilized means that there is an urgent need for improved detection methods. This is crucial for the food industry to provide safe seafood products and comply with international legislation. © 2020 Society of Chemical Industry.

Variability of allergens in commercial fish extracts for skin prick testing.

BACKGROUND: Commercial allergen extracts for allergy skin prick testing (SPT) are widely used for diagnosing fish allergy. However, there is currently no regulatory requirement for standardization of protein and allergen content, potentially impacting the diagnostic reliability of SPTs. We therefore sought to analyse commercial fish extracts for the presence and concentration of fish proteins and in vitro IgE reactivity using serum from fish-allergic patients. METHODS: Twenty-six commercial fish extracts from five different manufacturers were examined. The protein concentrations were determined, protein compositions analysed by mass spectrometry, followed by SDS-PAGE and subsequent immunoblotting with antibodies detecting 4 fish allergens (parvalbumin, tropomyosin, aldolase and collagen). IgE-reactive proteins were identified using serum from 16 children with confirmed IgE-mediated fish allergy, with focus on cod, tuna and salmon extracts. RESULTS: The total protein, allergen concentration and IgE reactivity of the commercial extracts varied over 10-fold between different manufacturers and fish species. The major fish allergen parvalbumin was not detected by immunoblotting in 6/26 extracts. In 7/12 extracts, five known fish allergens were detected by mass spectrometry. For cod and tuna, almost 70% of patients demonstrated the strongest IgE reactivity to collagen, tropomyosin, aldolase A or ß-enolase but not parvalbumin. CONCLUSIONS: Commercial fish extracts often contain insufficient amounts of important allergens including parvalbumin and collagen, resulting in low IgE reactivity. A comprehensive proteomic approach for the evaluation of SPT extracts for their utility in allergy diagnostics is presented. There is an urgent need for standardized allergen extracts, which will improve the diagnosis and management of fish allergy.

A cross-sectional, population-based study on the prevalence of food allergies among children in two different socio-economic regions of Vietnam.

BACKGROUND: There is a paucity of data on the prevalence of food allergy (FA) in Vietnam. A cross-sectional, population-based study was conducted to evaluate the current prevalence of FA among 2- to 6-year-old children in two different regions in Vietnam. METHOD: A structured, anonymous questionnaire, modified from published FA epidemiologic studies and based on EAACI guidelines, was distributed to parents/guardians of participating children in Hue City (urban area) and Tien Giang Province (rural area). Data collected from the survey were statistically analyzed to generate the prevalence of self-reported and doctor-diagnosed FA and overarching pattern of food allergens. RESULTS: A total of 8620 responses were collected (response rate 81.5%). Children in Tien Giang reported more than twice the food-induced adverse reactions seen in children in Hue (47.8% vs. 20.5%). In contrast, children in Hue showed higher self-reported FA (9.8%) and doctor-diagnosed FA rates (8.4%) than children in Tien Giang (7.9% and 5.0%, respectively). Crustacean was the predominant allergy-inducing food in both areas (330 of 580 cases, 56.9%), followed by fish, mollusk, beef, milk, and egg. However, substantial variations of FA patterns were seen between the study sites. Geographic location and co-morbidities of other allergic diseases were key risk factors for FA (P < 0.001). CONCLUSIONS: The prevalence of FA in Vietnamese children seems to be higher than previously reported from other Asian countries. Crustacean is the predominant allergy-inducing food among participating preschool children in Vietnam. The variation of reported food allergen sources across different socio-economic locations could imply different eating habits or the participation of indoor and outdoor allergen exposure.

Techno-functional properties and allergenicity of mung bean (Vigna radiata) protein isolates from Imara and KPS2 varieties.

Mung bean is an increasingly cultivated legume. This study compared mung bean varieties 'KPS2' from Thailand (Th) and 'Imara' from Tanzania (T) with a focus on protein composition, allergenicity, and techno-functional properties. Two rounds alkaline-acid extraction were performed to produce mung bean protein isolate (MBPI - Th1/T1 and Th2/T2), supernatant (S) and protein-poor residue (PPR). Mass spectrometric analysis revealed high abundance of 8 s-vicilin and 11 s-legumin in MBPI and S. Extraction removed considerable amounts of the seed albumin allergen but increased the relative abundance of cupins in MBPI. Higher vicilin levels were found in Th1 samples, contributed to increased protein solubility above pH 6.5. Th formed stronger gels which were more stable at higher frequencies. In contrast, T proteins were structurally more flexible, leading to its improved foaming ability. This study provides the knowledge and methods for appropriate selection of mung bean varieties for various food applications.

