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Sample records for major buckwheat allergen

  1. Allergenic potential and enzymatic resistance of buckwheat

    PubMed Central

    Lee, Sujin; Han, Youngshin; Do, Jeong-Ryong

    2013-01-01

    Buckwheat is known as a health food but is one of the major food allergens triggering potentially fatal anaphylaxis in Asia, especially in Japan and Korea. This study was conducted to investigate the characteristic of enzymatic resistance of buckwheat protein and allergenic potential. Enzymatic resistance of buckwheat protein was performed with in vitro digestibility test in simulated gastric fluid (SGF), pH 1.2, using pepsin and simulated intestinal fluid (SIF) using chymotrypsin. Reactivity of buckwheat proteins to human IgE was performed using six allergic patients sensitized to buckwheat. Buckwheat's IgE levels were measured using the Phadia UniCAP-system. Buckwheat protein, 16 kDa, still remained after 30 min treatment of pepsin on SDS-PAGE. Even though 16 kDa almost disappeared after 60 min treatment, two out of the six buckwheat patients' sera showed reactivity to hydrolysate after 60 min treatment, indicating that allergenicity still remained. In simulated intestinal fluid (SIF) using chymotrypsin, buckwheat protein, 24 kDa, showed resistance to hydrolysis with chymotrypsin on SDS-PAGE, and still had allergenicity based on the result of ELISA. Our results suggest that buckwheat proteins have strong resistance to enzyme degradation. This may be attributed in part to the allergenic potential of buckwheat. Further study should be continued regarding buckwheat allergy. PMID:23423876

  2. Expression of the immunoreactive buckwheat major allergenic storage protein in Lactococcus lactis.

    PubMed

    Shigemori, Suguru; Yonekura, Shinichi; Sato, Takashi; Otani, Hajime; Shimosato, Takeshi

    2013-04-01

    Proteins from buckwheat (Fagopyrum esculentum) are strong allergens that can cause serious symptoms, including anaphylaxis, in patients with hypersensitivity. In this study, we successfully developed a modified lactic acid bacterial vector (pNSH) and a recombinant strain of Lactococcus lactis NZ9000 (NZ9000) that produced a major allergenic storage protein of buckwheat, Fagag1 (61.2 kDa, GenBank accession number AF152003), with or without a green fluorescent protein (GFP) tag. GFP fluorescence allows for rapid, simple, and accurate measurement of target protein expression by microscopy or fluorimetry. We describe a convenient method for production of rGFP-Fagag1 fusion and rFagag1 proteins with a good yield in an advantageous probiotic host. We found that in vitro treatment of splenocytes isolated from buckwheat crude protein-immunized mice with rFagag1 increased the expression of allergic inflammation cytokines such as IL-4, IL-13, and IL-17 F. Because it was less antigenic, rGFP-Fagag1 protein from NZ9000 might be of limited use; however, rFagag1 from NZ9000 evoked a robust response as measured by induction of IL-4 and IL-17 F expression levels. The observed allergic activity is indicative of a Th2 cell-mediated immune response and is similar to the effects induced by exposure to buckwheat crude protein. Our results suggest that expression of rFagag1 in NZ9000 may facilitate in vivo applications of this system aimed at improving the specificity of immunological responses to buckwheat allergens.

  3. Buckwheat anaphylaxis: an unusual allergen in Taiwan.

    PubMed

    Wang, Tsung-Chi; Shyur, Shyh-Dar; Wen, Da-Chin; Kao, Yu-Hsuan; Huang, Li-Hsin

    2006-01-01

    IgE-mediated hypersensitivity to buckwheat is common in Korea, Japan, and some other Asian countries. However, buckwheat is not a common allergen in Taiwan. We report a woman with asthma who had anaphylactic shock, generalized urticaria, and an acute exacerbation of asthma five minutes after ingesting buckwheat. The patient underwent skin prick and Pharmacia CAP testing (Uppsala, Sweden) for specific IgE to buckwheat, white sesame and soybean as well as other common allergens in Taiwan including Dermatophagoides pteronyssinus (Dp), D. farinae (Df), cat and dog dander, cockroach, egg white, cow milk and codfish. The patient had a strongly positive skin prick test response to buckwheat and positive reactions to Dp and latex. Specific IgE results were class 6 for buckwheat, class 4 for Dp and Df, and class 2 for dog dander, wheat, sesame and soybean. Results of an open food challenge with white sesame and soybean were negative. Although buckwheat is a rare allergen in Taiwan, it can cause extremely serious reactions and should be considered in patients presenting with anaphylaxis after exposure to buckwheat.

  4. An autoclave treatment reduces the solubility and antigenicity of an allergenic protein found in buckwheat flour.

    PubMed

    Tomotake, Hiroyuki; Yamazaki, Rikio; Yamato, Masayuki

    2012-06-01

    The effects of an autoclave treatment of buckwheat flour on a 24-kDa allergenic protein were investigated by measuring reduction in solubility and antibody binding. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that the intensity of the major bands, including that of the 24-kDa allergen, was reduced by the autoclave treatment. The protein solubility in buckwheat flour was variably decreased by the autoclave treatment. Enzyme-linked immunosorbent assay analysis using a monoclonal antibody specific for buckwheat 24-kDa protein showed that the reactivity of protein extracts (10 μg/ml) from buckwheat flour was lowered by the autoclave treatment. The autoclave treatment may reduce the major allergen content of buckwheat. Future studies will determine if autoclaving treatments affect the allergenicity of the 24-kDa buckwheat protein.

  5. Effects of Maillard reaction on allergenicity of buckwheat allergen Fag t 3 during thermal processing.

    PubMed

    Yang, Zhen-Huang; Li, Chen; Li, Yu-Ying; Wang, Zhuan-Hua

    2013-04-01

    Fag t 3 is a major allergenic protein in tartary buckwheat. The Maillard reaction commonly occurs in food processing, but few studies have been conducted on the influence of thermal processing on the allergenic potential of buckwheat allergen. The aim of the present study was to investigate the effects of autologous plant polysaccharides on the immunoreactivity of buckwheat Fag t 3 (11S globulin) following the Maillard reaction. Fag t 3 and crude polysaccharides were prepared from tartary buckwheat (Fagopyrum tataricum) flour. After heating, the polysaccharides were covalently linked to Fag t 3 via a Maillard reaction, and the IgE/IgG-binding properties of Fag t 3 decreased dramatically, with significant changes also being observed in the electrophoretic mobility, secondary structure and solubility of the glycated Fag t 3. The great influence of glycation on IgE/IgG binding to Fag t 3 was correlated with a significant change in the structure and epitopes of the allergenic protein. These data indicated that conjugation of polysaccharides to Fag t 3 markedly reduced the allergen's immunoreactivity. Glycation that occurs via the Maillard reaction during the processing of buckwheat food may be an efficient method to reduce Fag t 3 allergenicity. © 2012 Society of Chemical Industry.

  6. Monoclonal Antibodies to Recombinant Fag e 3 Buckwheat Allergen and Development of a Two-site ELISA for Its Quantification.

    PubMed

    Jeong, Kyoung Yong; Park, Kyung Hee; Lee, Jae Hyun; Park, Jung Won

    2017-09-01

    Buckwheat is a major cause of anaphylaxis, and Fag e 3 is the key major allergen in buckwheat. However, an immunoassay system for the quantification of Fag e 3 has yet to be developed. We developed a 2-site enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies (mAbs) produced against recombinant Fag e 3. We applied this ELISA to quantify native Fag e 3 in total buckwheat extract. Four clones of mAbs were produced, and all recognized vicilin allergens not only from buckwheat, but also from peanut and walnut. However, the ELISA using these antibodies was only able to quantify Fag e 3 in the total extract after addition of 1% sodium dodecyl sulphate (SDS) and heating, which facilitated dissociation of the allergen. The detection limit of the developed 2-site ELISA was 0.8 μg/mL. The measurement of Fag e 3 in the total extract of buckwheat showed that approximately 12% of protein in total buckwheat extract was Fag e 3. We have developed an ELISA system for the quantification of the group 3 buckwheat allergen, Fag e 3, specifically. This assay will be useful for standardization of buckwheat allergens and monitoring of buckwheat contamination in foods.

  7. Anaphylaxis after eating Italian pizza containing buckwheat as the hidden food allergen.

    PubMed

    Heffler, E; Guida, G; Badiu, I; Nebiolo, F; Rolla, G

    2007-01-01

    A 20-year-old woman developed anaphylaxis after eating pizza on 4 different occasions in 2 restaurants. Both restaurants made their pizza dough with a mixture of wheat and buckwheat flours. A prick-to-prick test with buckwheat flour was positive. Skin prick tests and specific immunoglobulin E responses to soybean and peanut were weakly positive while the response to buckwheat was negative. We ruled out a pathogenic role for peanut and soybean because the patient usually eats both with no signs of allergic reaction. Double-blind, placebo-controlled food challenges with buckwheat flour were positive after the administration of a cumulative dose of 2.3 g of the culprit flour. To our knowledge, our report describes the first case of anaphylaxis after intake of buckwheat flour as the hidden allergen in pizza dough.

  8. Redefining the major peanut allergens

    PubMed Central

    Zhuang, Yonghua

    2015-01-01

    Food allergy has become a major public health concern in westernized countries, and allergic reactions to peanuts are particularly common and severe. Allergens are defined as antigens that elicit an IgE response, and most allergenic materials (e.g., pollens, danders, and foods) contain multiple allergenic proteins. This has led to the concept that there are “major” allergens and allergens of less importance. “Major allergens” have been defined as allergens that bind a large amount of IgE from the majority of patients and have biologic activity. However, the ability of an allergen to cross-link complexes of IgE and its high-affinity receptor FcεRI (IgE/FcεRI), which we have termed its allergic effector activity, does not correlate well with assays of IgE binding. To identify the proteins that are the most active allergens in peanuts, we and others have employed in vitro model assays of allergen-mediated cross-linking of IgE/FcεRI complexes and have demonstrated that the most potent allergens are not necessarily those that bind the most IgE. The importance of a specific allergen can be determined by measuring the allergic effector activity of that allergen following purification under non-denaturing conditions and by specifically removing the allergen from a complex allergenic extract either by chromatography or by specific immunodepletion. In our studies of peanut allergens, our laboratory has found that two related allergens, Ara h 2 and Ara h 6, together account for the majority of the effector activity in a crude peanut extract. Furthermore, murine studies demonstrated that Ara h 2 and Ara h 6 are not only the major elicitors of anaphylaxis in this system, but also can effectively desensitize peanut-allergic mice. As a result of these observations, we propose that the definition of a major allergen should be based on the potency of that allergen in assays of allergic effector activity and demonstration that removal of that allergen from an extract

  9. Endotoxin and house dust mite allergen levels on synthetic and buckwheat pillows.

    PubMed

    Nam, Hae-Seon; Park, Choon-Sik; Crane, Julian; Siebers, Rob

    2004-08-01

    Pillows are known to contain significant levels of indoor allergens and endotoxin, that are of importance to house dust mite sensitized asthmatics. Buckwheat pillows are commonly used in Korea. We studied the levels of the house dust mite allergen, Der f 1, and endotoxin on new synthetic and buckwheat pillows and their accumulation over three months. Endotoxin levels were significantly higher on new buckwheat pillows compared to synthetic pillows; geometric mean levels (95% CI) were 60,950 EU/g (30,270-122,700) and 4,887 EU/g (2,570-9,311) respectively (p<0.001). No Der f 1 was detected on the new pillows. After three months Der f 1 levels were similar on buckwheat and synthetic pillows, geometric mean levels (95% CI) were 1.16 microg/g (0.02-8.13) and 1.08 microg/g (0.19-1.68) respectively. These results indicate that buckwheat pillows are a source of very high endotoxin levels that may be of relevance to asthma severity of atopic asthmatics.

  10. IgE-binding epitopic peptide mapping on a three-dimensional model built for the 13S globulin allergen of buckwheat (Fagopyrum esculentum).

    PubMed

    Sordet, Camille; Culerrier, Raphaël; Granier, Claude; Didier, Alain; Rougé, Pierre

    2009-06-01

    The three-dimensional model built for the 13S globulin allergen of buckwheat (Fagopyrum esculentum) consists of three protomers exhibiting the cupin motif, arranged in a homotrimer around a three-fold symmetry axis. Using the SPOT technique, 11 continuous IgE-binding epitopic peptides were characterized on the molecular surface of the 13S globulin allergen of buckwheat. Except for one of them, they all correspond to well exposed regions containing electropositiveley and/or electronegatively charged residues, which cover up to 40% of the molecular surface of the allergen. Some of these epitopes come in close contact to probably create more extended discontinuous epitopes, especially those located on the edge of the 13S globulin homotrimer. Half of the identified epitope peptides remain unaltered in a core structure protected against hydrolysis by digestive proteases and are thus assumed to promote the allergenicity of the 13S globulin. In addition, a few of these epitopes coincide with sequential IgE-binding epitopes previously characterized in soybean 11S globulins, that could account for the IgE-binding cross-reactions observed between soybean and buckwheat in Western blot experiments.

  11. The major allergen of Dendropanax trifidus Makino.

    PubMed

    Oka, K; Saito, F; Yasuhara, T; Sugimoto, A

    1997-05-01

    Dendropanax trifidus Makino (family Araliaceae, syn. Gilibertia trifida Makino) has been reported as causing allergic contact dermatitis in Japan. To identify the major allergen, fractionated extracts of fresh leaves of Dendropanax trifidus were patch tested on 2 patients with hypersensitivity to the plant. Cis-9,17-octadecadiene-12,14-diyne-1, 16-diol (I), an analog of falcarinol, was identified as an active component. 18 normal control subjects were patch tested with the leaf of Dendropanax trifidus and I diluted to 0.05% in pet. 4 of them showed active sensitization to the leaf of Dendropanax trifidus and I. Our results suggest that I is the major allergen of Dendropanax trifidus and is a strong sensitizer. The results of patch testing on patients and control subjects with the leaves of Fatsia japonica Decne. et Planch. and Hedera helix L., which also belong to the Araliaceae family, and urushiol are also shown.

  12. Fish Allergens at a Glance: Variable Allergenicity of Parvalbumins, the Major Fish Allergens

    PubMed Central

    Kuehn, Annette; Swoboda, Ines; Arumugam, Karthik; Hilger, Christiane; Hentges, François

    2014-01-01

    Fish is a common trigger of severe, food-allergic reactions. Only a limited number of proteins induce specific IgE-mediated immune reactions. The major fish allergens are the parvalbumins. They are members of the calcium-binding EF-hand protein family characterized by a conserved protein structure. They represent highly cross-reactive allergens for patients with specific IgE to conserved epitopes. These patients might experience clinical reactions with various fish species. On the other hand, some individuals have IgE antibodies directed against unique, species-specific parvalbumin epitopes, and these patients show clinical symptoms only with certain fish species. Furthermore, different parvalbumin isoforms and isoallergens are present in the same fish and might display variable allergenicity. This was shown for salmon homologs, where only a single parvalbumin (beta-1) isoform was identified as allergen in specific patients. In addition to the parvalbumins, several other fish proteins, enolases, aldolases, and fish gelatin, seem to be important allergens. New clinical and molecular insights advanced the knowledge and understanding of fish allergy in the last years. These findings were useful for the advancement of the IgE-based diagnosis and also for the management of fish allergies consisting of advice and treatment of fish-allergic patients. PMID:24795722

  13. Fish allergens at a glance: variable allergenicity of parvalbumins, the major fish allergens.

    PubMed

    Kuehn, Annette; Swoboda, Ines; Arumugam, Karthik; Hilger, Christiane; Hentges, François

    2014-01-01

    Fish is a common trigger of severe, food-allergic reactions. Only a limited number of proteins induce specific IgE-mediated immune reactions. The major fish allergens are the parvalbumins. They are members of the calcium-binding EF-hand protein family characterized by a conserved protein structure. They represent highly cross-reactive allergens for patients with specific IgE to conserved epitopes. These patients might experience clinical reactions with various fish species. On the other hand, some individuals have IgE antibodies directed against unique, species-specific parvalbumin epitopes, and these patients show clinical symptoms only with certain fish species. Furthermore, different parvalbumin isoforms and isoallergens are present in the same fish and might display variable allergenicity. This was shown for salmon homologs, where only a single parvalbumin (beta-1) isoform was identified as allergen in specific patients. In addition to the parvalbumins, several other fish proteins, enolases, aldolases, and fish gelatin, seem to be important allergens. New clinical and molecular insights advanced the knowledge and understanding of fish allergy in the last years. These findings were useful for the advancement of the IgE-based diagnosis and also for the management of fish allergies consisting of advice and treatment of fish-allergic patients.

  14. Sensitive detection of major food allergens in breast milk: first gateway for allergenic contact during breastfeeding.

    PubMed

    Pastor-Vargas, C; Maroto, A S; Díaz-Perales, A; Villaba, M; Casillas Diaz, N; Vivanco, F; Cuesta-Herranz, J

    2015-08-01

    Food allergy is recognized as a major public health issue, especially in early childhood. It has been hypothesized that early sensitization to food allergens maybe due to their ingestion as components dissolved in the milk during the breastfeeding, explaining reaction to a food, which has never been taken before. Thus, the aim of this work has been to detect the presence of the food allergens in breast milk by microarray technology. We produced a homemade microarray with antibodies produced against major food allergens. The antibody microarray was incubated with breast milk from 14 women collected from Fundación Jiménez Díaz Hospital. In this way, we demonstrated the presence of major foods allergens in breast milk. The analysis of allergens presented in breast milk could be a useful tool in allergy prevention and could provide us a key data on the role of this feeding in tolerance induction or sensitization in children.

  15. Wild buckwheat is unlikely to pose a risk to buckwheat-allergic individuals.

    PubMed

    Nordlee, Julie A; Panda, Rakhi; Baumert, Joseph L; Goodman, Richard E; Taylor, Steve L

    2011-10-01

    Buckwheat (Fagopyrum esculentum) is a commonly allergenic food especially in Asia where buckwheat is more commonly consumed. Wild buckwheat (Polygonum convolvulus, recently changed to Fallopia convolvulus) is an annual weed prevalent in grain-growing areas of the United States. Wild buckwheat is not closely related to edible buckwheat although the seeds do have some physical resemblance. A large shipment of wheat into Japan was halted by the discovery of the adventitious presence of wild buckwheat seeds over possible concerns for buckwheat-allergic consumers. However, IgE-binding was not observed to an extract of wild buckwheat using sera from 3 buckwheat-allergic individuals either by radio-allergosorbent test inhibition or by immunoblotting after protein separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Furthermore, the extract of wild buckwheat was not detected in a buckwheat enzyme-linked immunosorbent assay developed with antisera against common buckwheat. Thus, wild buckwheat is highly unlikely to pose any risk to buckwheat-allergic individuals. The common names of plants should not be a factor in the risk assessment for possible cross-allergenicity. © 2011 Institute of Food Technologists®

  16. Allergenic components in three different species of Penicillium: crossreactivity among major allergens.

    PubMed

    Shen, H D; Lin, W L; Tsai, J J; Liaw, S F; Han, S H

    1996-04-01

    Penicillium species have been considered as important causative agents of extrinsic bronchial asthma. However, little is known about the allergens of these ubiquitous airborne fungal species. This study compares the allergenic profiles and allergenic crossreactivity among allergens of three prevalent airborne Penicillium species. IgE-binding Penicillium components were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)-immunoblotting using sera from 67 asthmatic patients. The presence of allergenic crossreactivity was analysed by immunoblot inhibition. Among the 67 serum samples tested, 15, 14 and 11 samples showed IgE reactivity to components of P. citrinum, P. notatum and P. brevicompactum, respectively. All 15 P. citrinum-positive serum samples showed IgE-binding to a 33 kDa extract component of this species. Thirteen (93%) of the 14 P. notatum-positive serum samples and 10 (91%) of the 11 P. brevicompactum-positive sera also showed IgE reactivity to components with a molecular weight of about 33 kDa in individual Penicillium species. All of the 10 P. brevicompactum 33 kDa component-positive serum samples showed IgE reactivity to the 33 kDa components of the other two Penicillium species tested. Dose-dependent inhibition of IgE-binding to these major allergens was observed when the positive serum sample was absorbed with different amounts of individual allergenic extract as well as with different amounts of extracts prepared from the other two Penicillium species. Although different allergenic profiles were observed in the three different Penicillium species tested, results showed that there was an IgE crossreactivity among the 33 kDa group major allergens of P. citrinum, P. notatum and P. brevicompactum.

  17. PCR method for detecting trace amounts of buckwheat (Fagopyrum spp.) in food.

    PubMed

    Hirao, Takashi; Imai, Shinsuke; Sawada, Hiroshi; Shiomi, Nobuo; Hachimura, Satoshi; Kato, Hisanori

    2005-04-01

    Buckwheat often causes severe allergic reactions, even when its ingestion level is extremely low. Therefore, buckwheat is listed in several countries as a common food allergen. In addition to common buckwheat and Tartarian buckwheat that are cultivated and consumed widely, wild buckwheat may be potentially allergenic. Food containing undeclared buckwheat poses a risk to patients with the buckwheat allergy. We describe in this report a PCR method to detect buckwheat DNA by using primers corresponding to the internal transcribed spacer region and the 5.8S rRNA gene. The method is buckwheat-specific and compatible with both cultivated and wild buckwheat of the Fagopyrum spp. Its sensitivity was sufficient to detect 1 ppm (w/w) of buckwheat DNA spiked in wheat DNA. This method should benefit food manufacturers, clinical doctors, and allergic patients by providing information on the presence of buckwheat contamination in food.

