IgE-binding Bands Research Articles

CHARACTERIZATION THE ALLERGENICITY AND STABILITY OF ALLERGENS OF FRESHWATER SNAIL, Pomacea canaliculata

Snail allergy is considered a serious form of food allergy with prevalence of 1.4 to 22% worldwide. However, allergy to Pomacea canaliculata, a commonly consumed local snail has not been well-described. Hence, this study aimed to characterize the allergenicity and stability of the allergenic proteins of P. canaliculata by proteomics approach. Snail flesh was treated with several thermal and non-thermal treatments prior to overnight protein extraction. The snail proteins were then subjected to SDS-PAGE, followed by immunoblotting, two-dimensional electrophoresis (2-DE), 2-DE immunoblotting and mass-spectrometry analysis. Raw snail demonstrated 31 protein bands between 10 to 250 kDa, with fewer protein bands in treated snails. Boiled snails had the most protein bands among thermally treated snails, while salted and dried snails showed more bands than pickled snails among non-thermal treatments. Immunoblotting of raw extract demonstrated 16 IgE-binding bands, with the 33 and 42 kDa protein bands were identified as the major. The 33 kDa allergen was highly stable to all treatments applied, while the 42 kDa was sensitive to thermal and pickling treatments. Fewer allergenic bands were present in treated snails, with allergenicity ranked as raw > boiled > roasted > fried for thermal treatments, and raw > salted > dried > pickled for non-thermal treatments. Mass spectrometry identified the 33 kDa and 42 kDa allergens as tropomyosin and actin, respectively. In conclusion, P. canaliculata has numerous allergenic proteins with varying stability.

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

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. 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. 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.

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.

Clinical Characteristics and Sensitization Profile of Patients Allergic to Cow Epithelium

Introduction: Cow epithelium allergy (CEA) has been described in workers highly exposed to cattle, such as farmers and veterinarians, being a health problem in this population since it is their main livelihood. This study aimed to characterize the main clinical manifestations and define the sensitization profile of the cow epithelium-allergic population treated in our health area. Methods: This is a retrospective study including a total of 34 patients with a clinical diagnosis of CEA, confirmed by skin tests, bovine epithelium-specific IgE levels and allergen-specific conjunctival challenge test in some cases. They were distributed by age, sex, profession, clinical symptoms, specific IgE levels to other mammalian epithelia, pollens, mites, and foods. Immunoblotting was performed with extracts from cow dander, cow body fluids (urine and saliva), bull urine, and 17 sera from immunotherapy-untreated CEA patients. Results: The mean age of the patients was 44 years, with a higher incidence in cattle farmers. Rhinoconjunctivitis occurred in 100% of cases, with 35% having monosensitization to cow epithelium. Sera from most patients detected a 20-kDa IgE-binding band in cow dander, cow saliva, cow urine, and bull urine, corresponding to the major allergen Bos d 2 (bovine lipocalin). In 70% of the patients, a 25-kDa band was detected in cow and bull urine extracts, whose identification by mass spectrometry and investigation with protein databases led to the identification of a Bos taurus lipocalin (UniProt protein ID: A0A3Q1LGU7_BOVIN). Conclusion: CEA should be considered in patients exposed to cattle and as a cause of occupational disease. The IgE immunodetection revealed sensitization to a protein present in cow and bull urine (odorant-binding protein) not previously described.

Allergen Profile of London Plane Tree Pollen: Clinical and Molecular Pattern in Central Spain.

Platanus acerifolia (London plane tree) is a deciduous tree of the Platanaceae family. Sensitization to this plant varies with geography. Madrid, located in central Spain, has one of the highest London plane tree pollen concentration levels on the Iberian Peninsula. We evaluated both the clinical characteristics and the molecular sensitization pattern of patients with allergy to London plane tree pollen in the region of Madrid. Thirty-eight patients allergic to London plane tree pollen were selected according to their clinical symptoms and positive results in skin prick testing and/or specific IgE determination. Serum was collected, and allergen components were evaluated using immunodetection techniques as well as ImmunoCAP. The IgE-binding proteins detected were identified and characterized using mass spectrometry. Analysis of serum samples from allergic patients revealed 9 IgE-binding bands in London plane tree pollen extract. Among these, the 45-kDa protein, which corresponded to Pla a 2, was detected in 76.3% of patients. However, the 18-kDa (Pla a 1) and 9-kDa (Pla a 3) bands were detected in 44.7% and 23.7% of sera, respectively. These results were confirmed using purified proteins. Characterization of the allergen revealed the 27-kDa protein to be glutathione-S-transferase. The molecular profile of patients sensitized to London plane tree pollen differs from that reported in studies from other locations. In the population we studied, the prevalence of Pla a 2 was higher than that of Pla a 1 and Pla a 3. In addition, the minor allergen previously referred to as Pla a 4 was characterized as glutathione-S-transferase.