Allergen Diversity and Abundance in Different Tissues of the Redclaw Crayfish (Cherax quadricarinatus).

Shellfish allergy affects ~2.5% of the global population and is a type I immune response resulting from exposure to crustacean and/or molluscan proteins. The Australian Redclaw crayfish (Cherax quadricarinatus) is a freshwater species endemic to and farmed in northern Australia and is becoming an aquaculture species of interest globally. Despite being consumed as food, allergenic proteins from redclaw have not been identified or characterised. In addition, as different body parts are often consumed, it is conceivable that redclaw tissues vary in allergenicity depending on tissue type and function. To better understand food-derived allergenicity, this study characterised allergenic proteins in various redclaw body tissues (the tail, claw, and cephalothorax) and how the stability of allergenic proteins was affected through cooking (raw vs. cooked tissues). The potential of redclaw allergens to cross-react and cause IgE-binding in patients allergic to other shellfish (i.e., shrimp) was also investigated. Raw and cooked extracts were prepared from each body part. SDS-PAGE followed by immunoblotting was performed to determine allergen-specific antibody reactivity to sarcoplasmic calcium-binding protein and hemocyanin, as well as to identify redclaw proteins binding to IgE antibodies from individual and pooled sera of shrimp-allergic patients. Liquid chromatography-mass spectrometry (LC/MS) was utilised to identify proteins and to determine the proportion within extracts. Known crustacean allergens were found in all tissues, with a variation in tissue distribution (e.g., higher levels of hemocyanin in the claw and cephalothorax than in the tail). The proportion of some allergens as a percentage of remaining heat-stable proteins increased in cooked tissues. Previously described heat-stable allergens (i.e., hemocyanin and sarcoplasmic calcium-binding protein) were found to be partially heat-labile. Immunoblotting indicated that shrimp-allergic patients cross-react to redclaw allergens. IgE-binding bands, analysed by LC/MS, identified up to 11 known shellfish allergens. The findings of this study provide fundamental knowledge into the diagnostic and therapeutic field of shellfish allergy.

The Allergen Profile of Two Edible Insect Species-Acheta domesticus and Hermetia illucens.

SCOPE: Edible insect proteins are increasingly introduced as an alternative sustainable food source to address the world's need to feed the growing population. Tropomyosin is the main insect allergen; however, additional potential allergens are not well characterized and the impact of extraction procedures on immunological reactivity is unknown. METHODS AND RESULTS: Proteins from different commercial food products derived from cricket (Acheta domesticus) and black soldier fly (BSF) (Hermetia illucens) are extracted using five different extraction buffers. The proteins are analyzed by SDS-PAGE and immunoblotting using allergen-specific antibodies and crustacean allergic patient sera. IgE binding bands are analyzed by mass spectrometry as well as the complete allergen profile of all 30 extracts. Urea-based buffers are most efficient in extracting insect allergens. Shrimp-specific antibody cross-reactivity to tropomyosin from cricket and BSF indicates high sequence and structural similarity between shrimp and insects. Additional unique allergens are identified in both species, including hemocyanin, vitellogenin, HSP20, apolipophorin-III, and chitin-binding protein. CONCLUSIONS: Identifying potential allergenic proteins and their isoforms in cricket and BSF requires specific extraction approaches using urea-based methods. While tropomyosin is the most abundant and immunoreactive allergen, seven unique allergens are identified, highlighting the need for insect species-specific allergen detection in food products.

Recombinant Tropomyosin from the Pacific Oyster (Crassostrea gigas) for Better Diagnosis.

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.

IgE and IgG4 epitopes revealed on the major fish allergen Lat c 1.