  18. Comparative and Evolutionary Analysis of Major Peanut Allergen Gene Families

    PubMed Central

    Ratnaparkhe, Milind B.; Lee, Tae-Ho; Tan, Xu; Wang, Xiyin; Li, Jingping; Kim, Changsoo; Rainville, Lisa K.; Lemke, Cornelia; Compton, Rosana O.; Robertson, Jon; Gallo, Maria; Bertioli, David J.; Paterson, Andrew H.

    2014-01-01

    Peanut (Arachis hypogaea L.) causes one of the most serious food allergies. Peanut seed proteins, Arah1, Arah2, and Arah3, are considered to be among the most important peanut allergens. To gain insights into genome organization and evolution of allergen-encoding genes, approximately 617 kb from the genome of cultivated peanut and 215 kb from a wild relative were sequenced including three Arah1, one Arah2, eight Arah3, and two Arah6 gene family members. To assign polarity to differences between homoeologous regions in peanut, we used as outgroups the single orthologous regions in Medicago, Lotus, common bean, chickpea, and pigeonpea, which diverged from peanut about 50 Ma and have not undergone subsequent polyploidy. These regions were also compared with orthologs in many additional dicot plant species to help clarify the timing of evolutionary events. The lack of conservation of allergenic epitopes between species, and the fact that many different proteins can be allergenic, makes the identification of allergens across species by comparative studies difficult. The peanut allergen genes are interspersed with low-copy genes and transposable elements. Phylogenetic analyses revealed lineage-specific expansion and loss of low-copy genes between species and homoeologs. Arah1 syntenic regions are conserved in soybean, pigeonpea, tomato, grape, Lotus, and Arabidopsis, whereas Arah3 syntenic regions show genome rearrangements. We infer that tandem and segmental duplications led to the establishment of the Arah3 gene family. Our analysis indicates differences in conserved motifs in allergen proteins and in the promoter regions of the allergen-encoding genes. Phylogenetic analysis and genomic organization studies provide new insights into the evolution of the major peanut allergen-encoding genes. PMID:25193311

  19. Pine nut allergy: clinical features and major allergens characterization

    USDA-ARS?s Scientific Manuscript database

    Pine nuts, the seeds of pine trees, are widely used for human consumption in Europe, America, and Asia. The aims of this study were to evaluate IgE-mediated hypersensitivity to pine nut in a large number of patients with details of clinical reactions, and to characterize major pine nut allergens. Th...

  20. Characterization of major allergens of royal jelly Apis mellifera.

    PubMed

    Rosmilah, M; Shahnaz, M; Patel, G; Lock, J; Rahman, D; Masita, A; Noormalin, A

    2008-12-01

    Royal jelly is widely consumed in the community and has perceived benefits ranging from promoting growth in children and improvement of general health status to enhancement of longevity for the elderly. However, royal jelly consumption has been linked to contact dermatitis, acute asthma, anaphylaxis and death. High prevalence of positive skin tests to royal jelly have been reported among atopic populations in countries with a high rate of royal jelly consumption. The present study is aimed to identify the major allergens of royal jelly. Royal jelly extract was separated by sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE) and 2-dimensional electrophoresis (2-D). Immunoblotting of the SDS-PAGE and 2-D profiles were performed to identify the allergenic spots. Spots were then excised from the 2-D gel, digested with trypsin and analyzed by mass spectrometry. The SDS-PAGE of royal jelly extract revealed 18 bands between 10 to 167 kD. Western blot of the fractionated proteins detected 15 IgE-binding bands between 14 to 127 kD with seven major allergens of 32, 40, 42, 49, 55, 60 and 67 kD using serum from 53 subjects with royal jelly allergy. The 2-D gel fractionated the royal jelly proteins to more than 50 different protein spots. Out of these, 30 spots demonstrated specific IgE affinity to the sera tested. Eight spots of the major royal jelly allergens were selected for mass-spectrometry analysis. Digested tryptic peptides of the spots were compared to the amino acid sequence search in protein databases which identified the fragments of royal jelly homologus to major royal jelly protein 1 (MRJ1) and major royal jelly protein 2 (MRJ2). In conclusion, the major allergens of royal jelly are MRJ1 and MRJ2 in our patients' population.

  1. Effect of Proteolysis with Alkaline Protease Following High Hydrostatic Pressure Treatment on IgE Binding of Buckwheat Protein.

    PubMed

    Lee, Chaeyoon; Lee, Wonhui; Han, Youngshin; Oh, Sangsuk

    2017-03-01

    Buckwheat is a popular food material in many Asian countries and it contains major allergenic proteins. This study was performed to analyze the effects of hydrolysis with alkaline protease following high hydrostatic pressure (HHP) treatment on the IgE binding of buckwheat protein. Extracted buckwheat protein was treated with HHP at 600 MPa for 30 min and hydrolyzed with alkaline protease for 240 min. IgE binding was examined using an enzyme-linked immunosorbent assay (ELISA) with serum samples from 14 patients who were allergic to buckwheat. Depending on the serum samples, HHP treatment of buckwheat protein without enzymatic hydrolysis decreased the IgE binding by 8.9% to 73.2% or increased by 31% to 78%. The IgE binding of buckwheat protein hydrolyzed with alkaline protease decreased by 73.8% to 100%. The IgE binding of buckwheat protein hydrolyzed with alkaline protease following HHP treatment decreased by 83.8% to 100%. This suggested that hydrolysis with alkaline protease following HHP treatment could be applied to reduce the IgE binding of buckwheat protein. © 2017 Institute of Food Technologists®.

  2. Reduction of the number of major representative allergens: from clinical testing to 3-dimensional structures.

    PubMed

    He, Ying; Liu, Xueting; Huang, Yuyi; Zou, Zehong; Chen, Huifang; Lai, He; Zhang, Lida; Wu, Qiurong; Zhang, Junyan; Wang, Shan; Zhang, Jianguo; Tao, Ailin; Sun, Baoqing

    2014-01-01

    Vast amounts of allergen sequence data have been accumulated, thus complicating the identification of specific allergenic proteins when performing diagnostic allergy tests and immunotherapy. This study aims to rank the importance/potency of the allergens so as to logically reduce the number of allergens and/or allergenic sources. Meta-analysis of 62 allergenic sources used for intradermal testing on 3,335 allergic patients demonstrated that in southern China, mite, sesame, spiny amaranth, Pseudomonas aeruginosa, and house dust account for 88.0% to 100% of the observed positive reactions to the 62 types of allergenic sources tested. The Kolmogorov-Smironov Test results of the website-obtained allergen data and allergen family featured peptides suggested that allergen research in laboratories worldwide has been conducted in parallel on many of the same species. The major allergens were reduced to 21 representative allergens, which were further divided into seven structural classes, each of which contains similar structural components. This study therefore has condensed numerous allergenic sources and major allergens into fewer major representative ones, thus allowing for the use of a smaller number of allergens when conducting comprehensive allergen testing and immunotherapy treatments.

  3. Glycosylation site of the major allergen from olive tree pollen. Allergenic implications of the carbohydrate moiety.

    PubMed

    Batanero, E; Villalba, M; Rodríguez, R

    1994-01-01

    The electrophoretic analysis of purified Ole e I, the major allergen from Olea europaea pollen, reveals the presence of two main variants, glycosylated (20.0 kDa) and non-glycosylated (18.5 kDa) components. The glycosylated variant has been identified as a concanavalin A-binding glycoprotein. Its carbohydrate moiety has a molecular mass of about 1.3 kDa (5% weight of the glycosylated allergen), based on mass spectrometry analysis. Enzymatic treatment of native Ole e I with the specific glycosidase PNGase F accounts for an oligosaccharide N-linked to the polypeptide chain. This treatment does not sensibly modify the secondary structure of the protein but diminishes the affinity of the allergen for specific IgE antibodies. Tryptic digestion of Ole e I reveals the presence of a single carbohydrate-containing peptide. This peptide was recognized by the sera of hypersensitive individuals. The amino acid sequence of this peptide is Phe-Lys-Leu-Asn-Thr-Val-Asn-Gly-Thr-Thr-Arg, asparagine at the seventh being the carbohydrate attaching site. The obtained data are discussed in terms of the potential role of the sugar moiety in the allergenic activity of Ole e I.

  4. 27 CFR 7.22b - Petitions for exemption from major food allergen labeling.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... from major food allergen labeling. 7.22b Section 7.22b Alcohol, Tobacco Products and Firearms ALCOHOL... BEVERAGES Labeling Requirements for Malt Beverages § 7.22b Petitions for exemption from major food allergen... contain allergenic protein derived from one of the foods identified in § 7.22(a)(1)(i), even though...

  5. 27 CFR 7.22b - Petitions for exemption from major food allergen labeling.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... from major food allergen labeling. 7.22b Section 7.22b Alcohol, Tobacco Products and Firearms ALCOHOL... BEVERAGES Labeling Requirements for Malt Beverages § 7.22b Petitions for exemption from major food allergen... contain allergenic protein derived from one of the foods identified in § 7.22(a)(1)(i), even though...

  6. 27 CFR 7.22b - Petitions for exemption from major food allergen labeling.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... from major food allergen labeling. 7.22b Section 7.22b Alcohol, Tobacco Products and Firearms ALCOHOL... BEVERAGES Labeling Requirements for Malt Beverages § 7.22b Petitions for exemption from major food allergen... contain allergenic protein derived from one of the foods identified in § 7.22(a)(1)(i), even though...

  7. 27 CFR 7.22b - Petitions for exemption from major food allergen labeling.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... from major food allergen labeling. 7.22b Section 7.22b Alcohol, Tobacco Products and Firearms ALCOHOL... BEVERAGES Labeling Requirements for Malt Beverages § 7.22b Petitions for exemption from major food allergen... contain allergenic protein derived from one of the foods identified in § 7.22(a)(1)(i), even though...

  8. 27 CFR 7.22b - Petitions for exemption from major food allergen labeling.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... BEVERAGES Labeling Requirements for Malt Beverages § 7.22b Petitions for exemption from major food allergen... from major food allergen labeling. 7.22b Section 7.22b Alcohol, Tobacco Products and Firearms ALCOHOL... contain allergenic protein derived from one of the foods identified in § 7.22(a)(1)(i), even though a...

  9. 27 CFR 5.32b - Petitions for exemption from major food allergen labeling.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... from major food allergen labeling. 5.32b Section 5.32b Alcohol, Tobacco Products and Firearms ALCOHOL... food allergen labeling. (a) Submission of petition. Any person may petition the appropriate TTB officer... human health; or (2) Does not contain allergenic protein derived from one of the foods identified in §...

  10. 27 CFR 5.32b - Petitions for exemption from major food allergen labeling.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... from major food allergen labeling. 5.32b Section 5.32b Alcohol, Tobacco Products and Firearms ALCOHOL... food allergen labeling. (a) Submission of petition. Any person may petition the appropriate TTB officer... human health; or (2) Does not contain allergenic protein derived from one of the foods identified in §...

  11. 27 CFR 5.32b - Petitions for exemption from major food allergen labeling.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... from major food allergen labeling. 5.32b Section 5.32b Alcohol, Tobacco Products and Firearms ALCOHOL... food allergen labeling. (a) Submission of petition. Any person may petition the appropriate TTB officer... human health; or (2) Does not contain allergenic protein derived from one of the foods identified in §...

  12. 27 CFR 5.32b - Petitions for exemption from major food allergen labeling.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... from major food allergen labeling. 5.32b Section 5.32b Alcohol, Tobacco Products and Firearms ALCOHOL... food allergen labeling. (a) Submission of petition. Any person may petition the appropriate TTB officer... human health; or (2) Does not contain allergenic protein derived from one of the foods identified in §...

  13. Periplaneta americana arginine kinase as a major cockroach allergen among Thai patients with major cockroach allergies.

    PubMed

    Sookrung, Nitat; Chaicumpa, Wanpen; Tungtrongchitr, Anchalee; Vichyanond, Pakit; Bunnag, Chaweewan; Ramasoota, Pongrama; Tongtawe, Pongsri; Sakolvaree, Yuwaporn; Tapchaisri, Pramuan

    2006-06-01

    Periplaneta americana is the predominant cockroach (CR) species and a major source of indoor allergens in Thailand. Nevertheless, data on the nature and molecular characteristics of its allergenic components are rare. We conducted this study to identify and characterize the P. americana allergenic protein. A random heptapeptide phage display library and monoclonal antibody (MAb) specific to a the P. americana component previously shown to be an allergenic molecule were used to identify the MAb-bound mimotope and its phylogenic distribution. Two-dimensional gel electrophoresis, liquid chromatography, mass spectrometry, peptide mass fingerprinting, and BLAST search were used to identify the P. americana protein containing the MAb-specific epitope. We studied the allergenicity of the native protein using sera of CR-allergic Thai patients in immunoassays. The mimotope peptide that bound to the MAb specific to P. americana was LTPCRNK. The peptide has an 83-100% identity with proteins of Anopheles gambiae, notch homolog scalloped wings of Lucilia cuprina, delta protein of Apis mellifera; neu5Ac synthase and tyrosine phosphatase of Drosophila melanogaster, and a putative protein of Drosophila pseudoobscura. This finding implies that the mimotope-containing molecule of P. americana is a pan-insect protein. The MAb-bound protein of P. americana was shown to be arginine kinase that reacted to IgE in the sera of all of the CR-allergic Thai patients by immunoblotting, implying its high allergenicity. In conclusion, our results revealed that P. americana arginine kinase is a pan-insect protein and a major CR allergen for CR-allergic Thai patients.

  14. Mouse allergen is the major allergen of public health relevance in Baltimore City.

    PubMed

    Ahluwalia, Sharon K; Peng, Roger D; Breysse, Patrick N; Diette, Gregory B; Curtin-Brosnan, Jean; Aloe, Charles; Matsui, Elizabeth C

    2013-10-01

    Cockroach and mouse allergens have both been implicated as causes in inner-city asthma morbidity in multicenter studies, but whether both allergens are clinically relevant within specific inner-city communities is unclear. Our study aimed to identify relevant allergens in Baltimore City. One hundred forty-four children (5-17 years old) with asthma underwent skin prick tests at baseline and had clinical data collected at baseline and 3, 6, 9, and 12 months. Home settled dust samples were collected at the same time points for quantification of indoor allergens. Participants were grouped based on their sensitization and exposure status to each allergen. All analyses were adjusted for age, sex, and serum total IgE level. Forty-one percent were mouse sensitized/exposed, and 41% were cockroach sensitized/exposed based on bedroom floor exposure data. Mouse sensitization/exposure was associated with acute care visits, decreased FEV1/forced vital capacity percentage values, fraction of exhaled nitric oxide levels, and bronchodilator reversibility. Cockroach sensitization/exposure was only associated with acute care visits and bronchodilator reversibility when exposure was defined by using bedroom floor allergen levels. Mouse-specific IgE levels were associated with poor asthma health across a range of outcomes, whereas cockroach-specific IgE levels were not. The relationships between asthma outcomes and mouse allergen were independent of cockroach allergen. Although sensitization/exposure to both mouse and cockroach was generally associated with worse asthma, mouse sensitization/exposure was the primary contributor to these relationships. In a community with high levels of both mouse and cockroach allergens, mouse allergen appears to be more strongly and consistently associated with poor asthma outcomes than cockroach allergen. Community-level asthma interventions in Baltimore should prioritize reducing mouse allergen exposure. Copyright © 2013 American Academy of Allergy

  15. 27 CFR 4.32b - Petitions for exemption from major food allergen labeling.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... from major food allergen labeling. 4.32b Section 4.32b Alcohol, Tobacco Products and Firearms ALCOHOL... Labeling Requirements for Wine § 4.32b Petitions for exemption from major food allergen labeling. (a... protein derived from one of the foods identified in § 4.32a(a)(1)(i), even though a major food...

  16. 27 CFR 4.32b - Petitions for exemption from major food allergen labeling.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... from major food allergen labeling. 4.32b Section 4.32b Alcohol, Tobacco Products and Firearms ALCOHOL... Labeling Requirements for Wine § 4.32b Petitions for exemption from major food allergen labeling. (a... protein derived from one of the foods identified in § 4.32a(a)(1)(i), even though a major food...

  17. 27 CFR 4.32b - Petitions for exemption from major food allergen labeling.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... from major food allergen labeling. 4.32b Section 4.32b Alcohol, Tobacco Products and Firearms ALCOHOL... Labeling Requirements for Wine § 4.32b Petitions for exemption from major food allergen labeling. (a... protein derived from one of the foods identified in § 4.32a(a)(1)(i), even though a major food...

  18. 27 CFR 4.32b - Petitions for exemption from major food allergen labeling.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... from major food allergen labeling. 4.32b Section 4.32b Alcohol, Tobacco Products and Firearms ALCOHOL... Labeling Requirements for Wine § 4.32b Petitions for exemption from major food allergen labeling. (a... protein derived from one of the foods identified in § 4.32a(a)(1)(i), even though a major food...

  19. Molecular and immunological characterization of the first allergenic lipocalin in hamster: the major allergen from Siberian hamster (Phodopus sungorus).

    PubMed

    Torres, José Alberto; de Las Heras, Manuel; Maroto, Aroa Sanz; Vivanco, Fernando; Sastre, Joaquín; Pastor-Vargas, Carlos

    2014-08-22

    The most frequent pet allergy is to cat and dog, but in recent years, it has become increasingly popular to have other pets, and the risk of exposure to new allergens is more prevalent. The list of new pets includes hamsters, and one of the most popular hamsters is the Siberian hamster (Phodopus sungorus). The aim of this study was the characterization and cloning of the major allergen from this hamster. The study of its allergenicity and cross-reactivity could improve the specific diagnosis and treatment for hamster-allergic patients. Thirteen Siberian hamster-allergic patients were recruited at the outpatient clinic. Protein extracts were prepared from the hair, urine, and salivary glands of four hamster species (European, golden, Siberian, and Roborovski). IgE-binding proteins were detected by immunoblotting and identified by mass spectrometry. The recombinant protein was produced in Escherichia coli and then purified by metal chelate affinity chromatography. The allergenic properties of the recombinant protein were tested by ELISA and immunoblotting, and biological activity was tested according to capacity for basophil activation. Three IgE-binding proteins were identified in extracts obtained from Siberian hamster hair, urine, and salivary glands. All proteins corresponded to the same protein, which was identified as a lipocalin. This lipocalin had no cross-reactivity with common and golden hamsters. The recombinant allergen was cloned and purified, showing similar IgE reactivity in vitro to Siberian hamster protein extracts. Also, the recombinant allergen was capable of producing biological activation in vivo. The major Siberian hamster allergen was cloned, and allergenic properties were characterized, providing a new tool for specific diagnosis of allergy to Siberian hamster. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Haemoglobin, a new major allergen of Anisakis simplex.