Identification of Common and Novel Major Crab Allergens in Scylla tranquebarica and the Allergen Stability in Untreated and Vinegar-treated Crab.

Crab allergy is reported as a serious form of food allergy in many countries. This study was aimed to identify the major allergens of the local mud crab, Scylla tranquebarica (S. tranquebarica), and subsequently, determine the effect of vinegar treatments on the crab allergens. Crab muscles were treated with synthetic and natural vinegar. Crab proteins were then extracted from the untreated and vinegar-treated crabs. All extracts were then fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and analyzed by immunoblotting; using sera from crab-allergic patients. The crab proteins were then further fractionated by two-dimensional electrophoresis (2-DE)and analyzed by mass spectrometry (MS). The untreated crab had 38 protein bands, while that was only a few bands between 18 to 73 kDa for the vinegar-treated crabs. Immunoblotting of untreated crab revealed 20 IgE-binding bands, whereas the vinegar-treated crabs could only retain a few IgE-binding bands. Five major allergens were identified with molecular weightsof38, 42, 49, 63, and 73 kDa in the untreated crab. In contrast, the vinegar-treated crabs had only a few major allergens with molecular weights of 38, 42, and 73 kDa. MS identified the 43 and 49 kDa as arginine kinase, while the 38, 63, and 73 kDa were identified as tropomyosin, actin, and hemocyanin, respectively. Inconclusion, we found three common major allergens for S. tranquebarica including tropomyosin, arginine kinase, and actin, and one novel allergen known as hemocyanin. All the major allergens could retain minimal allergenic capability in vinegar-treated crabs, suggesting that vinegar treatments might be useful to reduce crab allergenicity. These data would assist the clinicians in the management of crab-allergic patients worldwide.

Occupational respiratory allergy to lettuce in lettuce farmers.

Lettuce-associated respiratory allergy has never been reported before. The aim of this study was to clarify the clinical condition of lettuce-associated respiratory allergy and to identify the lettuce antigen which induces allergic symptoms. We distributed questionnaires to 1168 lettuce farmers and performed medical examinations in those who exhibited respiratory symptoms related to occupational exposure to lettuce. We analysed specific IgE-binding proteins in the sera of patients through immunoblotting analysis and determined molecular characterization of the IgE-binding bands using liquid chromatography-mass spectrometry. A total of 932 farmers (80%) responded to the questionnaire. Of those, 7% exhibited lettuce-associated respiratory symptoms, during harvesting and packaging. Thirteen patients were diagnosed with allergy to lettuce and agreed to undergo further examinations. The percentage of activated basophils in these patients was significantly higher compared with that reported in negative controls (P<.05). Lettuce-specific IgE (ImmunoCAP® ) and skin prick testing was positive in 46% and 62% of patients, respectively. Notably, occupational lettuce-allergic asthma was detected in one patient through specific bronchial provocation testing. The IgE-binding bands recognized in the sera of >50% of patients were identified as epidermis-specific secreted glycoprotein EP1-like (51kDa). The present analysis identified a novel lettuce allergen. This allergen may have clinically useful applications, such as specific IgE testing and allergen-specific immunotherapy.

Pru p 9, a new allergen eliciting respiratory symptoms in subjects sensitized to peach tree pollen.