BACKGROUND: The IgE- and IgG4-binding patterns of the major fish allergen parvalbumins are not clearly understood. IgE antibody-binding to parvalbumin from Asian seabass, Lat c 1.01, is implicated in up to 90 % of allergic reactions, although the region of IgE or IgG4 epitopes are unknown. In the present study, we characterized the specific IgE- and IgG4-binding regions of Lat c 1.01 using serum from pediatric and adult patients with clinically-confirmed fish allergy. METHODS: A comparative investigation of patient IgE- and IgG4-binding to recombinant Lat c 1.01 was performed by immunoblotting and indirect ELISA using serum from 15 children and eight adults with clinically confirmed IgE-mediated reactions to fish. The IgE- and IgG4-binding regions of Lat c 1.01 were determined by inhibition ELISA using seven overlapping peptides spanning the entire 102 amino acid sequence. Elucidated IgE-binding regions were modelled and compared to known antibody-binding regions of parvalbumins from five other fish species. RESULTS: Ninety five percent (22/23) patients demonstrated IgE-binding to rLat c 1.01, while fewer patients (10/15 children and 7/8 adults) demonstrated robust IgG4 binding when determined by immunoblots. IgE-binding for both cohorts was significantly higher compared to IgG4-binding by ELISA. All patients in this study presented individual IgE and IgG4 epitope-recognition profiles. In addition to these patient-specific antibody binding sites, general IgE epitopes were also identified at the C- and N-terminal regions of this major fish allergen. CONCLUSIONS AND CLINICAL RELEVANCE: Our findings demonstrate two specific IgE epitopes on parvalbumin from Asian seabass, while IgG4 binding is much lower and patient specific. This study highlights the importance of advancement in epitope analysis regardless of the age group for diagnostics and immunotherapies for fish allergy.

Effects of Extraction Buffer on the Solubility and Immunoreactivity of the Pacific Oyster Allergens.

Despite recent technological advances, novel allergenic protein discovery is limited by their low abundance, often due to specific physical characteristics restricting their recovery during the extraction process from various allergen sources. In this study, eight different extraction buffers were compared for their ability to recover proteins from Pacific oyster (Crassostrea gigas). The protein composition was investigated using high resolution mass spectrometry. The antibody IgE-reactivity of each extract was determined using a pool of serum from five shellfish-allergic patients. Most of the investigated buffers showed good capacity to extract proteins from the Pacific oyster. In general, a higher concentration of proteins was recovered using high salt buffers or high pH buffers, subsequently revealing more IgE-reactive bands on immunoblotting. In contrast, low pH buffers resulted in a poor protein recovery and reduced IgE-reactivity. Discovery of additional IgE-reactive proteins in high salt buffers or high pH buffers was associated with an increase in allergen abundance in the extracts. In conclusion, increasing the ionic strength and pH of the buffer improves the solubility of allergenic proteins during the extraction process for oyster tissue. This strategy could also be applied for other difficult-to-extract allergen sources, thereby yielding an improved allergen panel for increased diagnostic efficiency.

Exposure to Bioaerosols During Fish Processing on Board Norwegian Fishing Trawlers.

OBJECTIVES: The main objective was to gain more knowledge on exposure to bioaerosols in the processing area on board fishing trawlers. METHODS: Exposure sampling was carried out during the work shifts when processing fish in the processing area on board five deep-sea fishing trawlers (trawlers 1-5). Exposure samples were collected from 64 fishermen breathing zone and from stationary sampling stations on board five deep-sea fishing trawlers (1-5). Trawlers 2, 3, and 4 were old ships, not originally built for on board processing of the catch. Trawlers 1 and 5 were relatively new and built to accommodate processing machineries. On trawlers 1-4 round fish was produced; the head and entrails were removed before the fishes were frozen in blocks. Trawler 5 had the most extensive processing, producing fish fillets. Samples were analysed for total protein, trypsin activity, parvalbumin, and endotoxin. One side analysis of variance and Kruskal-Wallis H test were used to compare levels of exposure on the different trawlers. RESULTS: Personal exposure to total protein were higher on the three oldest trawlers (2, 3, and 4) compared with the two new trawlers (1 and 5). Highest activity of trypsin was detected on the four trawlers producing round fish (1-4). Parvalbumin was detected in 58% of samples from the fillet-trawler (5) compared with 13% of samples from the four trawlers producing round fish. The highest level of endotoxin was detected when using high-pressure water during cleaning machines and floors in the processing area. CONCLUSIONS: Fishermen in the processing area on board Norwegian trawlers are exposed to airborne bioaerosols as proteins, trypsin, fish allergen parvalbumin, and endotoxin. Levels varied between trawlers and type of production.

Undeclared allergens in imported packaged food for retail in Australia.