    PubMed

    González-Fernández, Juan; Daschner, Alvaro; Nieuwenhuizen, Natalie E; Lopata, Andreas L; Frutos, Consolación De; Valls, Ana; Cuéllar, Carmen

    2015-05-01

    Gastro-allergic anisakiasis and Anisakis sensitisation associated chronic urticaria are diseases which differ in their IgE and IgG4 responses against both crude extract and specific allergens. Anisakis and Ascaris are closely related nematodes that usually cause problems with specificity in immunodiagnostics. In this study we measured IgE and IgG4 antibodies against Anisakis simplex sensu lato (s. l.) and Ascaris suum haemoglobins in sera of 21 gastro-allergic anisakiasis and 23 chronic urticaria patients. We used a capture ELISA with the anti-Anisakis haemoglobin monoclonal antibody 4E8g, which also recognises Ascaris haemoglobin. In addition, we determined specific IgE and IgG4 to both nematodes by indirect ELISA and immunoblotting. Anti-A. simplex s. l. haemoglobin IgE and IgG4 levels were higher in gastro-allergic anisakiasis than in chronic urticaria patients (P=0.002 and 0.026, respectively). Surprisingly, no patient had detectable IgE levels against A. suum haemoglobin. Finally, we carried out an in silico study of the B-cell epitopes of both haemoglobin molecules. Five epitopes were predicted in Anisakis pegreffii and four in A. suum haemoglobin. The epitope propensity values of Anisakis haemoglobin in the equivalent IgE binding region of the allergenic haemoglobin Chi t 1 from Chironomus thummi, were higher those of the Ascaris haemoglobin. In conclusion, we describe A. simplex haemoglobin as a new major allergen (Ani s 13), being recognised by a large number (64.3%) of sensitised patients and up to 80.9% in patients with gastro-allergic anisakiasis. The presence of a specific epitope and the different values of epitope propensity between Anisakis and Ascaris haemoglobin could explain the lack of cross-reactivity between the two molecules. The absence of IgE reactivity to Ascaris haemoglobin in Anisakis patients makes Anisakis haemoglobin (Ani s 13) a potential candidate for developing more specific diagnosis tools. Copyright © 2015 Australian Society for

  1. Purification and molecular cloning of a major allergen from Anisakis simplex.

    PubMed

    Shimakura, Kuniyoshi; Miura, Hironori; Ikeda, Kaori; Ishizaki, Shoichiro; Nagashima, Yuji; Shirai, Toshihiro; Kasuya, Shiro; Shiomi, Kazuo

    2004-05-01

    A heat-stable allergen with a molecular weight of 21 k was purified from larvae of the nematode Anisakis simplex by gel filtration, anion-exchange FPLC and reverse-phase HPLC. When analyzed by immunoblotting and ELISA, seven of eight patient sera reacted to the 21 k allergen, demonstrating that this protein is a major allergen of A. simplex. A full-length cDNA encoding the 21 k allergen was cloned by a combination of 3'RACE and screening of an expression library with DIG-labeled DNA probes. The precursor of the 21 k allergen was judged to be composed of a signal peptide (23 residues) and a mature protein (171 residues). As compared to the N-terminal amino acid sequence (up to the 17th residue) of Ani s 1 previously identified as the major allergen, the 21 k allergen has only one replacement, suggesting that the 21 k allergen belongs to the same protein family of Ani s 1. Although the 21 k allergen was found to have 30-40% sequence identity with Kunitz-type trypsin inhibitor domain containing hypothetical proteins of Caenorhabditis elegans, it lacked inhibitory activity against trypsin. The 21 k allergen was successfully expressed in Escherichia coli as a GST-fusion protein showing reactivity with IgE in patient sera.

  2. Glucoamylase is a major allergen of Schizophyllum commune.

    PubMed

    Toyotome, T; Satoh, M; Yahiro, M; Watanabe, A; Nomura, F; Kamei, K

    2014-03-01

    Schizophyllum commune is one of the causative agents of basidiomycosis including disorders such as allergic bronchopulmonary mycosis, allergic fungal sinusitis, and mucoid impaction of bronchi, the incidence of those of which has been increasing. These mycoses are difficult to diagnose because only a limited number of diagnostic tools are currently available. The biggest problem is that no specific antigens of S. commune have been identified to enable serodiagnosis of the disease. In this study, we attempted to identify a major antigen of S. commune to establish a reliable serodiagnostic method. We used mass spectrometry to identify an antigen that reacted with the serum of a patient with allergic bronchopulmonary mycosis caused by S. commune. The protein was expressed in Escherichia coli, highly purified, and the patient sera IgG and IgE titres against the protein were determined by enzyme-linked immunosorbent assay. The protein identified as a major antigen of S. commune was named Sch c 1; it was a homolog of glucoamylase. The IgG and IgE titres against Sch c 1 in patient sera were significantly higher than those in healthy volunteer sera (P < 0.01). Sch c 1 is recognized by the host immune system of patients as an antigen/allergen. The purified glucoamylase Sch c 1 is a promising candidate antigen for the serodiagnosis of S. commune-induced mycosis. © 2013 John Wiley & Sons Ltd.

  3. Monoclonal antibodies against Olea europaea major allergen: allergenic activity of affinity-purified allergen and depleted extract and development of a radioimmunoassay for the quantitation of the allergen.

    PubMed

    Lombardero, M; Quirce, S; Duffort, O; Barber, D; Carpizo, J; Chamorro, M J; Lezaun, A; Carreira, J

    1992-04-01

    Several monoclonal antibodies (MAbs) were raised against Olea europaea pollen-extract components. Two of these antibodies, named OL 2 and OL 7, recognize two nonoverlapping, nonrepeating epitopes on the olive-allergen Ole e I, as demonstrated by different techniques. The allergen was purified in a single step by MAb-based affinity chromatography, and the allergen revealed a band at molecular weight 20 kd as well as a minor band at 18 kd on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The contribution of allergen Ole e I to the allergenic activity of O. europaea pollen extracts was determined from the effect of allergen depletion by affinity chromatography on skin reactivity and a histamine-release test. The removal of allergen caused a large reduction in the activity of the preparation in 25 monospecific olive-allergic patients. In agreement, the affinity-purified allergen demonstrated a similar response when it was compared with the whole extract in these assays. The results indicated that Ole e I is by far the most important olive-pollen allergen. A two-site solid-phase radioimmunoassay was developed for the quantitation of the allergen Ole e I in mass units. The assay was based on the MAbs, OL 2 and OL 7, and had a detection limit in the nanogram range. A good correlation was found between allergenic activity, as determined by RAST inhibition, and allergen content in 18 olive-pollen extracts. This result indicates that the assay can be a good alternative to RAST inhibition for the standardization of O. europaea extracts.

  4. Allergen

    MedlinePlus

    Common allergens include: Animal proteins and animal dander Dust Drugs (such as antibiotics or medicines you put on your skin) Foods (such as egg, peanut, milk, nuts, soy, fish, animal meat, and wheat) Fungal spores ...

  5. Identification of tropomyosin and arginine kinase as major allergens of Portunus pelagicus (blue swimming crab).

    PubMed

    Rosmilah, M; Shahnaz, M; Zailatul, H M Y; Noormalin, A; Normilah, I

    2012-09-01

    Crab is an important source of food allergen. Tropomyosin represents the main crab allergen and is responsible for IgE cross-reactivity between various species of crustaceans. Recently, other new crab allergens including arginine kinase have been identified. However, information on allergens of the local Portunidcrab is not available. Thus, the aim of this study was to identify the major allergens of Portunus pelagicus (blue swimming crab) using the allergenomics approach. Raw and cooked extracts of the crab were prepared from the crab meat. Protein profile and IgE binding pattern were demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting using sera from 30 patients with crab allergy. The major allergens of the crab were then identified by two-dimensional electrophoresis (2-DE), followed by mass spectrometry analysis of the peptide digests. The SDS-PAGE of raw extract revealed approximately 20 protein fractions over a wide molecular weight range, while cooked extract demonstrated fewer protein bands. The raw extract also demonstrated a higher number of IgE reactive bands than the cooked extract. A heat-resistant protein of 36 kDa has been identified as the major allergen in both raw and cooked extracts. In addition, a heat-sensitive protein of 41 kDa was also recognized as a major allergen in raw crab. The 2-DE gel profile of the raw extract demonstrated about >100 distinct proteins spots and immunoblotting of the 2-DE profile demonstrated at least 12 different major IgE reactive spots with molecular masses between 13 to 250 kDa and isoelectric point (pI) values ranging from 4.0 to 7.0. The 36 and 41 kDa proteins were identified as the crab tropomyosin and arginine kinase, respectively by mass spectrometry. Therefore, this study confirmed that tropomyosin and arginine kinase are the major allergens of the local Portunid crab, P. pelagicus.

  6. Dermatophagoides pteronyssinus major allergen 1 activates the innate immune response of the fruit fly Drosophila melanogaster.

    PubMed

    Warmbold, Christine; Uliczka, Karin; Rus, Fiorentina; Suck, Roland; Petersen, Arnd; Silverman, Neal; Ulmer, Artur J; Heine, Holger; Roeder, Thomas

    2013-01-01

    Some allergens with relevant protease activity have the potential to directly interact with host structures. It remains to be elucidated whether this activity is relevant for developing their allergenic properties. The major goal of this study was to elucidate whether allergens with a strong protease activity directly interact with modules of the innate immune system, thereby inducing an immune response. We chose Drosophila melanogaster for our experiments to prevent the results from being influenced by the adaptive immune system and used the armamentarium of methods available for the fly to study the underlying mechanisms. We show that Dermatophagoides pteronyssinus major allergen 1 (Der p 1), the major allergen of the house dust mite, efficiently activates various facets of the Drosophila innate-immune system, including both epithelial and systemic responses. These responses depend on the immune deficiency (IMD) pathway via activation of the NF-κB transcription factor Relish. In addition, the major pathogen associated molecular pattern recognizing receptor of the IMD pathway, peptidoglycan recognition protein-LC, was necessary for this response. We showed that Der p 1, which has cysteine protease activity, cleaves the ectodomain of peptidoglycan recognition protein-LC and, thus, activates the IMD pathway to induce a profound immune response. We conclude that the innate immune response to this allergen-mediated proteolytic cleavage represents an ancient type of danger signaling that may be highly relevant for the primary allergenicity of compounds such as Der p 1.

  7. The Anisakis simplex Ani s 7 major allergen as an indicator of true Anisakis infections

    PubMed Central

    Anadón, A M; Romarís, F; Escalante, M; Rodríguez, E; Gárate, T; Cuéllar, C; Ubeira, F M

    2009-01-01

    Ani s 7 is currently the most important excretory/secretory (ES) Anisakis simplex allergen, as it is the only one recognized by 100% of infected patients. The allergenicity of this molecule is due mainly to the presence of a novel CX17–25CX9–22CX8CX6 tandem repeat motif not seen in any previously reported protein. In this study we used this allergen as a model to investigate how ES allergens are recognized during Anisakis infections, and the usefulness of a recombinant fragment of Ani s 7 allergen (t-Ani s 7) as a marker of true Anisakis infections. The possible antigenic relationship between native Ani s 7 (nAni s 7) from Anisakis and Pseudoterranova decipensantigens was also investigated. Our results demonstrate that nAni s 7 is secreted and recognized by the immune system of rats only when the larvae are alive (i.e. during the acute phase of infection), and that this molecule is not present in, or is antigenically different from, Pseudoterranova allergens. The t-Ani s 7 polypeptide is a useful target for differentiating immunoglobulin E antibodies induced by true Anisakis infections from those induced by other antigens that may cross-react with Anisakis allergens, including P. decipiens. The results also support the hypothesis that the Ani s 7 major allergen does not participate in maintaining the antigenic stimulus during chronic infections. PMID:19438600

  8. Maillard reaction and enzymatic browning affect the allergenicity of Pru av 1, the major allergen from cherry (Prunus avium).

    PubMed

    Gruber, Patrick; Vieths, Stefan; Wangorsch, Andrea; Nerkamp, Jörg; Hofmann, Thomas

    2004-06-16

    The influence of thermal processing and nonenymatic as well as polyphenoloxidase-catalyzed browning reaction on the allergenicity of the major cherry allergen Pru av 1 was investigated. After thermal treatment of the recombinant protein rPru av 1 in the absence or presence of carbohydrates, SDS-PAGE, enzyme allergosorbent tests, and inhibition assays revealed that thermal treatment of rPru av 1 alone did not show any influence on the IgE-binding activity of the protein at least for 30 min, thus correlating well with the refolding of the allergen in buffer solution as demonstrated by CD spectroscopic experiments. Incubation of the protein with starch and maltose also showed no effect on IgE-binding activity, whereas reaction with glucose and ribose and, even more pronounced, with the carbohydrate breakdown products glyceraldehyde and glyoxal induced a strong decrease of the IgE-binding capacity of rPru av 1. In the second part of the study, the effect of polyphenoloxidase-catalyzed oxidation of polyphenols on food allergen activity was investigated. Incubation of rPru av 1 with epicatechin in the presence of tyrosinase led to a drastic decrease in IgE-binding activity of the protein. Variations of the phenolic compound revealed caffeic acid and epicatechin as the most active inhibitors of the IgE-binding activity of rPru av 1, followed by catechin and gallic acid, and, finally, by quercetin and rutin, showing significantly lower activity. On the basis of these data, reactive intermediates formed during thermal carbohydrate degradation as well as during enzymatic polyphenol oxidation are suggested as the active chemical species responsible for modifying nucleophilic amino acid side chains of proteins, thus inducing an irreversible change in the tertiary structure of the protein and resulting in a loss of conformational epitopes of the allergen.

  9. Identification and characterization of major cat allergen Fel d 1 mimotopes on filamentous phage carriers.

    PubMed

    Luzar, Jernej; Molek, Peter; Šilar, Mira; Korošec, Peter; Košnik, Mitja; Štrukelj, Borut; Lunder, Mojca

    2016-03-01

    Cat allergy is one of the most prevalent allergies worldwide and can lead to the development of rhinitis and asthma. Thus far, only allergen extracts from natural sources have been used for allergen-specific immunotherapy. However, extracts and whole allergens in immunotherapy present an anaphylaxis risk. Identification of allergen epitopes or mimotopes has an important role in development of safe and effective allergen-specific immunotherapy. Moreover, with a suitable immunogenic carrier, the absence of sufficient immune response elicited by short peptides could be surmounted. In this study, we identified five structural mimotopes of the major cat allergen Fel d 1 by immunoscreening with random peptide phage libraries. The mimotopes were computationally mapped to the allergen surface, and their IgE reactivity was confirmed using sera from cat-allergic patients. Importantly, the mimotopes showed no basophil activation of the corresponding cat-allergic patients, which makes them good candidates for the development of hypoallergenic vaccine. As bacteriophage particles are becoming increasingly recognized as immunogenic carriers, we constructed bacteriophage particles displaying multiple copies of each selected mimotope on major phage coat protein. These constructed phages elicited T cell-mediated immune response, which was predominated by the type 1 T cell response. Mimotopes alone contributed to the type 1 T cell response by promoting IL-2 production. Fel d 1 mimotopes, as well as their filamentous phage immunogenic carriers, represent promising candidates in the development of hypoallergenic vaccine against cat allergy.

  10. A B Cell Epitope Peptide Derived from the Major Grass Pollen Allergen Phl p 1 Boosts Allergen-Specific Secondary Antibody Responses without Allergen-Specific T Cell Help.

    PubMed

    Narayanan, Meena; Freidl, Raphaela; Focke-Tejkl, Margarete; Baranyi, Ulrike; Wekerle, Thomas; Valenta, Rudolf; Linhart, Birgit

    2017-02-15

    More than 40% of allergic patients suffer from grass pollen allergy. Phl p 1, the major timothy grass pollen allergen, belongs to the cross-reactive group 1 grass pollen allergens that are thought to initiate allergic sensitization to grass pollen. Repeated allergen encounter boosts allergen-specific IgE production and enhances clinical sensitivity in patients. To investigate immunological mechanisms underlying the boosting of allergen-specific secondary IgE Ab responses and the allergen epitopes involved, a murine model for Phl p 1 was established. A B cell epitope-derived peptide of Phl p 1 devoid of allergen-specific T cell epitopes, as recognized by BALB/c mice, was fused to an allergen-unrelated carrier in the form of a recombinant fusion protein and used for sensitization. This fusion protein allowed the induction of allergen-specific IgE Ab responses without allergen-specific T cell help. Allergen-specific Ab responses were subsequently boosted with molecules containing the B cell epitope-derived peptide without carrier or linked to other allergen-unrelated carriers. Oligomeric peptide bound to a carrier different from that which had been used for sensitization boosted allergen-specific secondary IgE responses without a detectable allergen-specific T cell response. Our results indicate that allergen-specific secondary IgE Ab responses can be boosted by repetitive B cell epitopes without allergen-specific T cell help by cross-linking of the B cell epitope receptor. This finding has important implications for the design of new allergy vaccines.

  11. A B Cell Epitope Peptide Derived from the Major Grass Pollen Allergen Phl p 1 Boosts Allergen-Specific Secondary Antibody Responses without Allergen-Specific T Cell Help

    PubMed Central

    Narayanan, Meena; Freidl, Raphaela; Focke-Tejkl, Margarete; Baranyi, Ulrike; Wekerle, Thomas; Valenta, Rudolf

    2017-01-01

    More than 40% of allergic patients suffer from grass pollen allergy. Phl p 1, the major timothy grass pollen allergen, belongs to the cross-reactive group 1 grass pollen allergens that are thought to initiate allergic sensitization to grass pollen. Repeated allergen encounter boosts allergen-specific IgE production and enhances clinical sensitivity in patients. To investigate immunological mechanisms underlying the boosting of allergen-specific secondary IgE Ab responses and the allergen epitopes involved, a murine model for Phl p 1 was established. A B cell epitope–derived peptide of Phl p 1 devoid of allergen-specific T cell epitopes, as recognized by BALB/c mice, was fused to an allergen-unrelated carrier in the form of a recombinant fusion protein and used for sensitization. This fusion protein allowed the induction of allergen-specific IgE Ab responses without allergen-specific T cell help. Allergen-specific Ab responses were subsequently boosted with molecules containing the B cell epitope–derived peptide without carrier or linked to other allergen-unrelated carriers. Oligomeric peptide bound to a carrier different from that which had been used for sensitization boosted allergen-specific secondary IgE responses without a detectable allergen-specific T cell response. Our results indicate that allergen-specific secondary IgE Ab responses can be boosted by repetitive B cell epitopes without allergen-specific T cell help by cross-linking of the B cell epitope receptor. This finding has important implications for the design of new allergy vaccines. PMID:28093528

  12. Crystal structure and immunologic characterization of the major grass pollen allergen Phl p 4.

    PubMed

    Zafred, Domen; Nandy, Andreas; Pump, Linda; Kahlert, Helga; Keller, Walter

    2013-09-01

    Phl p 4 is a major pollen allergen but exhibits lower allergenicity than other major allergens. The natural protein is glycosylated and shows cross-reactivity with related and structurally unrelated allergens. We sought to determine the high-resolution crystal structure of Phl p 4 and to evaluate the immunologic properties of the recombinant allergen in comparison with natural Phl p 4. Different isoallergens of Phl p 4 were expressed, and the nonglycosylated mutant was crystallized. The specific role of protein and carbohydrate epitopes for allergenicity was studied by using IgE inhibition and basophil release assays. The 3-dimensional structure was determined by using x-ray crystallography at a resolution of 1.9 Å. The allergen is a glucose dehydrogenase with a bicovalently attached flavin adenine dinucleotide. Glycosylated and nonglycosylated recombinant Phl p 4 showed identical inhibition of IgE binding, but compared with natural Phl p 4, all recombinant isoforms displayed a reduced IgE-binding inhibition. However, the recombinant protein exhibited an approximately 10-fold higher potency in basophil release assays than the natural protein. The crystal structure reveals the compact globular nature of the protein, and the observed binding pocket implies the size of the natural substrate. Plant-derived cross-reactive carbohydrate determinants (CCDs) appear to reduce the allergenicity of the natural allergen, whereas the Pichia pastoris-derived glycosylation does not. Our results imply yet undescribed mechanism of how CCDs dampen the immune response, leading to a novel understanding of the role of CCDs. Copyright © 2013 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

  13. Orthologous Allergens and Diagnostic Utility of Major Allergen Alt a 1

    PubMed Central

    Moreno, Antonio; Alcover, Javier; Rodríguez, David; Palacios, Ricardo; Martínez-Naves, Eduardo

    2016-01-01

    Purpose Hypersensitivity to fungi is associated with rhinoconjunctivitis and asthma. For some fungi, such as Alternaria alternata (A. alternata), the symptoms of asthma are persistent, increasing disease severity and the risk of fatal outcomes. There are a large number of species of fungi but knowledge of them remains limited. This, together with the difficulties in obtaining adequate standardized extracts, means that there remain significant challenges in the diagnosis and immunotherapy of allergy associated with fungi. The type of indoor fungi related to asthma/allergy varies according to geographic, climatic, and seasonal factors, making their study difficult. The aim of this study was to determine hypersensitivity to indoor fungi in a population from Cuenca, Spain. Methods Thirty-five patients with symptoms compatible with rhinitis or asthma who showed clear worsening of their symptoms in their homes or workplace were included. In vivo and in vitro tests were made with a battery of fungal allergens, including the species isolated in the home or workplace. Results Ulocladium botrytis (U. botrytis) and A. alternata were the most representative species as a source of home sensitization. These species showed very high concordance in skin tests, specific IgE, and histamine release. The allergen Alt a 1, which was recognized in all patients, was detected in A. alternata, U. botrytis, and Stemphylium botryosum (S. botryosum). Conclusions U. botrytis and A. alternata were the most representative species as a source of home sensitization. Alt a 1 was recognized in all patients and may be considered a non-species-specific allergen that could be used as a diagnostic source of sensitization to some species of the Pleosporaceae family. PMID:27334781

  14. Glycoproteomic Analysis of Seven Major Allergenic Proteins Reveals Novel Post-translational Modifications*

    PubMed Central

    Halim, Adnan; Carlsson, Michael C.; Madsen, Caroline Benedicte; Brand, Stephanie; Møller, Svenning Rune; Olsen, Carl Erik; Vakhrushev, Sergey Y.; Brimnes, Jens; Wurtzen, Peter Adler; Ipsen, Henrik; Petersen, Bent L.; Wandall, Hans H.