Peach tree (PT) pollen sensitization is highly prevalent in subjects living in areas where this tree is widely cultivated. None of the allergens responsible for these sensitizations have been identified so far. Our aim was to identify the most relevant PT pollen allergens and analyze their capacity for inducing respiratory symptoms. We studied sixty-two individuals sensitized to PT pollen who developed symptoms after its exposure. The IgE binding profile on peach pollen extract by means of immunoblotting using sera from these subjects was analyzed. Protein extract was fractionated by anion-exchange chromatography and HPLC, fractions run in SDS-PAGE and proteins were identified from IgE-binding bands by mass spectrometry. Several allergenic proteins in the PT pollen extract were recognized by patients' IgE: a glucan endo-1,3-beta-glucosidase-like, a polygalacturonase, an UTP-glucose-1-phosphate uridylyltransferase and a PR-1a protein. This PR-1a protein is a novel allergen frequently recognized with a molecular mass of 18 kDa, named as Pru p 9 following the WHO-IUIS nomenclature. Skin Prick Test (SPT) performed with this allergen was positive in 41% of the PT pollen-sensitized clinical cases. Most of them had rhinitis or rhinoconjunctivitis, but a significant percentage experienced asthma with seasonal symptoms during the period of PT flowering. Nasal Provocation test (NPT) with Pru p 9 was positive in all cases with positive SPT to this new allergen eliciting nasal symptoms similar to those challenged with PT pollen. We demonstrate that PT pollen can induce sensitization and allergy in an exposed population, being Pru p 9 a relevant allergen responsible of respiratory symptoms. Considering the extensive peach worldwide production with a large number of people involved, our results add a great value for the diagnosis and management of subjects allergic to this pollen.

Characterization of protein allergens in a mouse model of wheat-induced anaphylaxis

Abstract Wheat allergies are among the major types of food allergies that are potentially life-threatening because of anaphylaxis. Wheat contains 3 different types of protein allergens: water/saline-soluble albumins/globulins, alcohol-soluble gliadins and acid-soluble glutenins. Although gliadins have been linked to anaphylaxis, whether or not other proteins cause anaphylaxis is not well understood. Here, we sought to identify saline-soluble wheat proteins (SSWP) involved in anaphylaxis using a mouse model. Balb/cJ mice were sensitized to SSWP by four intraperitoneal (IP) injections along with alum as an adjuvant. Sensitization was confirmed by testing for SSWP-specific IgE using ELISA. Anaphylaxis was measured by hypothermia shock response. After multiple booster injections with SSWP, hyper-IgE plasma was collected and pooled to create a mini-plasma bank. Using hyper-IgE plasma, an IgE-Western blotting (WB) method was optimized. The IgE-binding proteins were identified by sequencing using LC/MS/MS method. The hyper-IgE plasma had an IgE titer of ~2560. The optimized IgE-WB included 3 overnight incubations followed by signal detection using an HRPO-based method. Sequencing of the IgE binding bands in WB identified the following proteins: globulin 3A (66 kDa), globulin 3B (57 kDa), serpin (43 kDa), glyceraldehyde-3-phosphate dehydrogenase (37 kDa), chitinase (28 kDa) and globulin 1 (25 kDa). This is the first study characterizing salt-soluble protein allergens in a mouse model of wheat-induced anaphylaxis. Funding: USDA, NIFA, Hatch project MICL02486 (Accession Number: 1012322), Agricultural and Food Research Initiative Competitive Program, grant number: 2018-67017-27876.

Chinese Birch Pollen Allergy and Immunotherapy in Mice

Birch pollen allergy is a common cause of spring pollinosis in China. However, there is little research on birch pollen allergen in China and only the major allergen (Bet v 1) has been fully characterized. Chinese birch pollen-induced airway inflammation models in BALB/c mice were developed and administered subcutaneous immunotherapy (SCIT). BALB/c mice were sensitized subcutaneously on days 1, 8, and 15 with 25 μg/μL birch pollen extract. On days 24–26, the mice were challenged with 0.1% birch pollen aerosol. To investigate the efficacy of SCIT, mice were subcutaneously injected 0.3 mg birch pollen extract (BPE) with or without being adsorbed to alum. Airway hyper-responsiveness (AHR) to methacholine and immunological parameters was detected. Western blot analysis was applied with mice serum and mass spectrometry was used to identify the IgE-binding bands in birch pollen. Compared with PBS group, birch pollen sensitization and challenge BALB/c mice developed AHR, and IL4, IL5, IL6, IL10, and IL17 were significantly higher. Mice sensitized by birch pollen showed increased plasma levels of anti-BPE IgE, IgG1, and IgG2a. Histologic analyses showed that mice had peribranchial infiltration of inflammatory cells and mucosal hyperplasia. After SCIT, allergic symptoms effectively alleviated and kept for a long time. Interestingly, mice serum pool showed strong reactions to 70-kDa proteins. Mass spectrometry data suggests that the 70-kDa protein belongs to the HSP 70 family. SCIT inhibited the inflammatory response in the long term and a 70-kDa protein potentially belonging to the HSP 70 family plays a significant role in Chinese birch pollen-induced mice model.