The Australia New Zealand Food Standards Code (the Code) requires a declaration of the presence of 11 different allergens made through the label on a food product. Most food recalls in Australia are now due to undeclared allergens . This survey determined the extent of undeclared allergens in imported food products on the Asian retail market in Australia. A total of 50 imported packaged foods were selectively purchased from local Asian grocery retail stores in Melbourne and the presence of undeclared gluten, milk, peanut and egg determined. Analysis was performed using commercial enzyme-linked immunosorbent assay (ELISA) (R-Biopharm). Thirty-seven undeclared allergens (gluten n = 12, milk n = 12, peanut n = 6, and egg n = 7) were detected in 23 of the 50 products analysed (46%), with 18% containing multiple undeclared allergens. The high number of undeclared allergens is alarming and in line with the increasing number of food recalls and anaphylaxis recorded in Australia.

Collagen-An Important Fish Allergen for Improved Diagnosis.

BACKGROUND: Fish collagen is widely used in medicine, cosmetics, and the food industry. However, its clinical relevance as an allergen is not fully appreciated. This is likely due to collagen insolubility in neutral aqueous solutions, leading to low abundance in commercially available in vitro and skin prick tests for fish allergy. OBJECTIVE: To investigate the relevance of fish collagen as an allergen in a large patient population (n = 101). METHODS: Acid-soluble collagen type I was extracted from muscle and skin of Atlantic salmon, barramundi, and yellowfin tuna. IgE binding to collagen was analyzed by ELISA for 101 fish-allergic patients. Collagen-sensitized patients' sera were tested for IgE binding to parvalbumin from the same fish species. IgE cross-linking was analyzed by rat basophil leukemia assay and basophil activation test. Protein identities were confirmed by mass spectrometry. RESULTS: Purified fish collagen contained type I α1 and α2 chains and their multimers. Twenty-one of 101 patients (21%) were sensitized to collagen. Eight collagen-sensitized patients demonstrated absence of parvalbumin-specific IgE to some fish species. Collagen induced functional IgE cross-linking, as shown by rat basophil leukemia assay performed using 6 patients' sera, and basophil activation test using fresh blood from 1 patient. Collagen type I α chains from barramundi and Atlantic salmon were registered at www.allergen.org as Lat c 6 and Sal s 6, respectively. CONCLUSIONS: IgE sensitization and IgE cross-linking capacity of fish collagen were demonstrated in fish-allergic patients. Inclusion of relevant collagen allergens in routine diagnosis is indicated to improve the capacity to accurately diagnose fish allergy.

Conservation Analysis of B-Cell Allergen Epitopes to Predict Clinical Cross-Reactivity Between Shellfish and Inhalant Invertebrate Allergens.

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.

Fish-derived low molecular weight components modify bronchial epithelial barrier properties and release of pro-inflammatory cytokines.

The prevalence of fish allergy among fish-processing workers is higher than in the general population, possibly due to sensitization via inhalation and higher exposure. However, the response of the bronchial epithelium to fish allergens has never been explored. Parvalbumins (PVs) from bony fish are major sensitizers in fish allergy, while cartilaginous fish and their PVs are considered less allergenic. Increasing evidence demonstrates that components other than proteins from the allergen source, such as low molecular weight components smaller than 3 kDa (LMC) from pollen, may act as adjuvants during allergic sensitization. We investigated the response of bronchial epithelial cells to PVs and to LMC from Atlantic cod, a bony fish, and gummy shark, a cartilaginous fish. Polarized monolayers of the bronchial epithelial cell line 16HBE14o- were stimulated apically with fish PVs and/-or the corresponding fish LMC. Barrier integrity, transport of PVs across the monolayers and release of mediators were monitored. Intact PVs from both the bony and the cartilaginous fish were rapidly internalized by the cells and transported to the basolateral side of the monolayers. The PVs did not disrupt the epithelial barrier integrity nor did they modify the release of proinflammatory cytokines. In contrast, LMC from both fish species modified the physical and immunological properties of the epithelial barrier and the responses differed between bony and cartilaginous fish. While the barrier integrity was lowered by cod LMC 24 h after cell stimulation, it was increased by up to 2.3-fold by shark LMC. Furthermore, LMC from both fish species increased basolateral and apical release of IL-6 and IL-8, while CCL2 release was increased by cod but not by shark LMC. In summary, our study demonstrated the rapid transport of PVs across the epithelium which may result in their availability to antigen presenting cells required for allergic sensitization. Moreover, different cell responses to LMC derived from bony versus cartilaginous fish were observed, which may play a role in different allergenic potentials of these two fish classes.