    2015-01-01

    Allergenic proteins such as grass pollen and house dust mite (HDM) proteins are known to trigger hypersensitivity reactions of the immune system, leading to what is commonly known as allergy. Key allergenic proteins including sequence variants have been identified but characterization of their post-translational modifications (PTMs) is still limited. Here, we present a detailed PTM1 characterization of a series of the main and clinically relevant allergens used in allergy tests and vaccines. We employ Orbitrap-based mass spectrometry with complementary fragmentation techniques (HCD/ETD) for site-specific PTM characterization by bottom-up analysis. In addition, top-down mass spectrometry is utilized for targeted analysis of individual proteins, revealing hitherto unknown PTMs of HDM allergens. We demonstrate the presence of lysine-linked polyhexose glycans and asparagine-linked N-acetylhexosamine glycans on HDM allergens. Moreover, we identified more complex glycan structures than previously reported on the major grass pollen group 1 and 5 allergens, implicating important roles for carbohydrates in allergen recognition and response by the immune system. The new findings are important for understanding basic disease-causing mechanisms at the cellular level, which ultimately may pave the way for instigating novel approaches for targeted desensitization strategies and improved allergy vaccines. PMID:25389185

  15. Purification, characterization and allergenicity assessment of 26kDa protein, a major allergen from Cicer arietinum.

    PubMed

    Verma, Alok Kumar; Sharma, Akanksha; Kumar, Sandeep; Gupta, Rinkesh Kumar; Kumar, Dinesh; Gupta, Kriti; Giridhar, B H; Das, Mukul; Dwivedi, Premendra D

    2016-06-01

    Chickpea (CP), a legume of the family Fabaceae, is an important nutrient-rich food providing protein, essential amino acids, vitamins, dietary fibre, and minerals. Unfortunately, several IgE-binding proteins in CP have been detected that are responsible for allergic manifestations in sensitized population. Therefore, the prevalence of CP induced allergy prompted us towards purification, characterization and allergenicity assessment of a major ∼26kDa protein from chickpea crude protein extract (CP-CPE). Purification of CP 26kDa protein was done using a combination of fractionation and anion exchange chromatography. This protein was further characterized as "Chain A, crystal structure of a plant albumin" from Cicer arietinum with Mol wt 25.8kDa by Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Further, allergenic potential of purified 25.8kDa protein was assessed using in vivo and in vitro model. Purified protein showed IgE-binding capacity with sensitized BALB/c mice and CP allergic patient's sera. Enhanced levels of specific and total IgE, MCP-1, MCPT-1, myeloperoxidase, histamine, prostaglandin D2, and cysteinyl leukotriene were found in sera of mice treated with CP ∼26kDa protein. Further, expressions of Th2 cytokines (i.e. IL-4, IL-5, IL-13), transcription factors (i.e. GATA-3, STAT-6, SOCS-3) and mast cell signaling proteins (Lyn, cFgr, Syk, PLC-γ2, PI-3K, PKC) were also found increased at mRNA and protein levels in the intestines of mice treated with CP ∼26kDa protein. In addition, enhanced release of β-hexosaminidase, histamine, cysteinyl leukotriene and prostaglandin D2 were observed in RBL2H3 cell line when treated (125μg) with CP 26kDa protein. Conclusively, in vivo and in vitro studies revealed the allergenic potential of purified CP 26kDa protein. Being a potential allergen, plant albumin may play a pivotal role in CP induced allergenicity. Current study will be helpful for better development of therapeutic approaches to

  16. IgE cross-reactivity between the major peanut allergen Ara h 2 and the non-homologous allergens Ara h 1 and Ara h 3

    USDA-ARS?s Scientific Manuscript database

    Ara h 1, a vicilin, Ara h 2, a 2S albumin, and Ara h 3, a legumin, are major33 peanut allergens. Ara h 2 is an important predictor of clinical reactivity to peanut, but co-sensitization to all three allergens is correlated with the severity of patients’ symptoms. We investigated whether co-sensitiza...

  17. Immunological cross-reactivity between olive and grass pollen: implication of major and minor allergens

    PubMed Central

    2014-01-01

    Background Grasses and olive trees are the most common sources of allergenic pollen worldwide. Although they share some allergens, there are few studies analyzing the in vitro cross-reactivity between them. The aim was to define the cross-reactivity between Olea europaea and Phleum pratense using well-characterized sera of allergic children from Madrid, Spain. Methods 66 patients (mean age 10.32+/−4.07 years) were included in the study. All suffered from rhinoconjuntivitis and/or asthma and had a positive skin test and/or specific IgE determination to olive and grass pollen. Serum sIgE to individual allergens was conducted and sIgE against different grass species and olive was also determined by ELISA. Inhibition assays were performed using two serum sources, containing, or not, sIgE to minor allergens. Mass spectrometry analysis was performed in both extracts. Results 59/66 (89.39%) children had a positive sIgE determination by ELISA to grasses and 57/66 (86.36%) to olive pollen. There was no significant correlation between sIgE levels to grass and olive. Inhibition assays demonstrated no cross-reactivity between P. pratense and olive pollen when using the pool containing mainly sIgE to major allergens, whereas minimal to moderate cross-reactivity was detected when the serum contained high sIgE titers to minor allergens. Proteomic analyses revealed the presence of 42 common proteins in grasses and olive pollens. Conclusion No in vitro cross-reactivity was observed when sIgE was mainly directed to major allergens. In our population, sensitization to olive and grasses is not due to cross-reactivity. The contribution of the major allergens seems to be determinant. PMID:24940475

  18. Fold stability during endolysosomal acidification is a key factor for allergenicity and immunogenicity of the major birch pollen allergen.

    PubMed

    Machado, Yoan; Freier, Regina; Scheiblhofer, Sandra; Thalhamer, Theresa; Mayr, Melissa; Briza, Peter; Grutsch, Sarina; Ahammer, Linda; Fuchs, Julian E; Wallnoefer, Hannes G; Isakovic, Almedina; Kohlbauer, Vera; Hinterholzer, Arthur; Steiner, Markus; Danzer, Martin; Horejs-Hoeck, Jutta; Ferreira, Fatima; Liedl, Klaus R; Tollinger, Martin; Lackner, Peter; Johnson, Christopher M; Brandstetter, Hans; Thalhamer, Josef; Weiss, Richard

    2016-05-01

    The search for intrinsic factors, which account for a protein's capability to act as an allergen, is ongoing. Fold stability has been identified as a molecular feature that affects processing and presentation, thereby influencing an antigen's immunologic properties. We assessed how changes in fold stability modulate the immunogenicity and sensitization capacity of the major birch pollen allergen Bet v 1. By exploiting an exhaustive virtual mutation screening, we generated mutants of the prototype allergen Bet v 1 with enhanced thermal and chemical stability and rigidity. Structural changes were analyzed by means of x-ray crystallography, nuclear magnetic resonance, and molecular dynamics simulations. Stability was monitored by using differential scanning calorimetry, circular dichroism, and Fourier transform infrared spectroscopy. Endolysosomal degradation was simulated in vitro by using the microsomal fraction of JAWS II cells, followed by liquid chromatography coupled to mass spectrometry. Immunologic properties were characterized in vitro by using a human T-cell line specific for the immunodominant epitope of Bet v 1 and in vivo in an adjuvant-free BALB/c mouse model. Fold stabilization of Bet v 1 was pH dependent and resulted in resistance to endosomal degradation at a pH of 5 or greater, affecting presentation of the immunodominant T-cell epitope in vitro. These properties translated in vivo into a strong allergy-promoting TH2-type immune response. Efficient TH2 cell activation required both an increased stability at the pH of the early endosome and efficient degradation at lower pH in the late endosomal/lysosomal compartment. Our data indicate that differential pH-dependent fold stability along endosomal maturation is an essential protein-inherent determinant of allergenicity. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  19. Molecular cloning, characterization, and expression of Cuc m 2, a major allergen in Cucumis melo

    PubMed Central

    Sankian, Mojtaba; Mahmoudi, Mahmoud; Varasteh, Abdol-Reza

    2013-01-01

    Background: Several studies reported the clinical features of IgE-mediated hypersensitivity after ingestion of melon. Melon allergy is a common IgE-mediated fruit allergy in Iran. This prompted us to investigate immunochemical and molecular properties of the major allergen in melon fruit, to compare the IgE-binding capacity of the natural protein with the recombinant allergen, and to determine cross-reactivity of the major allergen with closely-related allergens from other plants displaying clinical cross-reactivity with melon. Methods: Identification and molecular characterization of the major melon allergen were performed using IgE immunoblotting, allergen-specific ELISA, affinity-based purifications, cross-inhibition assays, cloning, and expression of the allergen in Escherichia coli. Results: Melon profilin was identified and isolated as a major IgE-binding component and designated as Cuc m 2. Sequencing corresponding cDNA revealed an open reading frame of 363 bp coding for 131 amino acid residues and two fragments of 171 bp and 383 bps for the 5’and 3’ UTRs, respectively. Significant cross-reactivity was found between melon profilin and Cynodon dactylon, tomato, peach, and grape profilins in cross-inhibition assays. Although the highest degree of amino acid identity was revealed with watermelon profilin, there was no significant cross-reactivity between melon and watermelon profilins. Conclusion: Melon profilin is the major IgE-binding component in melon extract, and the recombinant and natural forms exhibited similar IgE-binding capacities. A part of the fruit-fruit and pollen-fruit cross-reactions could be explained by the presence of this conserved protein; however, sequence homology provides insufficient information to predict IgE cross-reactivity of profilins. PMID:26989709

  20. Structural and immunologic characterization of Ara h 1 – a major peanut allergen

    USDA-ARS?s Scientific Manuscript database

    Allergic reactions to peanut and tree nuts is the major cause of anaphylaxis in the U.S. In this report; structural, immunologic, and bioinformatics analysis of natural, and a recombinant variant of the major peanut allergen Ara h 1 is presented. Small angle X-ray scattering studies show that natura...

  1. Identification of the major allergens of Charybdis feriatus (red crab) and its cross-reactivity with Portunus pelagicus (blue crab).

    PubMed

    Misnan, Rosmilah; Murad, Shahnaz; Yadzir, Zailatul Hani Mohd; Abdullah, Noormalin

    2012-12-01

    Tropomyosin and arginine kinase have been identified as the major allergens in multiple species of crab. Charybdis feriatus is an important commercial crab in this country. To characterize the major allergens of C. feriatus using a proteomics approach and subsequently to identify the allergens involved in cross-reactivity with Portunus pelagicus. Raw and boiled extracts of the crabs were prepared from crab meat. The protein profile of the extracts was determined by SDS-PAGE and two-dimensional electrophoresis (2-DE). Major allergens were identified by the immunoblotting test using sera from 50 patients with crab allergy. The major allergens were further identified by 2-DE immunoblotting. The major allergenic spots were then excised, digested by trypsin and identified by mass spectrometry analysis. The immunoblotting inhibition test was performed to study the crossreactivity between red crab and blue crab allergens using sera from 20 patients with allergy to both red and blue crabs. At least 20 protein bands between 13 to 250 kDa were detected in the SDS-PAGE gel of raw extract, while boiled extract procuced fewer protein bands. Proteins of 36 kDa and 41 kDa were recognized as the major allergens of the crab. The major allergenic spot sequences of the 36 and 41 kDa proteins were identified as crab tropomyosin and arginine kinase, respectively. All IgE-binding proteins, including both major allergens, were found to be cross-creative with P. pelagicus allergens. In addition to tropomyosin, arginine kinase was also identified as the major allergen of C. feriatus among our local crab-allergic patients. Cross-reactivity of this crab with P. pelagicus was demonstrated in this study.

  2. Interactions of epigallo-catechin 3-gallate and ovalbumin, the major allergen of egg white.

    PubMed

    Ognjenović, Jana; Stojadinović, Marija; Milčić, Miloš; Apostolović, Danijela; Vesić, Jelena; Stambolić, Ivan; Atanasković-Marković, Marina; Simonović, Miljan; Velickovic, Tanja Cirkovic

    2014-12-01

    Polyphenols, the potent plant secondary metabolites, have beneficial effects on human health, but the mechanism(s) by which these effects are exerted is not well understood. Here, we present the detailed analysis of the interactions between the major green tea catechin, epigallo-catechin 3-gallate (EGCG), and the major dietary protein and allergen, ovalbumin (OVA). We show that EGCG binds to the pocket that partly overlaps with the previously identified IgE-binding region in OVA, and that this interaction induces structural changes in the allergen. Moreover, our ex vivo studies reveal that OVA binds IgE and stimulates degranulation of basophils, and that its uptake by monocytes proceeds at a slower rate in the presence of EGCG. This study provides further evidence in support of the proposed mechanism by which EGCG interactions with the food allergens contribute to its diverse biological activities and may impair antigen uptake by antigen-presenting cells.

  3. Olive cultivar origin is a major cause of polymorphism for Ole e 1 pollen allergen

    PubMed Central

    Hamman-Khalifa, AbdelMounim; Castro, Antonio Jesús; Jiménez-López, José Carlos; Rodríguez-García, María Isabel; Alché, Juan de Dios

    2008-01-01

    Background Pollens from different olive (Olea europaea L.) cultivars have been shown to differ significantly in their content in Ole e 1 and in their overall allergenicity. This allergen is, in addition, characterized by a high degree of polymorphism in its sequence. The purpose of this study is to evaluate the putative presence of divergences in Ole e 1 sequences from different olive cultivars. Results RNA from pollen individually collected from 10 olive cultivars was used to amplify Ole e 1 sequences by RT-PCR, and the sequences were analyzed by using different bioinformatics tools. Numerous nucleotide substitutions were detected throughout the sequences, many of which resulted in amino acid substitutions in the deduced protein sequences. In most cases variability within a single variety was much lower than among varieties. Key amino acid changes in comparison with "canonical" sequences previously described in the literature included: a) the substitution of C19-relevant to the disulphide bond structure of the protein-, b) the presence of an additional N-glycosylation motif, and c) point substitutions affecting regions of Ole e 1 already described like relevant for the immunogenicity/allergenicity of the protein. Conclusion Varietal origin of olive pollen is a major factor determining the diversity of Ole e 1 variants. We consider this information of capital importance for the optimal design of efficient and safe allergen formulations, and useful for the genetic engineering of modified forms of the allergen among other applications. PMID:18218146

  4. Antisense-mediated silencing of a gene encoding a major ryegrass pollen allergen

    PubMed Central

    Bhalla, Prem L.; Swoboda, Ines; Singh, Mohan B.

    1999-01-01

    Type 1 allergic reactions, such as hay fever and allergic asthma, triggered by grass pollen allergens are a global health problem that affects ≈20% of the population in cool, temperate climates. Ryegrass is the dominant source of allergens because of its prodigious production of airborne pollen. Lol p 5 is the major allergenic protein of ryegrass pollen, judging from the fact that almost all of the individuals allergic to grass pollen show presence of serum IgE antibodies against this protein. Moreover, nearly two-thirds of the IgE reactivity of ryegrass pollen has been attributed to this protein. Therefore, it can be expected that down-regulation of Lol p 5 production can significantly reduce the allergic potential of ryegrass pollen. Here, we report down-regulation of Lol p 5 with an antisense construct targeted to the Lol p 5 gene in ryegrass. The expression of antisense RNA was regulated by a pollen-specific promoter. Immunoblot analysis of proteins with allergen-specific antibodies did not detect Lol p 5 in the transgenic pollen. The transgenic pollen showed remarkably reduced allergenicity as reflected by low IgE-binding capacity of pollen extract as compared with that of control pollen. The transgenic ryegrass plants in which Lol p 5 gene expression is perturbed showed normal fertile pollen development, indicating that genetic engineering of hypoallergenic grass plants is possible. PMID:10500236

  5. Antisense-mediated silencing of a gene encoding a major ryegrass pollen allergen.

    PubMed

    Bhalla, P L; Swoboda, I; Singh, M B

    1999-09-28

    Type 1 allergic reactions, such as hay fever and allergic asthma, triggered by grass pollen allergens are a global health problem that affects approximately 20% of the population in cool, temperate climates. Ryegrass is the dominant source of allergens because of its prodigious production of airborne pollen. Lol p 5 is the major allergenic protein of ryegrass pollen, judging from the fact that almost all of the individuals allergic to grass pollen show presence of serum IgE antibodies against this protein. Moreover, nearly two-thirds of the IgE reactivity of ryegrass pollen has been attributed to this protein. Therefore, it can be expected that down-regulation of Lol p 5 production can significantly reduce the allergic potential of ryegrass pollen. Here, we report down-regulation of Lol p 5 with an antisense construct targeted to the Lol p 5 gene in ryegrass. The expression of antisense RNA was regulated by a pollen-specific promoter. Immunoblot analysis of proteins with allergen-specific antibodies did not detect Lol p 5 in the transgenic pollen. The transgenic pollen showed remarkably reduced allergenicity as reflected by low IgE-binding capacity of pollen extract as compared with that of control pollen. The transgenic ryegrass plants in which Lol p 5 gene expression is perturbed showed normal fertile pollen development, indicating that genetic engineering of hypoallergenic grass plants is possible.

  6. HLA-DR and HLA-DP genotypes and immunoglobulin E responses to common major allergens.

    PubMed

    Young, R P; Dekker, J W; Wordsworth, B P; Schou, C; Pile, K D; Matthiesen, F; Rosenberg, W M; Bell, J I; Hopkin, J M; Cookson, W O

    1994-05-01

    In order to test for human histocompatibility leucocyte antigens (HLA) class II restriction of IgE responses, 431 subjects from 83 families were genotyped at the HLA-DR and HLA-DP loci and serotyped for IgE responses to six major allergens from common aero-allergen sources. A possible excess of HLA-DR1 was found in subjects who were responsive to Fel d I compared with those who were not (Odds Ratio (OR) = 2, P = 0.002), and a possible excess of HLA-DR4 was found in subjects responsive to Alt a I (OR = 1.9, P = 0.006). Increased sharing of HLA-DR/DP haplotypes was seen in sibling pairs responding to both allergens. Der p I, Der p II, Phl p V and Can f I were not associated with any definite excess of HLA-DR alleles. No significant correlations were seen with HLA-DP genotype and reactivity to any of the allergens. The results suggest class II HLA restriction is insufficient to account for individual differences in reactivity to common allergens.

  7. Identification and characterization of an arginine kinase as a major allergen from silkworm (Bombyx mori) larvae.

    PubMed

    Liu, Zhigang; Xia, Lixin; Wu, Yulan; Xia, Qingyou; Chen, Jiajie; Roux, Kenneth H

    2009-01-01

    The silkworm, Bombyx mori, is an important insect in the textile industry and its pupa are used in Chinese cuisine and traditional Chinese medicine. The silk, urine and dander of silkworms is often the cause of allergies in sericulture workers and the pupa has been found to be a food allergen in China. Recent studies have focused on reporting cases of silkworm allergies, but only a few studies have addressed the specific allergens present in the B. mori silkworm. We collected sera from 10 patients with a positive skin prick test to silkworm crude extract (SCE) and analyzed these samples by Western blot and ELISA. The cDNA of arginine kinase from the B. mori silkworm was also cloned and expressed in high yield in Escherichia coli. Allergenicity and cross-allergenicity of the recombinant B. mori arginine kinase (rBmAK) were investigated by ELISA inhibition assay. Collected sera all reacted to a 42-kDa protein in a Western blot with SCE as the antigen. Preincubation of sera with rBmAK eliminated the reactivity of the patients' sera to this 42-kDa band. All patient sera also exhibited positive reactivity to SCE in an ELISA assay. BmAK also demonstrated cross-reactivity with a recombinant AK from cockroach. Arginine kinase from the B. mori silkworm is a major allergen and crossreacts with cockroach AK. Copyright 2009 S. Karger AG, Basel.

  8. 27 CFR 5.32b - Petitions for exemption from major food allergen labeling.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2013-04-01 2013-04-01 false Petitions for exemption from major food allergen labeling. 5.32b Section 5.32b Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE TREASURY ALCOHOL LABELING AND ADVERTISING...

  9. 27 CFR 4.32b - Petitions for exemption from major food allergen labeling.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2013-04-01 2013-04-01 false Petitions for exemption from major food allergen labeling. 4.32b Section 4.32b Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE TREASURY ALCOHOL LABELING AND ADVERTISING OF...

  10. Ligand binding modulates the structural dynamics and compactness of the major birch pollen allergen.

    PubMed

    Grutsch, Sarina; Fuchs, Julian E; Freier, Regina; Kofler, Stefan; Bibi, Marium; Asam, Claudia; Wallner, Michael; Ferreira, Fátima; Brandstetter, Hans; Liedl, Klaus R; Tollinger, Martin

    2014-12-16

    Pathogenesis-related plant proteins of class-10 (PR-10) are essential for storage and transport of small molecules. A prominent member of the PR-10 family, the major birch pollen allergen Bet v 1, is the main cause of spring pollinosis in the temperate climate zone of the northern hemisphere. Bet v 1 binds various ligand molecules to its internal cavity, and immunologic effects of the presence of ligand have been discussed. However, the mechanism of binding has remained elusive. In this study, we show that in solution Bet v 1.0101 is conformationally heterogeneous and cannot be represented by a single structure. NMR relaxation data suggest that structural dynamics are fundamental for ligand access to the protein interior. Complex formation then leads to significant rigidification of the protein along with a compaction of its 3D structure. The data presented herein provide a structural basis for understanding the immunogenic and allergenic potential of ligand binding to Bet v 1 allergens.