CHARACTERIZATION OF MAJOR ALLERGENS OF MACROBRACHIUM ROSENBERGII (GIANT RIVER PRAWN) BY ALLERGENOMIC APPROACH AND THEIR THERMOSTABILITY PROPERTIES

Prawn allergy is a common cause of allergic reactions in countries where crustacean is a famous seafood. Macrobrachium rosenbergii (giant river prawn) is declared as a local major food allergens, causes severe symptoms like asthma and anaphylactic shock. Therefore, the goal concerning this research is to identify the allergenicity of heat treated of M. rosenbergii. Prawn extracts have been prepared using fresh raw prawn and three heat-treated prawn flesh (boiled, steamed, fried), then afterward analyzed by the usage of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in conformity with their protein profiles. Allergenic proteins were identified by means of immunoblotting by the use of sera from 30 prawn-allergic patients. The identities of selective major allergens were then determined by mass-spectrometry analysis. The raw prawn has 27 protein fractions between 6 to 207 kDa, while the heat-treated prawns have reduced number of protein bands. Six prominent bands at 72, 65, 48, 38, 36, and 30 kDa were considered as the major allergens of raw extract. Meanwhile, after heat remedies applied, most of the IgE-binding bands were disappeared except for three major allergens at 38, 36 or 30 kDa which were still remain to be seen, suggesting as highly thermostable allergens. Among heat-treated prawns, steamed and boiled elicited more allergenic bands, contrast to fried prawn extract. Mass spectrometry analysis identified two selected major allergens at 36 and 48 kDa as tropomyosin and arginine kinase, respectively. As a conclusion, this study indicated that both thermostable and thermolabile proteins from M. rosenbergii were allergenic. Tropomyosin and arginine kinase were identified as the most important major allergens. Thus, the knowledge on thermostability of prawn allergens are crucial for improving diagnosis and management of prawn allergic patients in this county.

A 70-KD PROTEIN MAY PLAY A ROLE IN CHINESE BIRCH POLLEN ALLERGY

Introduction Birch pollen allergy is a common cause of spring pollinosis in China. Only the major allergen (Bet v 1) has been fully characterized and there is little research on birch pollen allergen in China. Methods Human serum was collected from patients with a documented clinical allergy in the Allergy Department. Birch pollen induced airway inflammation models in BALB/c mice were developed. Birch pollen extract was analyzed by SDS-PAGE. Western blot analysis was applied to both mice and human serum. Mass spectrometry was used to identify the IgE-binding bands in birch pollen. Results Chinese birch pollen extract (BPE) showed multiple protein bands clear at 17 kDa, 28 kDa, 36 kDa and 70 kDa. Patient serum exhibited IgE antibody binding against 17 kDa, 36 kDa and 70 kDa proteins, while healthy control serum reacted no bands. Interestingly, we established a birch pollen induced mice model. Compared with a PBS control group, birch pollen sensitized and challenged BALB/c mice developed airway responsiveness. In the bronchoalveolar lavage fluid (BALF), IL4, IL10 and IL17 were significantly higher. The levels of anti-BPE IgE, IgG1 and IgG2a also increased significantly. Histologic analyses showed that these mice had peribronchial infiltration of inflammatory cells and mucosal hyperplasia when challenged with BPE. Mice serum pool showed strong reactions to 17 kDa, 70 kDa and 100 kDa proteins. Mass spectrometry data suggests that the 70k Da protein may belong to the HSP 70 family. Conclusions A 70 kDa protein potentially belonging to the HSP 70 family plays a significant role in Chinese birch pollen allergy.