  11. Tropomyosin and Actin Identified as Major Allergens of the Carpet Clam (Paphia textile) and the Effect of Cooking on Their Allergenicity

    PubMed Central

    Mohamad Yadzir, Zailatul Hani; Misnan, Rosmilah; Bakhtiar, Faizal; Abdullah, Noormalin; Murad, Shahnaz

    2015-01-01

    Objectives. To identify the major allergenic proteins of clam (Paphia textile) and to investigate the effect of different cooking methods on the allergenicity of these identified proteins. Methods. Clam protein extracts were separated by denaturing polyacrylamide gel electrophoresis. IgE reactive proteins were then analyzed by immunoblotting with sera from patients with positive skin prick tests (SPT) to the raw clam extract. Mass spectrometry was used to identify the major allergenic proteins of this clam. Results. Raw extract showed 12 protein bands (18–150 kDa). In contrast, fewer protein bands were seen in the boiled extract; those ranging from 40 to 150 kDa were denatured. The protein profiles were similarly altered by frying or roasting. The immunoblots of raw and boiled extracts yielded 10 and 2 IgE-binding proteins, respectively. The fried and roasted extracts showed only a single IgE-binding protein at 37 kDa. Mass spectrometry analysis of the 37 and 42 kDa major allergens indicated that these spots were tropomyosin and actin, respectively. Conclusion. The two major allergens of Paphia textile were identified as the thermostable tropomyosin and a new thermolabile allergen actin. PMID:26413512

  12. Tropomyosin and Actin Identified as Major Allergens of the Carpet Clam (Paphia textile) and the Effect of Cooking on Their Allergenicity.

    PubMed

    Mohamad Yadzir, Zailatul Hani; Misnan, Rosmilah; Bakhtiar, Faizal; Abdullah, Noormalin; Murad, Shahnaz

    2015-01-01

    To identify the major allergenic proteins of clam (Paphia textile) and to investigate the effect of different cooking methods on the allergenicity of these identified proteins. Clam protein extracts were separated by denaturing polyacrylamide gel electrophoresis. IgE reactive proteins were then analyzed by immunoblotting with sera from patients with positive skin prick tests (SPT) to the raw clam extract. Mass spectrometry was used to identify the major allergenic proteins of this clam. Raw extract showed 12 protein bands (18-150 kDa). In contrast, fewer protein bands were seen in the boiled extract; those ranging from 40 to 150 kDa were denatured. The protein profiles were similarly altered by frying or roasting. The immunoblots of raw and boiled extracts yielded 10 and 2 IgE-binding proteins, respectively. The fried and roasted extracts showed only a single IgE-binding protein at 37 kDa. Mass spectrometry analysis of the 37 and 42 kDa major allergens indicated that these spots were tropomyosin and actin, respectively. The two major allergens of Paphia textile were identified as the thermostable tropomyosin and a new thermolabile allergen actin.

  13. 2-D DIGE analysis of the proteome of extracts from peanut variants reveals striking differences in major allergen contents.

    PubMed

    Schmidt, Hendrik; Gelhaus, Christoph; Latendorf, Ties; Nebendahl, Melanie; Petersen, Arnd; Krause, Susanne; Leippe, Matthias; Becker, Wolf-Meinhard; Janssen, Ottmar

    2009-07-01

    Over the last decade, an increasing prevalence of peanut allergies was observed worldwide. Peanuts are meanwhile categorized among the most dangerous food allergens. This is particularly relevant since peanut-derived ingredients are widely used in industrial food production. To minimize the problem of hidden food allergens causing severe anaphylactic reactions, pre-packaged food containing peanut components needs to be classified according to European ruling since 2005. Food companies search for strategies to reduce the allergenicity of peanut-derived food additives either by genetically altering the allergen content or by identifying peanut varieties with low levels of major allergens. In our study, we focused on peanut extracts from Indonesia that apparently contain lower levels of the major Arachis hypogaea allergen 1 (Ara h 1). Basic extracts of Virginia-type and Indonesian peanuts were compared by 1- and 2-DE. We identified more than hundred individual components in these extracts by MS and provide a high-resolution allergen map that also includes so far unknown fragments of major peanut allergens. The reduced level of Ara h 1 associated with a significantly lower abundance of the most potent peanut allergen Ara h 2 in various Indonesian peanuts was also confirmed by Western blotting with monoclonal antibodies and sera of allergic patients.

  14. Ana o 2, a major cashew (Anacardium occidentale L.) nut allergen of the legumin family.

    PubMed

    Wang, Fang; Robotham, Jason M; Teuber, Suzanne S; Sathe, Shridhar K; Roux, Kenneth H

    2003-09-01

    We recently cloned and described a vicilin and showed it to be a major cashew allergen. Additional IgE-reactive cashew peptides of the legumin group and 2S albumin families have also been reported. Here, we attempt to clone, express and characterize a second major cashew allergen. A cashew cDNA library was screened with human IgE and rabbit IgG anti-cashew extract antisera, and a reactive nonvicilin clone was sequenced and expressed as a fusion protein in Escherichia coli. Immunoblotting was used to screen for reactivity with patients' sera, and inhibition of immunoblotting was used to identify the corresponding native peptides in cashew nut extract. The identified allergen was subjected to linear epitope mapping using SPOTs solid-phase synthetic peptide technology. Sequence analysis showed the selected clone, designated Ana o 2, to encode for a member of the legumin family (an 11S globulin) of seed storage proteins. By IgE immunoblotting, 13 of 21 sera (62%) from cashew-allergic patients were reactive. Immunoblot inhibition data showed that the native Ana o 2 constitutes a major band at approximately 33 kD and a minor band at approximately 53 kD. Probing of overlapping synthetic peptides with pooled human cashew-allergic sera identified 22 reactive peptides, 7 of which gave strong signals. Several Ana o 2 epitopes were shown to overlap those of the peanut legumin group allergen, Ara h 3, in position but with little sequence similarity. Greater positional overlap and identity was observed between Ana o 2 and soybean glycinin epitopes. We conclude that this legumin-like protein is a major allergen in cashew nut. Copyright 2003 S. Karger AG, Basel

  15. Comparison of four variants of a major allergen in hazelnut (Corylus avellana) Cor a 1.04 with the major hazel pollen allergen Cor a 1.01.

    PubMed

    Lüttkopf, D; Müller, U; Skov, P S; Ballmer-Weber, B K; Wüthrich, B; Skamstrup Hansen, K; Poulsen, L K; Kästner, M; Haustein, D; Vieths, S

    2002-01-01

    The aim of this study was to produce the Bet v 1-related major hazelnut allergen Cor a 1.0401 and variants thereof as recombinant allergens, and to compare their immuno-reactivity with the major hazel pollen allergen using sera of patients whose hazelnut allergy recently was confirmed by double-blind placebo-controlled food challenges (DBPCFC) in a multicenter study. Total RNA was isolated from immature hazelnuts and transcribed into cDNA. Full length coding DNA obtained by PCR-strategy was subcloned into pTYB11 vector and expressed in E. coli ER2566 cells. Native non-fusion target proteins were purified by DTT-induced self-cleavage of the intein-tagged N-terminal fusion proteins. IgE reactivity of the recombinant allergens was tested by enzyme allergosorbent test (EAST), EAST-inhibition, immunoblot-inhibition and histamine release assays. Four recombinant allergens were produced showing deduced amino acid sequence identities among each other of 97-99%, and were considered as variants Cor a 1.0401 (GenBank Accession no.: AF136945), Cor a 1.0402 (AF323973), Cor a 1.0403 (AF323974) and Cor a 1.0404 (AF323975). Cor a 1.0402 and 03 only differed in a C4S exchange. Cor a 1.0404 had a unique proline residue in position 99. Surprisingly, only 63% identity was revealed with hazel pollen Cor a 1. EAST with 43 sera of patients with positive DBPCFC to hazelnut indicated IgE reactivity to Cor a 1.0401 in 95% of the sera, to Cor a 1.0402 in 93%, to Cor a 1.0403 in 91%, and in only 74% of the sera to the proline variant Cor a 1.0404. The allergenic activity of the four variants was confirmed by histamine release assays in 15 hazelnut-allergic patients stimulated with the four variants and controls. Eleven sera were positive with extract from native hazelnut, 13 with rCor a 1.0401, 12 with rCor a 1.0402, 11 with rCor a 1.0403, and only two with rCor a 1.0404 containing the proline exchange. The high IgE binding variant Cor a 1.0401 showed only partial IgE cross-reactivity with

  16. Changes in phenols contents from buckwheat sprouts during growth stage.

    PubMed

    Koyama, Masahiro; Nakamura, Chiho; Nakamura, Kozo

    2013-02-01

    Germinated buckwheat is buckwheat seeds soaked in water just until it begins to bud. Buckwheat sprouts are seedling plants of buckwheat grown up to 10-15 cm. The purpose of this study was to determine the optimal growth period for accumulating the most abundant functional phenol(s) in germinated buckwheat that had been soaked in darkness and buckwheat sprouts cultivated by hydroponic culture. The rutin contained in germinated buckwheat was analyzed by CE (capillary electrophoresis). Phenols, including isoorientin, orientin, isovitexin, vitexin, and rutin were separated from buckwheat sprouts by HPLC and identified by LC-MS. The highest rutin content in germinated buckwheat was found to be 15.8 mg/100 g DW at 20 h after germination. Buckwheat sprouts contained five kinds of major phenols. The highest amounts of isoorientin, orientin, isovitexin, and vitexin were measured at day 3, with the exception of rutin, and then a gradual decrease was observed as the sprouts grew. The quantities of isoorientin, orientin, isovitexin, and vitexin at day 3 were 5.8, 11.7, 26.2, and 28.9 mg/100 g FW, respectively. The rutin content rapidly increased to 109.0 mg/100 g FW until day 6. The highest total phenols in buckwheat sprouts were 162.9 mg/100 g FW at day 6. Germinated buckwheat soaked for 20 h and buckwheat sprouts cultivated for 6 days were rich in dietary phenol(s), which makes these plants a valuable functional food for human consumption.

  17. Structure of the Major Apple Allergen Mal d 1.

    PubMed

    Ahammer, Linda; Grutsch, Sarina; Kamenik, Anna S; Liedl, Klaus R; Tollinger, Martin

    2017-03-01

    More than 70% of birch pollen-allergic patients develop allergic cross-reactions to the major allergen found in apple fruits (Malus domestica), the 17.5 kDa protein Mal d 1. Allergic reactions against this protein result from initial sensitization to the major allergen from birch pollen, Bet v 1. Immunologic cross-reactivity of Bet v 1-specific IgE antibodies with Mal d 1 after apple consumption can subsequently provoke severe oral allergic syndromes. This study presents the three-dimensional NMR solution structure of Mal d 1 (isoform Mal d 1.0101, initially cloned from 'Granny Smith' apples). This protein is composed of a seven-stranded antiparallel β-sheet and three α-helices that form a large internal cavity, similar to Bet v 1 and other cross-reactive food allergens. The Mal d 1 structure provides the basis for elucidating the details of allergic cross-reactivity between birch pollen and apple allergens on a molecular level.

  18. Structure of the Major Apple Allergen Mal d 1

    PubMed Central

    2017-01-01

    More than 70% of birch pollen-allergic patients develop allergic cross-reactions to the major allergen found in apple fruits (Malus domestica), the 17.5 kDa protein Mal d 1. Allergic reactions against this protein result from initial sensitization to the major allergen from birch pollen, Bet v 1. Immunologic cross-reactivity of Bet v 1-specific IgE antibodies with Mal d 1 after apple consumption can subsequently provoke severe oral allergic syndromes. This study presents the three-dimensional NMR solution structure of Mal d 1 (isoform Mal d 1.0101, initially cloned from ‘Granny Smith’ apples). This protein is composed of a seven-stranded antiparallel β-sheet and three α-helices that form a large internal cavity, similar to Bet v 1 and other cross-reactive food allergens. The Mal d 1 structure provides the basis for elucidating the details of allergic cross-reactivity between birch pollen and apple allergens on a molecular level. PMID:28161953

  19. Multiple B- and T-cell epitopes on a major allergen of Kentucky Bluegrass pollen.

    PubMed Central

    Zhang, L; Yang, M; Chong, P; Mohapatra, S S

    1996-01-01

    The B- and T-cell epitopes of a recombinant grass allergen, rKBG60, were delineated using a set of overlapping synthetic peptides. Direct binding by enzyme-linked immunosorbent assay (ELISA) utilizing serum pools led to the identification of 13 murine immunoglobulin-, and nine to 13 human IgG- and five to seven human IgE-reactive overlapping peptides. Of the peptides which bound to human IgE antibodies, all but three peptides bound to human and/or murine IgG antibodies. Furthermore, eight out of 12 synthetic peptides induced antigen-specific antibodies in mice, suggesting that these peptides contained epitopes that recognized and/or induced T cells. These results, in conjunction with cross-recognition of different peptides at the C-terminus of rKBG60 by antibodies to neighbouring or non-overlapping peptides suggest that the C-terminus of this antigen represents a dominant antigenic and allergenic site. Peripheral blood mononuclear cell (PBMC) proliferation studies using these synthetic peptides for 13 grass allergic individuals indicated that seven potential human T-cell epitopes exist on this allergen. Taken together, the results demonstrate that multiple B- and T-cell epitopes exist on this major group of grass allergens, the majority of which are localized at the C-terminus of this antigen. PMID:8698392

  20. Structural and Immunologic Characterization of Ara h 1, a Major Peanut Allergen*

    PubMed Central

    Chruszcz, Maksymilian; Maleki, Soheila J.; Majorek, Karolina A.; Demas, Matthew; Bublin, Merima; Solberg, Robert; Hurlburt, Barry K.; Ruan, Sanbao; Mattisohn, Christopher P.; Breiteneder, Heimo; Minor, Wladek

    2011-01-01

    Allergic reactions to peanuts and tree nuts are major causes of anaphylaxis in the United States. We compare different properties of natural and recombinant versions of Ara h 1, a major peanut allergen, through structural, immunologic, and bioinformatics analyses. Small angle x-ray scattering studies show that natural Ara h 1 forms higher molecular weight aggregates in solution. In contrast, the full-length recombinant protein is partially unfolded and exists as a monomer. The crystal structure of the Ara h 1 core (residues 170–586) shows that the central part of the allergen has a bicupin fold, which is in agreement with our bioinformatics analysis. In its crystalline state, the core region of Ara h 1 forms trimeric assemblies, while in solution the protein exists as higher molecular weight assemblies. This finding reveals that the residues forming the core region of the protein are sufficient for formation of Ara h 1 trimers and higher order oligomers. Natural and recombinant variants of proteins tested in in vitro gastric and duodenal digestion assays show that the natural protein is the most stable form, followed by the recombinant Ara h 1 core fragment and the full-length recombinant protein. Additionally, IgE binding studies reveal that the natural and recombinant allergens have different patterns of interaction with IgE antibodies. The molecular basis of cross-reactivity between vicilin allergens is also elucidated. PMID:21917921

  1. Identification of IgE-binding proteins from Lepidoglyphus destructor and production of monoclonal antibodies to a major allergen.

    PubMed

    Ventas, P; Carreira, J; Polo, F

    1991-08-01

    The allergen composition of one of the most important storage mites, Lepidoglyphus destructor, has been studied by immunodetection after SDS-PAGE with individual patient sera. An allergenic polypeptide of 14 kDa was identified with 95% of the sera. This major allergen was isolated in the supernatant of 60% ammonium sulfate salt precipitation of the whole extract, which was subsequently used to immunize BALB/c mice so as to produce monoclonal antibodies. Four mAbs recognizing molecules with IgE-binding ability were obtained. The specificity of the mAbs was assayed against different allergenic extracts, and the molecules recognized by them were characterized by immunoblotting. Two mAbs (Le5B5 and Le9E4) were directed to the 14-kDa allergen; the other two to several proteins of lesser allergenic significance.

  2. Comparison of the digestibility of the major peanut allergens in thermally processed peanuts and in pure form

    USDA-ARS?s Scientific Manuscript database

    It has been suggested that boiling or frying of peanuts lead to less allergenic products than roasting. Here, we have compared the digestibility of the major peanut allergens in the context of peanuts subjected to boiling, frying, or roasting, and in purified form. The soluble peanut extracts and ...

  3. Functional compounds in fermented buckwheat sprouts.

    PubMed

    Maejima, Yasunori; Nakatsugawa, Hiroki; Ichida, Daiki; Maejima, Mayumi; Aoyagi, Yasuo; Maoka, Takashi; Etoh, Hideo

    2011-01-01

    Fermented buckwheat sprouts (FBS) are used as multifunctional foods. Their production process includes fermentation with lactic acid bacteria. The major strains were found to include Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus pentosus, Lactococcus lactis subsp. lactis, and Pediococcus pentosaceus in an investigation of the lactic acid bacteria. We searched for the functional components, and nicotianamine (NA) and 2″-hydroxynicotianamine (HNA) were identified as angiotensin I-converting enzyme (ACE) inhibitors. NA and HNA increased during fermentation. Indole-3-ethanol was identified as an antioxidant (a SOD active substance), and may have been generated from tryptophan during fermentation because it was not contained in green buckwheat juice. A safety test demonstrated that FBS contained were safe functional food components, showing negative results in buckwheat allergy tests. Any buckwheat allergy substances might have been degraded during the fermentation process.

  4. Crystal structure of prunin-1, a major component of the almond (Prunus dulcis) allergen amandin.

    PubMed

    Jin, Tengchuan; Albillos, Silvia M; Guo, Feng; Howard, Andrew; Fu, Tong-Jen; Kothary, Mahendra H; Zhang, Yu-Zhu

    2009-09-23

    Seed storage proteins are accumulated during seed development and act as a reserve of nutrition for seed germination and young sprout growth. Plant seeds play an important role in human nutrition by providing a relatively inexpensive source of protein. However, many plant foods contain allergenic proteins, and the number of people suffering from food allergies has increased rapidly in recent years. The 11S globulins are the most widespread seed storage proteins, present in monocotyledonous and dicotyledonous seeds as well as in gymnosperms (conifers) and other spermatophytes. This family of proteins accounts for a number of known major food allergens. They are of interest to both the public and industry due to food safety concerns. Because of the interests in the structural basis of the allergenicity of food allergens, we sought to determine the crystal structure of Pru1, the major component of the 11 S storage protein from almonds. The structure was refined to 2.4 A, and the R/Rfree for the final refined structure is 17.2/22.9. Pru1 is a hexamer made of two trimers. Most of the back-to-back trimer-trimer association was contributed by monomer-monomer interactions. An alpha helix (helix 6) at the C-terminal end of the acidic domain of one of the interacting monomers lies at the cleft of the two protomers. The residues in this helix correspond to a flexible region in the peanut allergen Ara h 3 that encompasses a previously defined linear IgE epitope.

  5. Hypoallergenic derivatives of the major birch pollen allergen Bet v 1 obtained by rational sequence reassembly.

    PubMed

    Campana, Raffaela; Vrtala, Susanne; Maderegger, Bernhard; Jertschin, Peter; Stegfellner, Gottfried; Swoboda, Ines; Focke-Tejkl, Margarete; Blatt, Katharina; Gieras, Anna; Zafred, Domen; Neubauer, Angela; Valent, Peter; Keller, Walter; Spitzauer, Susanne; Valenta, Rudolf

    2010-11-01

    At least 100 million patients suffer from birch pollen allergy. Rational design of recombinant derivatives of the major birch pollen allergen, Bet v 1, characterized by reduced IgE reactivity, preservation of sequences relevant for the induction of allergen-specific blocking IgG, and maintenance of T-cell epitopes for immunotherapy of birch pollen allergy. Three recombinant mosaic proteins derived from Bet v 1 were generated by reassembly of codon-optimized genes coding for Bet v 1 fragments containing the elements for the induction of allergen-specific blocking IgG antibodies and the major T-cell epitopes. The proteins were expressed in Escherichia coli as recombinant mosaic molecules and compared with the Bet v 1 wild-type protein by chemical and structural methods, regarding IgE-binding and IgG-binding capacity, in basophil activation assays and tested for the in vivo induction of IgG responses. Three recombinant Bet v 1 (rBet v 1) mosaic proteins with strongly reduced IgE reactivity and allergenic activity were expressed and purified. Immunization with the recombinant hypoallergens induced IgG antibodies that inhibited IgE reactivity of patients with allergy to Bet v 1 comparable to those induced with the rBet v 1 wild-type allergen. We report the generation and preclinical characterization of 3 hypoallergenic rBet v 1 derivatives with suitable properties for immunotherapy of birch pollen allergy. Copyright © 2010 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

  6. Crystal Structure of Prunin-1, a Major Component of the Almond (Prunus dulcis) Allergen Amandin

    SciTech Connect

    Jin, Tengchuan; Albillos, Silvia M.; Guo, Feng; Howard, Andrew; Fu, Tong-Jen; Kothary, Mahendra H.; Zhang, Yu-Zhu

    2010-10-28

    Seed storage proteins are accumulated during seed development and act as a reserve of nutrition for seed germination and young sprout growth. Plant seeds play an important role in human nutrition by providing a relatively inexpensive source of protein. However, many plant foods contain allergenic proteins, and the number of people suffering from food allergies has increased rapidly in recent years. The 11S globulins are the most widespread seed storage proteins, present in monocotyledonous and dicotyledonous seeds as well as in gymnosperms (conifers) and other spermatophytes. This family of proteins accounts for a number of known major food allergens. They are of interest to both the public and industry due to food safety concerns. Because of the interests in the structural basis of the allergenicity of food allergens, we sought to determine the crystal structure of Pru1, the major component of the 11 S storage protein from almonds. The structure was refined to 2.4 {angstrom}, and the R/Rfree for the final refined structure is 17.2/22.9. Pru1 is a hexamer made of two trimers. Most of the back-to-back trimer-trimer association was contributed by monomer-monomer interactions. An {alpha} helix (helix 6) at the C-terminal end of the acidic domain of one of the interacting monomers lies at the cleft of the two protomers. The residues in this helix correspond to a flexible region in the peanut allergen Ara h 3 that encompasses a previously defined linear IgE epitope.

  7. Molecular Cloning and Expression of a New Major Allergen, Ani s 14, from Anisakis simplex.

    PubMed

    Kobayashi, Yukihiro; Kakemoto, Seiko; Shimakura, Kuniyoshi; Shiomi, Kazuo

    2015-01-01

    The nematode Anisakis simplex is a representative parasite infecting marine animals. When third stage larvae of A. simplex infecting fish and squids are ingested by humans, individuals previously sensitized by this parasite may experience IgE-mediated allergic reactions. So far, as many as 13 kinds of proteins (Ani s 1-13) have been identified as A. simplex allergens but several more unknown allergens are suggested to exist. In this study, therefore, chemiluminescent immunoscreening of an expression cDNA library constructed from the third stage larvae was conducted to identify a new allergen. As a result, an IgE-positive clone coding for a 23.5 kDa protein (named Ani s 14) composed of 217 amino acid residues was isolated. The regions 4-147 and 34-123 of Ani s 14 share 31% identity with the region 796-940 of Ani s 7 and 32% identity with the region 2-91 of Ani s 12, respectively. Recombinant Ani s 14 was successfully expressed in Escherichia coli as a His-tagged protein and shown to be IgE reactive to 14 (54%) of 26 sera from Anisakis-allergic patients. In conclusion, Ani s 14 is a new major allergen of A. simplex that is specific to Anisakis-allergic patients.

  8. Identification of the cysteine protease Amb a 11 as a novel major allergen from short ragweed.

    PubMed

    Bouley, Julien; Groeme, Rachel; Le Mignon, Maxime; Jain, Karine; Chabre, Henri; Bordas-Le Floch, Véronique; Couret, Marie-Noëlle; Bussières, Laetitia; Lautrette, Aurélie; Naveau, Marie; Baron-Bodo, Véronique; Lombardi, Vincent; Mascarell, Laurent; Batard, Thierry; Nony, Emmanuel; Moingeon, Philippe

    2015-10-01

    Allergy to pollen from short ragweed (Ambrosia artemisiifolia) is a serious and expanding health problem in the United States and in Europe. We sought to investigate the presence of undescribed allergens in ragweed pollen. Ragweed pollen proteins were submitted to high-resolution gel electrophoresis and tested for IgE reactivity by using sera from 92 American or European donors with ragweed allergy. Pollen transcriptome sequencing, mass spectrometry (MS), and recombinant DNA technologies were applied to characterize new IgE-binding proteins. High-resolution IgE immunoblotting experiments revealed that 50 (54%) of 92 patients with ragweed allergy were sensitized to a 37-kDa allergen distinct from Amb a 1. The full-length cDNA sequence for this molecule was obtained by means of PCR cloning after MS sequencing of the protein combined with ragweed pollen RNA sequencing. The purified allergen, termed Amb a 11, was fully characterized by MS and confirmed to react with IgEs from 66% of patients. This molecule is a 262-amino-acid thiol protease of the papain family expressed as a combination of isoforms and glycoforms after proteolytic removal of N- and C-terminal propeptides from a proform. Three-dimensional modeling revealed a high structural homology with known cysteine proteases, including the mite Der p 1 allergen. The protease activity of Amb a 11, as well as its capacity to activate basophils from patients with ragweed allergy, were confirmed. The production of a nonglycosylated recombinant form of Amb a 11 in Escherichia coli established that glycosylation is not required for IgE binding. We identified the cysteine protease Amb a 11 as a new major allergen from ragweed pollen. Given the similar physicochemical properties shared by the 2 major allergens, we hypothesize that part of the allergenic activity previously ascribed to Amb a 1 is rather borne by Amb a 11. Copyright © 2015 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All

  9. The lipid interaction capacity of Sin a 2 and Ara h 1, major mustard and peanut allergens of the cupin superfamily, endorses allergenicity.

    PubMed

    Angelina, A; Sirvent, S; Palladino, C; Vereda, A; Cuesta-Herranz, J; Eiwegger, T; Rodríguez, R; Breiteneder, H; Villalba, M; Palomares, O

    2016-09-01

    Sin a 2 (11S globulin) and Ara h 1 (7S globulin) are major allergens from yellow mustard seeds and peanut, respectively. The ability of these two allergens to interact with lipid components remains unknown. To study the capacity of Sin a 2 and Ara h 1 to interact with lipid components and the potential effects of such interaction in their allergenic capacity. Spectroscopic and SDS-PAGE binding assays of Sin a 2 and Ara h 1 with different phospholipid vesicles and gastrointestinal and endolysosomal digestions in the presence or absence of lipids were performed. The capacity of human monocyte-derived dendritic cells (hmoDCs) to capture food allergens in the presence or absence of lipids, the induced cytokine signature, and the effect of allergens and lipids to regulate TLR2-L-induced NF-kB/AP-1 activation in THP1 cells were analyzed. Sin a 2 and Ara h 1 bind phosphatidylglycerol (PG) acid but not phosphatidylcholine (PC) vesicles in a pH-dependent manner. The interaction of these two allergens with lipid components confers resistance to gastrointestinal digestion, reduces their uptake by hmoDCs, and enhances their stability to microsomal degradation. Mustard and peanut lipids favor a proinflammatory environment by increasing the IL-4/IL-10 ratio and IL-1β production by hmoDCs. The presence of mustard lipids and PG vesicles inhibits TLR2-L-induced NF-kB/AP-1 activation in THP1 cells. Sin a 2 and Ara h 1 interact with lipid components, which might well contribute to explain the potent allergenic capacity of these two clinically relevant allergens belonging to the cupin superfamily. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  10. Nonanaphylactic synthetic peptides derived from B cell epitopes of the major grass pollen allergen, Phl p 1, for allergy vaccination.

    PubMed

    Focke, M; Mahler, V; Ball, T; Sperr, W R; Majlesi, Y; Valent, P; Kraft, D; Valenta, R

    2001-09-01

    Worldwide more than 200 million individuals are allergic to group 1 grass pollen allergens. We have used the major timothy grass pollen allergen Phl p 1, which cross-reacts with most grass-, corn-, and monocot-derived group 1 allergens to develop a generally applicable strategy for the production of hypoallergenic allergy vaccines. On the basis of the experimentally determined B cell epitopes of Phl p 1, we have synthesized five synthetic peptides. These peptides are derived from the major Phl p 1 IgE epitopes and were between 28-32 amino acids long. We demonstrate by nuclear magnetic resonance that the peptides exhibit no secondary and tertiary structure and accordingly failed to bind IgE antibodies from grass pollen allergic patients. The five peptides, as well as an equimolar mixture thereof, lacked allergenic activity as demonstrated by basophil histamine release and skin test experiments in grass pollen allergic patients. When used as immunogens in mice and rabbits, the peptides induced protective IgG antibodies, which recognized the complete Phl p 1 wild-type allergen and group 1 allergens from other grass species. Moreover, peptide-induced antibodies inhibited the binding of grass pollen allergic patients IgE antibodies to the wild-type allergen. We thus demonstrate that synthetic hypoallergenic peptides derived from B cell epitopes of major allergens represent safe vaccine candidates for the treatment of IgE- mediated allergies.

  11. Antibody reactivity to the major fish allergen parvalbumin is determined by isoforms and impact of thermal processing.

    PubMed

    Saptarshi, Shruti R; Sharp, Michael F; Kamath, Sandip D; Lopata, Andreas L

    2014-04-01

    The EF-hand calcium binding protein, parvalbumin, is a major fish allergen. Detection of this allergen is often difficult due to its structural diversity among various fish species. The aim of this study was to evaluate the cross-reactivity of parvalbumin in a comprehensive range of bony and cartilaginous fish, from the Asia-Pacific region, and conduct a molecular analysis of this highly allergenic protein. Using the monoclonal anti-parvalbumin antibody PARV-19, we demonstrated the presence of monomeric and oligomeric parvalbumin in all fish analysed, except for gummy shark a cartilaginous fish. Heat processing of this allergen greatly affected its antibody reactivity. While heating caused a reduction in antibody reactivity to multimeric forms of parvalbumins for most bony fish, a complete loss of reactivity was observed for cartilaginous fish. Molecular analysis demonstrated that parvalbumin cross-reactivity, among fish species, is due to the molecular phylogenetic association of this major fish allergen.

  12. Purification of the major group 1 allergen from Bahia grass pollen, Pas n 1.

    PubMed

    Drew, Alexander C; Davies, Janet M; Dang, Thanh D; Rolland, Jennifer M; O'Hehir, Robyn E

    2011-01-01

    Group 1 grass pollen allergens are glycoproteins of the β-expansin family. They are a predominant component of pollen and are potent allergens with a high frequency of serum IgE reactivity in grass pollen-allergic patients. Bahia grass is distinct from temperate grasses and has a prolonged pollination period and wide distribution in warmer climates. Here we describe the purification of the group 1 pollen allergen, Pas n 1, from Bahia grass (Paspalum notatum), an important subtropical aeroallergen source. Pas n 1 was purified from an aqueous Bahia grass pollen extract by ammonium sulphate precipitation, hydrophobic interaction and size exclusion chromatography, and assessed by one- and two-dimensional gel electrophoresis, immunoblotting and ELISA. Pas n 1 was purified to a single 29-kDa protein band containing two dominant isoforms detected by an allergen-specific monoclonal antibody and serum IgE of a Bahia grass pollen-allergic donor. The frequency of serum IgE reactivity with purified Pas n 1 in 51 Bahia grass pollen-allergic patients was 90.6%. Serum IgE reactivity with purified Pas n 1 was highly correlated with serum IgE reactivity with Bahia grass pollen extract and recombinant Pas n 1 (r = 0.821 and 0.913, respectively). Pas n 1 is a major allergen reactive at high frequency with serum IgE of Bahia grass pollen-allergic patients. Purified natural Pas n 1 has utility for improved specific diagnosis and immunotherapy for Bahia grass pollen allergy. Copyright © 2010 S. Karger AG, Basel.

  13. MALDI-TOF MS analysis of labile Lolium perenne major allergens in mixes.

    PubMed

    Irañeta, S G; Acosta, D M; Duran, R; Apicella, C; Orlando, U D; Seoane, M A; Alonso, A; Duschak, V G

    2008-08-01

    It is well known that allergen extracts used for specific therapy of allergic disorders are commonly stored as mixtures, causing an alteration of its stability. The aim of this report is to identify pollen allergens susceptible to degradation during storage of mixtures containing different sources of proteases in the absence of glycerol as a preserving agent. Mixes containing Lolium perenne (Lol p) pollen extract with either Aspergillus fumigatus or Periplaneta americana extracts were prepared and co-incubated for 90 days at 4 degrees C. Samples were taken off at fixed times and comparatively tested by in vitro and in vivo assays with atopic patients. Selected pollinic allergens were subjected to MALDI-TOF MS analysis. ELISA inhibition evidenced the loss of potency from ryegrass extract, and immunoblotting assays showed the degradation of specific pollinic allergens during storage of mixtures containing protease-rich sources. An in vivo intradermal skin assay confirmed the gradual loss of the biological activity of L. perenne pollen extract co-incubated with non-related protease-rich extracts in comparison with that of the control pollen extract. MALDI-TOF MS analysis allowed us to determine that Lol p 1 and Lol p 5 are susceptible to proteolysis whereas Lol p 4 was found to be resistant to degradation during storage. Lol p 1 and Lol p 5 degradation is responsible for the loss of the biological activity of L. perenne pollen extract when co-incubated with protease-rich fungal and cockroach extracts in the same vial for months in the absence of glycerol as a preserving agent. The integrity of these major allergens must be preserved to increase the vaccine stability and to assure efficacy when mixes are used for immunotherapy.

  14. NMR resonance assignments of the major apple allergen Mal d 1.

    PubMed

    Ahammer, Linda; Grutsch, Sarina; Tollinger, Martin

    2016-10-01

    The major apple allergen Mal d 1 is the predominant cause of apple (Malus domestica) allergies in large parts of Europe and Northern America. Allergic reactions against this 17.5 kDa protein are the consequence of initial sensitization to the structurally homologous major allergen from birch pollen, Bet v 1. Consumption of apples can subsequently provoke immunologic cross-reactivity of Bet v 1-specific antibodies with Mal d 1 and trigger severe oral allergic syndroms, affecting more than 70 % of all individuals that are sensitized to birch pollen. While the accumulated immunological data suggest that Mal d 1 has a three-dimensional fold that is similar to Bet v 1, experimental structural data for this protein are not available to date. In a first step towards structural characterization of Mal d 1, backbone and side chain (1)H, (13)C and (15)N chemical shifts of the isoform Mal d 1.0101 were assigned. The NMR-chemical shift data show that this protein is composed of seven β-strands and three α-helices, which is in accordance with the reported secondary structure of the major birch pollen allergen, indicating that Mal d 1 and Bet v 1 indeed have similar three-dimensional folds. The next stage in the characterization of Mal d 1 will be to utilize these resonance assignments in solving the solution structure of this protein.

  15. Molecular basis of IgE-recognition of Lol p 5, a major allergen of rye-grass pollen.

    PubMed

    Suphioglu, C; Blaher, B; Rolland, J M; McCluskey, J; Schäppi, G; Kenrick, J; Singh, M B; Knox, R B

    1998-04-01

    Grass pollen, especially of rye-grass (Lolium perenne). represents an important cause of type I allergy. Identification of IgE-binding (allergenic) epitopes of major grass pollen allergens is essential for understanding the molecular basis of interaction between allergens and human IgE antibodies and therefore facilitates the devising of safer and more effective diagnostic and immunotherapy reagents. The aim of this study was to identify the allergenic epitopes of Lol p 5, a major allergen of rye-grass pollen, immunodissect these epitopes further so that the amino acid residues critical for antibody binding can be determined and investigate the conservation and nature of these epitopes within the context of the natural grass pollen allergens. Peptides, 12-13 amino acid residues long and overlapping each other by 4 amino acid residues, based on the entire deduced amino acid sequence of the coding region of Lol p 5, were synthesised and assayed for IgE-binding. Two strong IgE-binding epitopes (Lol p 5 (49-60) and (265-276), referred to as peptides 7 and 34, respectively) were identified. These epitopes were further resolved by truncated peptides and amino acid replacement studies and the amino acid residues critical for IgE-binding determined (Lol p 5 (49-60) residue Lys57 and (265-276) residue Lys275). Sequences of these epitopes were conserved in related allergens and may form the conserved allergenic domains responsible for the cross-reactivity observed between pollen allergens of taxonomically related grasses. Furthermore, due to its strong IgE-reactivity, synthetic peptide Lol p 5 (265-276) was used to affinity-purify specific IgE antibodies which recognised proteins of other clinically important grass pollens. further indicating presence of allergenic cross-reactivity at the level of allergenic epitope. Moreover, Lol p 5 (265 276) demonstrated a strong capacity to inhibit IgE-binding to natural rye-grass pollen proteins highlighting the antibody accessibility

  16. Purification and characterization of Lep d I, a major allergen from the mite Lepidoglyphus destructor.

    PubMed

    Ventas, P; Carreira, J; Polo, F

    1992-04-01

    A major allergen of the storage mite Lepidoglyphus destructor (Lep d I) has been purified by affinity chromatography using an anti-Lep d I monoclonal antibody. The purity of the protein obtained by this procedure was assessed by reverse-phase HPLC. Lep d I displayed a molecular weight of 14 kD on SDS-PAGE under non-reducing conditions, and 16 kD in the presence of a reducing agent. Analytical IEF revealed a little charge microheterogeneity, showing three bands with pIs 7.6-7.8. Purified Lep d I retained IgE-binding ability, as proved by immunoblotting experiments after SDS-PAGE and RAST with individual sera from L. destructor-sensitive patients. Results from the latter technique demonstrated that 87% of L. destructor-allergic patients had specific IgE to Lep d I, and a good correlation between IgE reactivity with L. destructor extract and Lep d I was found. In addition, RAST inhibition experiments showed that IgE-binding sites on Lep d I are major L. destructor-allergenic determinants, since Lep d I could inhibit up to 75% the binding of specific IgE to L. destructor extract; on the other hand, Lep d I did not cross-react with D. pteronyssinus allergens.

  17. A proteomic study of the major allergens from yellow jacket venoms.

    PubMed

    Kolarich, Daniel; Loos, Andreas; Léonard, Renaud; Mach, Lukas; Marzban, Gorji; Hemmer, Wolfgang; Altmann, Friedrich

    2007-05-01

    The venoms of stinging insects belong to the most dangerous allergen sources and can cause fatal anaphylactic reactions. Reliable prediction of a patient's risk to anaphylactic reactions is vital, and diagnosis requires the knowledge of the relevant allergens. Recently, a new hyaluronidase -like glycoprotein from Vespula vulgaris (Ves v 2b) was identified. This led us to investigate hyaluronidases and also other major allergens from V. germanica and four additional Vespula species. By MALDI-Q-TOF-MS, the new hyaluronidase-like protein was shown to be the major component of the 43-kDa band in all Vespula species studied. LC-ESI-Q-TOF-MS/MS sequencing of Ves g 2a and Ves g 2b facilitated the cloning of their cDNA. Ves v 2b and Ves g 2b turned out to be essentially identical on protein level. Whereas the less abundant "a" form displayed enzymatic activity, the new "b" homologue did not. This is probably caused by amino acid exchanges in the active site, and it raises questions about the physiological role of this protein. Sequence comparisons by MS/MS of antigen 5 and phospholipases from V. vulgaris, germanica, maculifrons, pensylvanica, flavopilosa and squamosa revealed the latter as a taxonomic outlier and led to the discovery of several not previously reported amino acid differences.

  18. Identification of major rice allergen and their clinical significance in children

    PubMed Central

    Jeon, You Hoon; Oh, Se Jo; Yang, Hyeon Jong; Lee, Soo Young

    2011-01-01

    Purpose Recently, an increase in the number of patients sensitized to rice allergen with or without clinical symptoms has been reported. This study was designed to determine the major allergens in rice and their clinical significance. Methods Twenty-four children (15 boys and 9 girls; mean age, 16.3 months) with allergic disease, who were sensitized to rice antigen (by UniCAP) in the Pediatric Allergy Respiratory Center at Soonchunhyang University Hospital, were enrolled in this study. The allergenicity of various types of rice (raw, cooked, and heat-treated, simulated gastric fluid [SGF], and simulated intestinal fluid [SIF]) was investigated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoglobulin E (IgE) immunoblots. The patients' medical records, including laboratory data and allergy symptoms after ingestion of rice were reviewed. Results Patients were sensitized to an average of 13.5 food antigens and their mean total IgE was 6,888.7 kU/L. In SDS-PAGE, more than 16 protein bands were observed in the raw rice, whereas only 14-16 kDa and 31-35 kDa protein bands were observed in cooked rice. The common SDS-PAGE protein bands observed in SGF-, SIF-, and heat-treated rice were 9, 14, and 31 kDa. In a heated-rice IgE immunoblot, protein bands of 9, 14, and 31-33 kDa were found in 27.8%, 38.9%, and 38.9% of all sera, respectively, and in 50%, 50%, and 75%, of ser a from the 4 symptomatic patients, respectively. Conclusion The 9-, 14-, and 31-kDa protein bands appeared to be the major allergens responsible for rice allergy symptoms. PMID:22232624

  19. High-resolution crystal structure and IgE recognition of the major grass pollen allergen Phl p 3.

    PubMed

    Devanaboyina, S C; Cornelius, C; Lupinek, C; Fauland, K; Dall'Antonia, F; Nandy, A; Hagen, S; Flicker, S; Valenta, R; Keller, W

    2014-12-01

    Group 2 and 3 grass pollen allergens are major allergens with high allergenic activity and exhibit structural similarity with the C-terminal portion of major group 1 allergens. In this study, we aimed to determine the crystal structure of timothy grass pollen allergen, Phl p 3, and to study its IgE recognition and cross-reactivity with group 2 and group 1 allergens. The three-dimensional structure of Phl p 3 was solved by X-ray crystallography and compared with the structures of group 1 and 2 grass pollen allergens. Cross-reactivity was studied using a human monoclonal antibody which inhibits allergic patients' IgE binding and by IgE inhibition experiments with patients' sera. Conformational Phl p 3 IgE epitopes were predicted with the algorithm SPADE, and Phl p 3 variants containing single point mutations in the predicted IgE binding sites were produced to analyze allergic patients' IgE binding. Phl p 3 is a globular β-sandwich protein showing structural similarity to Phl p 2 and the Phl p 1-C-terminal domain. Phl p 3 showed IgE cross-reactivity with group 2 allergens but not with group 1 allergens. SPADE identified two conformational IgE epitope-containing areas, of which one overlaps with the epitope defined by the monoclonal antibody. The mutation of arginine 68 to alanine completely abolished binding of the blocking antibody. This mutation and a mutation of D13 in the predicted second IgE epitope area also reduced allergic patients' IgE binding. Group 3 and group 2 grass pollen allergens are cross-reactive allergens containing conformational IgE epitopes. They lack relevant IgE cross-reactivity with group 1 allergens and therefore need to be included in diagnostic tests and allergen-specific treatments in addition to group 1 allergens. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  20. Standardization of allergen products: 3. Validation of candidate European Pharmacopoeia standard methods for quantification of major birch allergen Bet v 1.