Towards complete identification of allergens in Jack Jumper (Myrmecia pilosula) ant venom and their clinical relevance: An immunoproteomic approach.

The venomous stings of Jack Jumper ant (JJA; species of the Myrmecia pilosula taxonomic group) are a significant public health issue in parts of south-eastern and south-western Australia, causing anaphylaxis in approximately 3% of the population. Three allergenic peptides, Myr p 1, Myr p 2 and Myr p 3, and one histamine-releasing peptide, pilosulin 5, have been fully described, but there are at least 5 additional high molecular weight IgE-binding components that have not been identified. To identify IgE-binding components in JJA venom (JJAV) and to relate the IgE recognition of these components to relevant clinical parameters. Identification of IgE-binding components and determination of their sensitizing prevalence was performed using SDS-PAGE immunoblot assay and sera from 90 patients with confirmed allergy to JJAV. Tandem mass spectrometry was used for identification of novel JJAV components fractionated by size exclusion chromatography (SEC) and SDS-PAGE. Using SDS-PAGE immunoblot, 10 IgE-binding bands were identified in JJAV, two of which were recognized by 81% and 47% of the population studied. Mass spectrometry identified 17 novel JJAV proteins, including 2 glycoproteins, and confirmed the presence of 4 known Myr p and pilosulin peptides in JJAV. Most of the newly identified IgE-binding proteins were enzymes, including phospholipase A2 , hyaluronidase, arginine kinase and dipeptidyl peptidase IV. Correlations were found between recognition of certain IgE-binding bands with JJAV-specific IgE titre by ImmunoCAP, intradermal test threshold and treatment-related issues. This study has for the first time revealed the identity of various proteins with IgE-binding capacity in the venom of JJA and demonstrated their clinical relevance in the diagnosis and treatment of JJAV allergy.

Pork‐cat syndrome?

Pork–cat syndrome was first described by Drouet et al, in 1994. It is a rare pathology, seen in patients sensitized to cat epithelium, which present symptoms suggestive of IgE-mediated hypersensitivity upon ingestion of pork meat. These symptoms range from urticaria with/without angioedema, to potentially fatal anaphylaxis. This rare syndrome is a result of a cross-reactivity of a protein of approximately 66kDa, identified as cat serum albumin (SA), which is highly homologous with porcine SA. In some patients, this cross-reactivity seems to extend to other mammals meat. In this study, we aim to present a case of a 76 year-old patient, just known to be sensitized to cat epithelium, that immediately after the intake of rice and beef, started with generalized urticaria and aqueous, limited and non hematic diarrhea. He was assisted at the emergency department, with an excellent response to oral corticosteroid and antihistamines, without any residual damage. After a two months period of totally avoiding red meat, the patient presented an anaphylatic shock, in an inadvertent ingestion of beef, pork and sausages at a barbecue. After being again assisted at the hospital he was referred for our immunoallergology department. We performed - (1) skin prick tests to aeroallergens and commercial food extracts: positive to milk, cat and dog epithelium; (2) laboratory tests: blood count (normal); total IgE (559KUA/L - Phadia ImmunoCAP®) and specific IgE for cat epithelium (4.82KUA/L), dog epithelium (1.04KUA/L), milk (2.72KUA/L), beef (8.6KUA/L), pork (6.67KUA/L) and for cat serum albumin that revealed to be also positive. It was carried out a cow-milk oral challenge test with a cumulative dose of 200ml that was negative. SDS PAGE studies IgE-immunoblotting were also performed using extracts of pork, cow and cat epithelium that showed a common IgE binding band with a molecular weight of 67kDa. This study describe a rare case of meat allergy with probably cross reactivity to a cat origin protein (primary sensitization), extended not only to pork meet but also to cow meat. This hypothesis is reinforced by the presence of a 67kDa protein which may correspond to the cat SA already described in the literature found in cow, pork and cat extracts. The patient is keeping red meat eviction diet (for 8 months now), and ever since, no reactions were reported.