    PubMed

    Kaul, S; Zimmer, J; Dehus, O; Costanzo, A; Daas, A; Buchheit, K H; Asturias, J A; Barber, D; Carnés, J; Chapman, M; Dayan-Kenigsberg, J; Döring, S; Führer, F; Hanschmann, K M; Holzhauser, T; Ledesma, A; Moingeon, P; Nony, E; Pini, C; Plunkett, G; Reese, G; Sandberg, E; Sander, I; Strecker, D; Valerio, C; van Ree, R; Vieths, S

    2016-10-01

    The BSP090 project aims at establishing European Pharmacopoeia Reference Substances in combination with the corresponding ELISA methods for the quantification of major allergens in allergen products. Two sandwich ELISAs proved suitable for quantification of Bet v 1, the major birch pollen allergen, in preceding phases of BSP090. Two Bet v 1-specific ELISA systems were compared with respect to accuracy and precision in a ring trial including 13 laboratories. Model samples containing recombinant rBet v 1.0101 as well as native birch pollen extracts were measured independently at least three times in each facility. The assessment was completed with a comparative quantification of Bet v 1 in 30 marketed birch allergen products in one laboratory, simulating the future use as reference method. In the collaborative study, both candidate ELISAs confirmed their suitability to quantify recombinant and native Bet v 1. ELISA-A showed higher precision and lower interlaboratory variability, yet ELISA-B exhibited slightly higher accuracy. Subsequent parallel measurement of Bet v 1 in a panel of 'real-life' birch allergen products indicated better repeatability of ELISA-B. Both systems detected substantial differences in Bet v 1 content between allergen products, but the effect was more pronounced using ELISA-B due to persistently higher values compared to ELISA-A. In the collaborative study, no deciding differences were observed between the two candidate ELISAs. Further comparison under conditions simulating the intended use combined with the criterion of long-term availability enabled the selection of one Bet v 1-specific ELISA for proposal as European Pharmacopoeia standard method. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  1. Ligand Binding Modulates the Structural Dynamics and Compactness of the Major Birch Pollen Allergen

    PubMed Central

    Grutsch, Sarina; Fuchs, Julian E.; Freier, Regina; Kofler, Stefan; Bibi, Marium; Asam, Claudia; Wallner, Michael; Ferreira, Fátima; Brandstetter, Hans; Liedl, Klaus R.; Tollinger, Martin

    2014-01-01

    Pathogenesis-related plant proteins of class-10 (PR-10) are essential for storage and transport of small molecules. A prominent member of the PR-10 family, the major birch pollen allergen Bet v 1, is the main cause of spring pollinosis in the temperate climate zone of the northern hemisphere. Bet v 1 binds various ligand molecules to its internal cavity, and immunologic effects of the presence of ligand have been discussed. However, the mechanism of binding has remained elusive. In this study, we show that in solution Bet v 1.0101 is conformationally heterogeneous and cannot be represented by a single structure. NMR relaxation data suggest that structural dynamics are fundamental for ligand access to the protein interior. Complex formation then leads to significant rigidification of the protein along with a compaction of its 3D structure. The data presented herein provide a structural basis for understanding the immunogenic and allergenic potential of ligand binding to Bet v 1 allergens. PMID:25517162

  2. Physicochemical properties and thermal stability of Lep w 1, the major allergen of whiff.

    PubMed

    Griesmeier, Ulrike; Bublin, Merima; Radauer, Christian; Vázquez-Cortés, Sonia; Ma, Yan; Fernández-Rivas, Montserrat; Breiteneder, Heimo

    2010-06-01

    Whiff (Lepidorhombus whiffiagonis) is a fish frequently consumed in Spain. Lep w 1, its major allergen, is a calcium-binding beta-parvalbumin. The resistance of Lep w 1 to heat denaturation and to digestion were studied by circular dichroism spectroscopy and by in vitro gastric digestion systems. Purified Lep w 1 was thermally stable up to 65 degrees C at neutral pH. Calcium depletion resulted in a change of its structure as determined by circular dichroism spectroscopy. A partial loss of structure was also observed at acidic pH; however, the allergen retained its full IgE-binding ability. The partially denatured Lep w 1 was easily digested by pepsin within 2 min. Further, the IgE reactivity of proteins extracted from cooked fish and their stability to proteolysis were analyzed. The extract revealed a higher number of IgE reactive bands than an extract from uncooked fish. IgE binding to these proteins could not be inhibited by an extract from uncooked fish. In contrast to a raw fish extract, the cooked extract showed higher resistance to pepsinolysis. The stability of Lep w 1 to thermal denaturation and digestion explains the high allergenicity of whiff.

  3. Ligand Recognition of the Major Birch Pollen Allergen Bet v 1 is Isoform Dependent

    PubMed Central

    Seutter von Loetzen, Christian; Jacob, Thessa; Hartl-Spiegelhauer, Olivia; Vogel, Lothar; Schiller, Dirk; Spörlein-Güttler, Cornelia; Schobert, Rainer; Vieths, Stefan; Hartl, Maximilian Johannes; Rösch, Paul

    2015-01-01

    Each spring millions of patients suffer from allergies when birch pollen is released into the air. In most cases, the major pollen allergen Bet v 1 is the elicitor of the allergy symptoms. Bet v 1 comes in a variety of isoforms that share virtually identical conformations, but their relative concentrations are plant-specific. Glycosylated flavonoids, such as quercetin-3-O-sophoroside, are the physiological ligands of Bet v 1, and here we found that three isoforms differing in their allergenic potential also show an individual, highly specific binding behaviour for the different ligands. This specificity is driven by the sugar moieties of the ligands rather than the flavonols. While the influence of the ligands on the allergenicity of the Bet v 1 isoforms may be limited, the isoform and ligand mixtures add up to a complex and thus individual fingerprint of the pollen. We suggest that this mixture is not only acting as an effective chemical sunscreen for pollen DNA, but may also play an important role in recognition processes during pollination. PMID:26042900

  4. Generation and purification of monoclonal antibodies against Der f 2, a major allergen from Dermatophagoides farinae.

    PubMed

    Chen, Huifang; Zhang, Kejun; Wang, Shan; Xu, Chenxia; Zou, Zehong; Tao, Ailin

    Monoclonal antibodies (mAbs) are needed for the quantitation of environmental allergens for precise diagnosis and immunotherapy. In this study, we produced and purified monoclonal antibodies against Der f 2, one of the major allergens of the house dust mite Dermatophagoides farina, in order to develop an assay for the detection of this allergen. BALB/c mice were immunized four times with the protein Der f 2 together with an adjuvant after which splenocytes were collected and fused with SP2/0 (myeloma cells) in the presence of polyethylene glycol (PEG). The fused cells were selected in the presence of Hypoxanthine-Aminopterin-Thymidine (HAT) and then Hypoxanthine-Thymidine (HT) medium. Positive cells were screened with ELISA and subcloned by limited dilution at least three times to achieve stable mAb-producing clones. Four stable mAb-producing clones were obtained. One clone with IgG1 isotype and another with IgG2b isotype were chosen to produce large amounts of mAb by inoculation of the cells into the abdominal cavity of mice. Ascites were collected and the mAbs were purified using protein A affinity chromatography. Testing of the ascites by ELISA showed the titration of IgG1 and IgG2b to be higher than 1/10(6) dilution. The specificity of both antibodies was confirmed by immunoblotting. Thus, we produced two mAb clones against Der f 2 that can be used to create a precise quantitative method to identify allergen components in dust samples and facilitate further study in Der f 2 component-resolved diagnosis (CRD).

  5. Purification of the major allergen of red soft coral (Dendronephthya nipponica).

    PubMed

    Onizuka, R; Kamiya, H; Muramoto, K; Goto, R; Inoue, K; Kumamoto, K; Nakajima, Y; Iida, S; Ishigami, F

    2001-06-01

    Red soft coral (RSC; Dendronephthya nipponica, a marine coelenterate) causes spiny lobster fishermen living along the Pacific coast of Miyazaki Prefecture in Japan to develop occupational allergies, such as conjunctivitis, rhinitis, dermatitis and bronchial asthma. The aim of this study was to purify and to characterize RSC allergen, which causes occupational asthma in spiny lobster fishermen. The allergic responsiveness of spiny lobster fishermen to RSC was examined. The examinations included specific IgE production, skin test responses, lymphocyte stimulation tests and specific IgG production. We found that RSC has a strong sensitizing activity in humans at a molecular weight of 10 kD or more, while it has no IgE-producing activity at a molecular weight of less than 10 kD. Neither the nonatopic controls nor the atopic non-coral-allergic controls exhibited any RAST-binding activity to any fraction. For the purification and the identification of this new allergen component, repeated gel filtration of the RSC extract was performed on a Sephacryl S-200 column, followed by gel filtration on a Superose-6 column. The purified major allergen component Den n 1, which is separated on a Mono-Q column, showed intradermal responses, lymphocyte stimulating activity and specific IgG-producing activity in RSC-induced bronchial asthma patients. The 53-kD component was electroblotted on a polyvinylidene difluoride membrane. The N-terminal amino acid sequence of this new allergen component (Den n 1) was determined as Asp-Asp-Ile-Asn-Arg-Tyr-Ala-Phe-Asp-Asn-Lys-Ile-Asn- Asp-Lys-Leu-Phe-Asp-His-Trp-Gln-Ser. Copyright 2001 S. Karger AG, Basel

  6. Phytochemical and Pharmacological Profiles of Three Fagopyrum Buckwheats.

    PubMed

    Jing, Rui; Li, Hua-Qiang; Hu, Chang-Ling; Jiang, Yi-Ping; Qin, Lu-Ping; Zheng, Cheng-Jian

    2016-04-19

    The genus Fagopyrum (Polygonaceae), currently comprising 15 species of plants, includes three important buckwheat species: Fagopyrum esculentum (F. esculentum) Moench. (common buckwheat), Fagopyrum tataricum (F. tataricum) (L.) Gaertn. (tartary buckwheat) and Fagopyrum dibotrys (F. dibotrys) (D. Don) Hara. (perennial buckwheat), which have been well explored due to their long tradition of both edible and medicinal use. This review aimed to present an up-to-date and comprehensive analysis of the phytochemistry and pharmacology of the three Fagopyrum buckwheats. In addition, the scope for future research was also discussed. All available references included in this paper were compiled from major databases, such as MEDLINE, Pubmed, Scholar, Elsevier, Springer, Wiley and CNKI. A total of 106 compounds isolated from three Fagopyrum buckwheats can be mainly divided into six classes: flavonoids, phenolics, fagopyritols, triterpenoids, steroids and fatty acids. Flavonoids and phenolic compounds were considered to be the major active components. Considerable pharmacological experiments both in vitro and in vivo have validated that Fagopyrum buckwheats possess antitumor, anti-oxidant, anti-inflammatory, hepatoprotective, anti-diabetic activities, etc. All reported data lead us to conclude that Fagopyrum buckwheats have convincing medicinal potential. However, further research is needed to explore its bioactive constituents, the relationship to their structural activities and the molecular mechanisms of action.

  7. Phytochemical and Pharmacological Profiles of Three Fagopyrum Buckwheats

    PubMed Central

    Jing, Rui; Li, Hua-Qiang; Hu, Chang-Ling; Jiang, Yi-Ping; Qin, Lu-Ping; Zheng, Cheng-Jian

    2016-01-01

    The genus Fagopyrum (Polygonaceae), currently comprising 15 species of plants, includes three important buckwheat species: Fagopyrum esculentum (F. esculentum) Moench. (common buckwheat), Fagopyrum tataricum (F. tataricum) (L.) Gaertn. (tartary buckwheat) and Fagopyrum dibotrys (F. dibotrys) (D. Don) Hara. (perennial buckwheat), which have been well explored due to their long tradition of both edible and medicinal use. This review aimed to present an up-to-date and comprehensive analysis of the phytochemistry and pharmacology of the three Fagopyrum buckwheats. In addition, the scope for future research was also discussed. All available references included in this paper were compiled from major databases, such as MEDLINE, Pubmed, Scholar, Elsevier, Springer, Wiley and CNKI. A total of 106 compounds isolated from three Fagopyrum buckwheats can be mainly divided into six classes: flavonoids, phenolics, fagopyritols, triterpenoids, steroids and fatty acids. Flavonoids and phenolic compounds were considered to be the major active components. Considerable pharmacological experiments both in vitro and in vivo have validated that Fagopyrum buckwheats possess antitumor, anti-oxidant, anti-inflammatory, hepatoprotective, anti-diabetic activities, etc. All reported data lead us to conclude that Fagopyrum buckwheats have convincing medicinal potential. However, further research is needed to explore its bioactive constituents, the relationship to their structural activities and the molecular mechanisms of action. PMID:27104519

  8. Optimization of allergen standardization.

    PubMed

    Jeong, Kyoung Yong; Hong, Chein-Soo; Lee, Joo-Shil; Park, Jung-Won

    2011-05-01

    Preparation of high quality allergen extracts is essential for the diagnosis and immunotherapy of allergic disorders. Standardization of allergen extracts concerns determination of the allergen unit, development of reference material and measurement of the overall IgE binding capacity of an allergen extract. Recently, quantification of individual allergens has been the main focus of allergen standardization because the allergenicity of most allergen extracts is known to be mainly dependent on the content of a small number of allergen molecules. Therefore, characterization of major allergens will facilitate the standardization of allergens. In this article, we review the current state of allergen standardization. In addition, we briefly summarize the components of allergen extracts that should be under control for the optimization of allergen standardization, since its adjuvant-like activities could play an important role in allergic reactions even though the molecule itself does not bind to the IgE antibodies from subjects.

  9. Homology modelling of the major peanut allergen Ara h 2 and surface mapping of IgE-binding epitopes.

    PubMed

    Barre, Annick; Borges, Jean-Philippe; Culerrier, Raphaël; Rougé, Pierre

    2005-09-15

    Three-dimensional models built for the peanut Ara h 2 allergen and other structurally-related 2S albumin allergens of dietary nuts exhibited an overall three-dimensional fold stabilized by disulphide bridges well conserved among all the members of the 2S albumin superfamily. Conformational analysis of the linear IgE-binding epitopes mapped on the molecular surface of Ara h 2 showed no structural homology with the corresponding regions of the walnut Jug r 1, the pecan nut Car i 1 or the Brazil nut Ber e 1 allergens. The absence of epitopic community does not support the allergenic cross-reactivity observed between peanut and walnut or Brazil nut, which presumably depends on other ubiquitous seed storage protein allergens, namely the vicilins. However, the major IgE-binding epitope identified on the molecular surface of the walnut Jug r 1 allergen shared a pronounced structural homology with the corresponding region of the pecan nut Car i 1 allergen. With the exception of peanut, 2S albumins could thus account for the IgE-binding cross-reactivity observed between some other dietary nuts, e.g. walnut and pecan nut.

  10. IgE cross-reactivity between the major peanut allergen Ara h 2 and the nonhomologous allergens Ara h 1 and Ara h 3.

    PubMed

    Bublin, Merima; Kostadinova, Maria; Radauer, Christian; Hafner, Christine; Szépfalusi, Zsolt; Varga, Eva-Maria; Maleki, Soheila J; Hoffmann-Sommergruber, Karin; Breiteneder, Heimo

    2013-07-01

    Ara h 1, a vicilin; Ara h 2, a 2S albumin; and Ara h 3, a legumin, are major peanut allergens. Ara h 2 is an important predictor of clinical reactivity to peanut, but cosensitization to all 3 allergens is correlated with the severity of patients' symptoms. We investigated whether cosensitization to these 3 allergens is caused by IgE cross-reactivity, despite the fact that they do not display obvious structural or sequence similarities. IgE cross-inhibitions were performed with purified Ara h 1, Ara h 2, and Ara h 3 and IgG-depleted sera from 10 patients with peanut allergy. After an in silico search for similar peptides, IgE ELISA inhibition assays with synthetic peptides were performed. Ara h 2 inhibited IgE binding to Ara h 1 (average, 86% ± 13%) and Ara h 3 (average, 96% ± 6%). IgE binding to Ara h 2 was inhibited by Ara h 1 by 78% ± 15% and by Ara h 3 by 80% ± 6%. A subsequent sequence comparison showed that these nonhomologous allergens contained several similar surface-exposed peptides. IgE binding to Ara h 2-derived peptides was completely inhibited by Ara h 1 and Ara h 3. A mixture of these peptides reduced IgE binding to Ara h 1 and Ara h 3 by 20% to 60% and to Ara h 2 by 49% to 89%. Occurrence of similar sequences in the 3 major peanut allergens accounts for the high extent of cross-reactivity among them. Copyright © 2013 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

  11. Antioxidant activity of tartary (Fagopyrum tataricum (L.) Gaertn.) and common (Fagopyrum esculentum moench) buckwheat sprouts.

    PubMed

    Liu, Chia-Ling; Chen, Yih-Shyuan; Yang, Joan-Hwa; Chiang, Been-Huang

    2008-01-09

    This study compared the differences of two types of buckwheat sprouts, namely, common buckwheat ( Fagopyrum esculentum Moench) and tartary buckwheat ( Fagopyrum tataricum (L.) Gaertn.), in general composition, functional components, and antioxidant capacity. The ethanol extracts of tartary buckwheat sprouts (TBS) had higher reducing power, free radical scavenging activity, and superoxide anion scavenging activity than those of common buckwheat sprouts (CBS). As for chelating effects on ferrous ions, CBS had higher values than TBS. Rutin was the major flavonoid found in these two types of buckwheat sprouts, and TBS was 5 fold higher in rutin than CBS. The antioxidant effects of buckwheat sprouts on human hepatoma HepG2 cells revealed that both of TBS and CBS could decrease the production of intracellular peroxide and remove the intracellular superoxide anions in HepG2 cells, but TBS reduced the cellular oxidative stress more effectively than CBS, possibly because of its higher rutin (and quercetin) content.

  12. Alpha amylase is a major allergenic component in occupational asthma patients caused by porcine pancreatic extract.

    PubMed

    Park, Hae-Sim; Kim, Hee-Yeon; Suh, You-Jin; Lee, Soo-Jin; Lee, Soo-Keol; Kim, Sun-Sin; Nahm, Dong-Ho

    2002-09-01

    Porcine pancreatic extracts (PPE) are composed of alpha-amylase and lipase, which are common components of digestive enzymes. They have been known to cause occupational asthma in exposed workers in pharmaceutical and baking industries, as well as in a laboratory technician, but there has been no report of PPE-induced occupational asthma in medical personnel and their IgE binding components to each component. Four asthmatic subjects showing positive results on PPE-bronchoprovocation testing were enrolled. All of them were nurses working in a university hospital. Their job included grinding and mixing PPE powder for admitted patients. Serum-specific IgE antibodies to PPE, alpha-amylase, and lipase were measured by enzyme linked immunosorbent assay (ELISA). To confirm specificity of IgE binding and cross-allergenicity among the three extracts, ELISA inhibition tests were performed. In order to characterize allergenic components within these three extracts, SDS-PAGE and IgE immunoblot analysis were done. Specific IgE antibodies to PPE, alpha-amylase, and lipase were detectable by ELISA in all study subjects. An alpha-amylase ELISA inhibition test showed significant inhibitions by amylase and PPE, and minimal inhibition by lipase. However, a lipase ELISA inhibition test showed significant inhibitions by alpha-amylase and PPE with a lesser degree of inhibition by lipase. Furthermore, IgE immunoblot analysis showed one IgE binding component (55 kDa) within PPE, six components (55 kDa, 43 kDa, 41 kDa, 32 kDa, 31 kDa, 29 kDa) within alpha-amylase and two components (31 kDa, 29 kDa) within lipase extracts. Thesefindings suggest that inhalation of PPE powder can induce IgE-mediated bronchoconstriction in exposed nurses. Alpha-amylase is a major allergenic component within PPE.