Anaphylaxis caused by flaxseed

Methods and results Prick-prick carried out with flaxseed showed a strong positivity. Skin prick test to inhaled allergens were positive to mites, dog and cat danders, plain tree and grasses. Skin prick test to food allergens were positive to almond, hazelnut, peanut, chestnut, walnut, mustard, lentils, soybeans, rice, oat, corn and lupine. Total IgE was: 291 UI/ml. Specific IgE (CAP, ThermoFisher Scientific) to flaxseed was 2.68 kU peach 7.65 kU/L, peanut 2.01 kU / L , lettuce 0.92 kU / L , lupin 3.76 kU / L. Corn IgE 2.71 kU / L , Pru p3 7.62 kU / L , Cor a8 0.68 kU / L. ISAC, ImmunoCAP was positive to Fel d1, Phl p1, Der f1, Der f2, Der p1, Der p2, Pla a3, Ara h9, Cor a8, Jug r3, Pru p3 and Art v3. ISAC performed after inhibit patients’s serum (ISAC inhibition) with a complete flaxseed extract (f233:Linum usitatissium from ImmunoCAP, ThermoFisher Scientific) did not reduced the positivity to non-specific lipid transfer proteins (nsLTP). ISAC inhibition with a complete mite extract (d1: D. pteronyssinus from ImmunoCAP, ThermoFisher Scientific) reduces exclusively the signal of mites (Der f1, Der f2, Der p1, Der p2) in >83%.An immunoblot performed with flaxseed extracts (soluble and liposoluble), showed the presence of an IgE-binding band of about 18 kDa in the liposoluble fraction and a 20 kDa band in the soluble one.

In-Vitro Simulated Gastric Fluid Digestion and Immunogenicity of Different Crustacean Protein Extracts

To compare the immunogenicity and digestibility in-vitro simulated gastric fluid, the protein extracts of white-leg shrimp, Chinese shrimp (female and male shrimp were separated), Southern rough shrimp, Acetes chinensis, Mantis shrimp and crawfish were analyzed by SDS-PAGE, indirect ELISA and Western blotting. The results showed that except for Acetes chinensis, the larger proportion of molecular mass were near to 25 kDa (sarcoplasmic calcium-binding protein) and 38 kDa (tropomyosin). White-leg shrimp protein and Southern rough shrimp protein had a maximum similarity of 68.2%. White-leg shrimp protein had higher immunogenicity than others. There were much more IgE-binding bands in male Chinese shrimp protein, while Crawfish just had only one IgE-binding band (38 kDa, tropomyosin). In simulated gastric fluid system, the protein such as 25 kDa was rapidly degraded within 0.5 mins, while tropomyosin (38 kDa) was relatively resistant to pepsin digestion. The Western blotting results indicated that the allergenicity of those crustacean protein extracts still remained even they were digested with simulated gastric fluid for 60 min.

Molecular characterization of contact urticaria in patients with melon allergy

The relevance of contact allergy to plant-related food has recently emerged. Oral allergy syndrome is one of the most characteristic symptoms of fruit allergy, although it also causes systemic reactions. Plant-food allergy is increasing at the same time as pollen allergy, and fruit-induced allergic contact urticaria could be rising as well. The present study was carried out in order to investigate whether one particular primary melon-peel allergen is responsible for contact urticaria. Fourteen patients presenting with contact urticaria after touching melon peel were evaluated. A melon-peel extract was prepared and analysed by immunoblotting using the patients' sera. Molecular characterization of IgE-binding bands was performed using mass spectrometry. Melon-peel lipid transfer protein (LTP) was purified. Inhibition studies and contact challenge with the protein were performed to confirm IgE reactivity to the purified allergen. An IgE-binding band of ~8-9 kDa was observed in an immunoblotting assay with all the patients' sera and was identified as an LTP. The melon-peel LTP was purified in two chromatography steps. Inhibition studies confirmed LTP as a major allergen in patients with melon-peel contact urticaria. Contact challenge with melon-peel LTP was performed in five patients, all of whom had positive results, exhibiting itchy erythema and hives in the area of contact. This study confirmed our previous findings that melon-peel LTP is a major allergen and is responsible for contact allergy. This knowledge may be used to improve both diagnosis and treatment of patients allergic to melon.

Occupational Rhinoconjunctivitis due to Maize in a Snack Processor: A Cross-Reactivity Study Between Lipid Transfer Proteins From Different Cereals and Peach.

We report the case of a snack processor who developed occupational rhinoconjunctivitis due to maize brand exposure during the extrusion process, and who experienced abdominal pain upon drinking beer. The allergens implicated and the cross-reactivity between non-specific lipid transfer proteins (LTPs) from different cereals and peach were investigated. Skin prick tests and specific IgE to cereal flours, pulmonary functions tests and specific conjunctival and inhalation challenges to maize extract were performed. In vitro studies included IgE immunoblotting and ELISA inhibition assays. Skin prick tests with maize flour, maize brand and wheat flour extracts were positive, whereas serum specific IgE was positive only to maize flour. Specific inhalation challenge (SIC) to maize flour did not elicit an asthmatic reaction; however, conjunctival challenge test with the same extract was positive. Patient's serum recognized IgE-binding bands in the maize and beer extracts corresponding to LTPs. In the ELISA inhibition assays, a significant degree of allergenic cross-reactivity was found between maize and beer LTPs, whereas no cross-reactivity was observed between maize LTP and wheat and peach LTPs.

Identification of Parvalbumin and Two New Thermolabile Major Allergens of Thunnus tonggol Using a Proteomics Approach

Background: The longtail tuna (Thunnus tonggol) is widely consumed in Asia. Parvalbumin, the main major allergen of fish, has been well identified in multiple fish species, yet little is known about the allergenic proteins in T. tonggol. Thus, the aim of this study was to characterize the major allergens of T. tonggol using a proteomics approach. Methods: Raw and boiled extracts of the fish were prepared. Fish proteins were separated by means of SDS-PAGE and two-dimensional (2-DE) electrophoresis. 1-DE immunoblotting of raw extract was performed with sera from fish-allergic patients. Ten sera were further analysed by 2-DE immunoblotting. Selected major allergenic protein spots were excised, trypsin digested and analysed by means of mass spectrometry. Results: SDS-PAGE of raw extract revealed 26 protein fractions, while boiled extract demonstrated fewer bands. The 2-DE gel profile of the raw extract further fractionated the protein bands to more than 100 distinct protein spots. 1-DE immunoblotting of raw extract exhibited two thermolabile protein fractions of 42 and 51 kDa as the major allergens, while the boiled extract only revealed a single IgE-binding band at 151 kDa. 2-DE immunoblotting of raw extract further detected numerous major IgE-reactive spots of 11-13, 42 and 51 kDa. Mass spectrometry analysis of the peptides generated from the 12, 42 and 51 kDa digested spots indicated that these spots were parvalbumin, creatine kinase and enolase, respectively. Conclusions: In addition to parvalbumin, two new thermolabile allergens were identified as major allergenic proteins of T. tonggol. This study proved that both thermostable and thermolabile proteins are important in local tuna allergy and should be included in diagnostic strategies.

Identification of thaumatin-like protein and aspartyl protease as new major allergens in lettuce (Lactuca sativa).

Today, about 2-8% of the population of Western countries exhibits some type of food allergy whose impact ranges from localized symptoms confined to the oral mucosa to severe anaphylactic reactions. Consumed worldwide, lettuce is a Compositae family vegetable that can elicit allergic reactions. To date, however, only one lipid transfer protein has been described in allergic reaction to lettuce. The aim of this study was to identify potential new allergens involved in lettuce allergy. Sera from 42 Spanish lettuce-allergic patients were obtained from patients recruited at the outpatient clinic. IgE-binding proteins were detected by SDS-PAGE and immunoblotting. Molecular characterization of IgE-binding bands was performed by MS. Thaumatin was purified using the Agilent 3100 OFFGEL system. The IgE-binding bands recognized in the sera of more than 50% of patients were identified as lipid transfer protein (9 kDa), a thaumatin-like protein (26 kDa), and an aspartyl protease (35 and 45 kDa). ELISA inhibition studies were performed to confirm the IgE reactivity of the purified allergen. Two new major lettuce allergens-a thaumatin-like protein and an aspartyl protease-have been identified and characterized. These allergens may be used to improve both diagnosis and treatment of lettuce-allergic patients.