  13. Immune recognition of novel isoforms and domains of the mugwort pollen major allergen Art v 1.

    PubMed

    Dedic, Azra; Gadermaier, Gabriele; Vogel, Lothar; Ebner, Christof; Vieths, Stefan; Ferreira, Fátima; Egger, Matthias

    2009-01-01

    Allergen isoforms can differ in their IgE and T cell recognition patterns, and thus might have an impact on the selection of candidates for molecule-based diagnostic and therapeutic approaches. The present study aimed at the identification and characterization of isoforms of Art v 1, the mugwort pollen major allergen. In addition, single Art v 1 domains were physicochemically and immunologically characterized. For this purpose, the Art v 1 cDNA was radiolabeled and used to screen a mugwort pollen cDNA library. Positive clones were sequenced and used for the production of recombinant proteins in Escherichia coli using the pHIS-Parallel2 vector. Protein purification was performed by affinity- and ion exchange chromatography. Antibody binding to the recombinant proteins was determined by immunoblot, ELISA, cross-inhibition experiments, and mediator release assays. We could identify 7 Art v 1 isoforms differing in 1-6 amino acid residues. Interestingly, all amino acid variations were restricted to the proline domain carrying the molecule's post-translational modifications. No significant difference in IgG or IgE reactivity could be observed between Art v 1 isoforms and the defensin domain produced in E. coli. When expressed in E. coli, the proline domain was not recognized by Art v 1-specific antibodies. Our results demonstrated that the relevant IgE epitopes of Art v 1 are located on the defensin domain and suggest the involvement of carbohydrates in the allergenicity of natural Art v 1. Plant-based expression systems could help to reveal possibly different glycosylation patterns and IgE binding properties of Art v 1 isoforms. These findings have direct implications on the development of novel tools for mugwort pollen allergy diagnosis and therapy.

  14. Changes in Major Peanut Allergens Under Different pH Conditions

    PubMed Central

    Kim, Jihyun; Lee, Jeongok; Seo, Won Hee; Ahn, Kangmo; Lee, Sang-Il

    2012-01-01

    Regional dietary habits and cooking methods affect the prevalence of specific food allergies; therefore, we determined the effects of various pH conditions on major peanut allergens. Peanut kernels were soaked overnight in commercial vinegar (pH 2.3) or acetic acid solutions at pH 1.0, 3.0, or 5.0. Protein extracts from the sera of seven patients with peanut-specific IgE levels >15 kUA/L were analyzed by SDS-PAGE and immunolabeling. A densitometer was used to quantify and compare the allergenicity of each protein. The density of Ara h 1 was reduced by treatment with pH 1.0, 3.0, or 5.0 acetic acid, or commercial vinegar. Ara h 2 remained largely unchanged after treatment with pH 5.0 acetic acid, and was decreased following treatment with pH 1.0, 2.3, or 3.0 acetic acid. Ara h 3 and Ara h 6 appeared as a thick band after treatment with pH 1.0 acetic acid and commercial vinegar. IgE-binding intensities to Ara h 1, Ara h 2, and Ara h 3 were significantly reduced after treatment with pH 1.0 acetic acid or commercial vinegar. These data suggest that treatment with acetic acid at various pH values affects peanut allergenicity and may explain the low prevalence of peanut allergy in Korea. PMID:22548209

  15. Search for Allergens from the Pollen Proteome of Sunflower (Helianthus annuus L.): A Major Sensitizer for Respiratory Allergy Patients

    PubMed Central

    Saha, Bodhisattwa; Pandey, Naren; Gupta Bhattacharya, Swati

    2015-01-01

    Background Respiratory allergy triggered by pollen allergens is increasing at an alarming rate worldwide. Sunflower pollen is thought to be an important source of inhalant allergens. Present study aims to identify the prevalence of sunflower pollinosis among the Indian allergic population and characterizes the pollen allergens using immuno-proteomic tools. Methodology Clinico-immunological tests were performed to understand the prevalence of sensitivity towards sunflower pollen among the atopic population. Sera from selected sunflower positive patients were used as probe to detect the IgE-reactive proteins from the one and two dimensional electrophoretic separated proteome of sunflower pollen. The antigenic nature of the sugar moiety of the glycoallergens was studied by meta-periodate modification of IgE-immunoblot. Finally, these allergens were identified by mass-spectrometry. Results Prevalence of sunflower pollen sensitization was observed among 21% of the pollen allergic population and associated with elevated level of specific IgE and histamine in the sera of these patients. Immunoscreening of sunflower pollen proteome with patient sera detected seven IgE-reactive proteins with varying molecular weight and pI. Hierarchical clustering of 2D-immunoblot data highlighted three allergens characterized by a more frequent immuno-reactivity and increased levels of IgE antibodies in the sera of susceptible patients. These allergens were considered as the major allergens of sunflower pollen and were found to have their glycan moiety critical for inducing IgE response. Homology driven search of MS/MS data of these IgE-reactive proteins identified seven previously unreported allergens from sunflower pollen. Three major allergenic proteins were identified as two pectate lyases and a cysteine protease. Conclusion Novelty of the present report is the identification of a panel of seven sunflower pollen allergens for the first time at immuno-biochemical and proteomic level

  16. Fusion proteins of flagellin and the major birch pollen allergen Bet v 1 show enhanced immunogenicity, reduced allergenicity, and intrinsic adjuvanticity.

    PubMed

    Kitzmüller, Claudia; Kalser, Julia; Mutschlechner, Sonja; Hauser, Michael; Zlabinger, Gerhard J; Ferreira, Fatima; Bohle, Barbara

    2017-04-26

    Recombinant fusion proteins of flagellin and antigens have been demonstrated to induce strong innate and adaptive immune responses. Such fusion proteins can enhance the efficacy of allergen-specific immunotherapy. We sought to characterize different fusion proteins of flagellin and the major birch pollen allergen Bet v 1 for suitability as allergy vaccines. A truncated version of flagellin (NtCFlg) was genetically fused to the N- or C-terminus of Bet v 1. Toll-like receptor (TLR) 5 binding was assessed with HEK293 cells expressing TLR5. Upregulation of CD40, CD80, CD83, and CD86 on monocyte-derived dendritic cells from allergic patients was analyzed by using flow cytometry. The T cell-stimulatory capacity of the fusion proteins was assessed with naive and Bet v 1-specific T cells. IgE binding was tested in inhibition ELISAs and basophil activation tests. Mice were immunized with the fusion proteins in the absence and presence of aluminum hydroxide. Cellular and antibody responses were monitored. Murine antibodies were tested for blocking capacity in basophil activation tests. Both fusion proteins matured monocyte-derived dendritic cells through TLR5. Compared with Bet v 1, the fusion proteins showed stronger T cell-stimulatory and reduced IgE-binding capacity and induced murine Bet v 1-specific antibodies in the absence of aluminum hydroxide. However, only antibodies induced by means of immunization with NtCFlg fused to the C-terminus of Bet v 1 inhibited binding of patients' IgE antibodies to Bet v 1. Bet v 1-flagellin fusion proteins show enhanced immunogenicity, reduced allergenicity, and intrinsic adjuvanticity and thus represent promising vaccines for birch pollen allergen-specific immunotherapy. However, the sequential order of allergen and adjuvant within a fusion protein determines its immunologic characteristics. Copyright © 2017 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  17. Vacuolar Serine Protease Is a Major Allergen of Fusarium proliferatum and an IgE-Cross Reactive Pan-Fungal Allergen

    PubMed Central

    Yeh, Chang-Ching; Tai, Hsiao-Yun; Chou, Hong; Wu, Keh-Gong

    2016-01-01

    Purpose Fusarium species are among prevalent airborne fungi and causative agents of human respiratory atopic disorders. We previously identified a 36.5-kDa F. proliferatum component recognized by IgE antibodies in 9 (53%) of the 17 F. proliferatum-sensitized atopic serum samples. The purpose of this study is to characterize the 36.5-kDa allergen of F. proliferatum. Methods Characterization of allergens and determination of IgE cross-reactivity were performed by cDNA cloning/expression and immunoblot inhibition studies. Results Based on the finding that the 36.5-kDa IgE-binding component reacted with the mouse monoclonal antibody FUM20 against fungal vacuolar serine protease allergens, the cDNA of F. proliferatum vacuolar serine protease (Fus p 9.0101) was subsequently cloned. Nine serum samples from respiratory atopic patients with IgE binding to the vacuolar serine protease allergen of Penicillium chrysogenum (Pen ch 18) also showed IgE-immunoblot reactivity to rFus p 9.0101. The purified rFus p 9.0101 can inhibit IgE and FUM20 binding to the 36.5-kDa component of F. proliferatum. Thus, a novel and important Fus p 9.0101 was identified. The rPen ch 18 can inhibit IgE binding to Fus p 9.0101. It indicates that IgE cross-reactivity between Fus p 9.0101 and Pen ch 18 also exists. Furthermore, neither rFus p 9.0101 K88A nor rPen ch 18 K89A mutants inhibited IgE binding to rFus p 9.0101. Lys88 was considered a critical core amino acid in IgE binding to r Fus p 9.0101 and a residue responsible for IgE cross-reactivity between Fus p 9.0101 and Pen ch 18 allergens. Conclusions Results obtained from this study indicate that vacuolar serine protease may be a major allergen of F. proliferatum and an important IgE cross-reactive pan-fungal allergen, and provide important bases for clinical diagnosis of fungal allergy. PMID:27334782

  18. Creation of an engineered APC system to explore and optimize the presentation of immunodominant peptides of major allergens

    PubMed Central

    Rosskopf, Sandra; Jutz, Sabrina; Neunkirchner, Alina; Candia, Martín R.; Jahn-Schmid, Beatrice; Bohle, Barbara; Pickl, Winfried F.; Steinberger, Peter

    2016-01-01

    We have generated engineered APC to present immunodominant peptides derived from the major aero-allergens of birch and mugwort pollen, Bet v 1142–153 and Art v 125–36, respectively. Jurkat-based T cell reporter lines expressing the cognate allergen-specific T cell receptors were used to read out the presentation of allergenic peptides on the engineered APC. Different modalities of peptide loading and presentation on MHC class II molecules were compared. Upon exogenous loading with allergenic peptides, the engineered APC elicited a dose-dependent response in the reporter T cells and the presence of chemical loading enhancers strongly increased reporter activation. Invariant chain-based MHC class II targeting strategies of endogenously expressed peptides resulted in stronger activation of the reporters than exogenous loading. Moreover, we used Bet v 1 as model allergen to study the ability of K562 cells to present antigenic peptides derived from whole proteins either taken up or endogenously expressed as LAMP-1 fusion protein. In both cases the ability of these cells to process and present peptides derived from whole proteins critically depended on the expression of HLA-DM. We have identified strategies to achieve efficient presentation of allergenic peptides on engineered APC and demonstrate their use to stimulate T cells from allergic individuals. PMID:27539532

  19. Targeting allergenic fungi in agricultural environments aids the identification of major sources and potential risks for human health.

    PubMed

    Weikl, F; Radl, V; Munch, J C; Pritsch, K

    2015-10-01

    Fungi are, after pollen, the second most important producers of outdoor airborne allergens. To identify sources of airborne fungal allergens, a workflow for qPCR quantification from environmental samples was developed, thoroughly tested, and finally applied. We concentrated on determining the levels of allergenic fungi belonging to Alternaria, Cladosporium, Fusarium, and Trichoderma in plant and soil samples from agricultural fields in which cereals were grown. Our aims were to identify the major sources of allergenic fungi and factors potentially influencing their occurrence. Plant materials were the main source of the tested fungi at and after harvest. Amounts of A. alternata and C. cladosporioides varied significantly in fields under different management conditions, but absolute levels were very high in all cases. This finding suggests that high numbers of allergenic fungi may be an inevitable side effect of farming in several crops. Applied in large-scale studies, the concept described here may help to explain the high number of sensitization to airborne fungal allergens. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. The Impact of Nitration on the Structure and Immunogenicity of the Major Birch Pollen Allergen Bet v 1.0101

    PubMed Central

    Ackaert, Chloé; Kofler, Stefan; Horejs-Hoeck, Jutta; Zulehner, Nora; Asam, Claudia; von Grafenstein, Susanne; Fuchs, Julian E.; Briza, Peter; Liedl, Klaus R.; Bohle, Barbara; Ferreira, Fátima; Brandstetter, Hans; Oostingh, Gertie J.; Duschl, Albert

    2014-01-01

    Allergy prevalence has increased in industrialized countries. One contributing factor could be pollution, which can cause nitration of allergens exogenously (in the air) or endogenously (in inflamed lung tissue). We investigated the impact of nitration on both the structural and immunological behavior of the major birch pollen allergen Bet v 1.0101 to determine whether nitration might be a factor in the increased incidence of allergy. Bet v 1.0101 was nitrated with tetranitromethane. Immune effects were assessed by measuring the proliferation of specific T-cell lines (TCLs) upon stimulation with different concentrations of nitrated and unmodified allergen, and by measurement of cytokine release of monocyte-derived dendritic cells (moDCs) and primary DCs (primDCs) stimulated with nitrated versus unmodified allergen. HPLC-MS, crystallography, gel electrophoresis, amino acid analysis, size exclusion chromatography and molecular dynamics simulation were performed to characterize structural changes after nitration of the allergen. The proliferation of specific TCLs was higher upon stimulation with the nitrated allergen in comparison to the unmodified allergen. An important structural consequence of nitration was oligomerization. Moreover, analysis of the crystal structure of nitrated Bet v 1.0101 showed that amino acid residue Y83, located in the hydrophobic cavity, was nitrated to 100%. Both moDCs and primDCs showed decreased production of TH1-priming cytokines, thus favoring a TH2 response. These results implicate that nitration of Bet v 1.0101 might be a contributing factor to the observed increase in birch pollen allergy, and emphasize the importance of protein modifications in understanding the molecular basis of allergenicity. PMID:25126882

  1. The impact of nitration on the structure and immunogenicity of the major birch pollen allergen Bet v 1.0101.

    PubMed

    Ackaert, Chloé; Kofler, Stefan; Horejs-Hoeck, Jutta; Zulehner, Nora; Asam, Claudia; von Grafenstein, Susanne; Fuchs, Julian E; Briza, Peter; Liedl, Klaus R; Bohle, Barbara; Ferreira, Fátima; Brandstetter, Hans; Oostingh, Gertie J; Duschl, Albert

    2014-01-01

    Allergy prevalence has increased in industrialized countries. One contributing factor could be pollution, which can cause nitration of allergens exogenously (in the air) or endogenously (in inflamed lung tissue). We investigated the impact of nitration on both the structural and immunological behavior of the major birch pollen allergen Bet v 1.0101 to determine whether nitration might be a factor in the increased incidence of allergy. Bet v 1.0101 was nitrated with tetranitromethane. Immune effects were assessed by measuring the proliferation of specific T-cell lines (TCLs) upon stimulation with different concentrations of nitrated and unmodified allergen, and by measurement of cytokine release of monocyte-derived dendritic cells (moDCs) and primary DCs (primDCs) stimulated with nitrated versus unmodified allergen. HPLC-MS, crystallography, gel electrophoresis, amino acid analysis, size exclusion chromatography and molecular dynamics simulation were performed to characterize structural changes after nitration of the allergen. The proliferation of specific TCLs was higher upon stimulation with the nitrated allergen in comparison to the unmodified allergen. An important structural consequence of nitration was oligomerization. Moreover, analysis of the crystal structure of nitrated Bet v 1.0101 showed that amino acid residue Y83, located in the hydrophobic cavity, was nitrated to 100%. Both moDCs and primDCs showed decreased production of TH1-priming cytokines, thus favoring a TH2 response. These results implicate that nitration of Bet v 1.0101 might be a contributing factor to the observed increase in birch pollen allergy, and emphasize the importance of protein modifications in understanding the molecular basis of allergenicity.

  2. The major cockroach allergen Bla g 4 binds tyramine and octopamine.

    PubMed

    Offermann, Lesa R; Chan, Siew Leong; Osinski, Tomasz; Tan, Yih Wan; Chew, Fook Tim; Sivaraman, J; Mok, Yu-Keung; Minor, Wladek; Chruszcz, Maksymilian

    2014-07-01

    Bla g 4 is a male cockroach specific protein and is one of the major allergens produced by Blattella germanica (German cockroach). This protein belongs to the lipocalin family that comprises a set of proteins that characteristically bind small hydrophobic molecules and play a role in a number of processes such as: retinoid and pheromone transport, prostaglandin synthesis and mammalian immune response. Using NMR and isothermal titration calorimetry we demonstrated that Bla g 4 binds tyramine and octopamine in solution. In addition, crystal structure analysis of the complex revealed details of tyramine binding. As tyramine and octopamine play important roles in invertebrates, and are counterparts to vertebrate adrenergic transmitters, we speculate that these molecules are physiological ligands for Bla g 4. The nature of binding these ligands to Bla g 4 sheds light on the possible biological function of the protein. In addition, we performed a large-scale analysis of Bla g 4 and Per a 4 (an allergen from American cockroach) homologs to get insights into the function of these proteins. This analysis together with a structural comparison of Blag 4 and Per a 4 suggests that these proteins may play different roles and most likely bind different ligands. Accession numbers: The atomic coordinates and the structure factors have been deposited to the Protein Data Band under accession codes: 4N7C for native Bla g 4 and 4N7D for the Se-Met Bla g 4 structure.

  3. Production and characterization of monoclonal antibodies against major allergens of American cockroach.

    PubMed

    Wu, C H; Chiang, B T; Fann, M C; Lan, J L

    1990-11-01

    From several fusion experiments between spleen cells obtained from BALB/c mice immunized with partially purified Cr-PI of American cockroach and NS-1 cells, growth was observed in many wells. Seven stable subclones secreting monoclonal antibodies (mAbs) against Cr-PI, as determined by enzyme-linked immunosorbent assay (ELISA) with high absorbance values and immunoblot analysis, were obtained. All seven mAbs were characterized as IgG1 subclass by immunodiffusion, and reacted strongly with 72 kilodaltons (kD) of Cr-PI which have been identified as a major allergen of American cockroach. Six mAbs were found to have similar epitope specificities against Cr-PI by ELISA. The remaining mAb was found to have different epitope specificities with others. Interestingly, all mAbs did not react with any components of crude extracts of Oriental and German cockroaches as determined by immunoblot analysis and ELISA. A mAb-based double-antibody sandwich ELISA was developed, and the ELISA was dose-dependent and capable of detecting as little as 140 ng of Cr-PI allergen.

  4. NMR resonance assignments of a hypoallergenic isoform of the major birch pollen allergen Bet v 1.

    PubMed

    Ahammer, Linda; Grutsch, Sarina; Wallner, Michael; Ferreira, Fatima; Tollinger, Martin

    2017-08-14

    In Northern America and Europe a great number of people are suffering from birch pollen allergy and pollen related food allergies. The trigger for these immunological reactions is the 17.5 kDa major birch pollen allergen Bet v 1, which belongs to the family of PR-10 (pathogenesis-related) proteins. In nature, Bet v 1 occurs as a mixture of various isoforms that possess different immunological properties despite their high sequence identities. Bet v 1.0102 (Bet v 1d), which is investigated here, is a hypoallergenic isoform of Bet v 1 and a potential candidate for allergen-specific immunotherapy. We assigned the backbone and side chain (1)H, (13)C and (15)N resonances of this protein and predicted its secondary structure. The NMR-chemical shift data indicate that Bet v 1.0102 is composed of three α-helices and a seven stranded β-sheet, in agreement with the known structure of the hyperallergenic isoform Bet v 1.0101 (Bet v 1a). Our resonance assignments create the foundation for detailed characterization of the dynamic properties of Bet v 1 isoforms by NMR relaxation measurements.

  5. The major cockroach allergen Bla g 4 binds tyramine and octopamine

    PubMed Central

    Offermann, Lesa R.; Chan, Siew Leong; Osinski, Tomasz; Tan, Yih Wan; Chew, Fook Tim; Sivaraman, Jayaraman; Mok, Yu-Keung; Minor, Wladek; Chruszcz, Maksymilian

    2014-01-01

    Bla g 4 is a male cockroach specific protein and is one of the major allergens produced by Blattella germanica (German cockroach). This protein belongs to the lipocalin family that comprises a set of proteins that characteristically bind small hydrophobic molecules and play a role in a number of processes such as: retinoid and pheromone transport, prostaglandin synthesis and mammalian immune response. Using NMR and Isothermal Titration Calorimetry we demonstrated that Bla g 4 binds tyramine and octopamine in solution. In addition, crystal structure analysis of the complex revealed details of tyramine binding. As tyramine and octopamine play important roles in invertebrates, and are counterparts to vertebrate adrenergic transmitters, we speculate that these molecules are physiological ligands for Bla g 4. The nature of binding these ligands to Bla g 4 sheds light on the possible biological function of the protein. In addition, we performed a large-scale analysis of Bla g 4 and Per a 4 (an allergen from American cockroach) homologs to get insights into the function of these proteins. This analysis together with a structural comparison of Blag 4 and Per a 4 suggests that these proteins may play different roles and most likely bind different ligands. PMID:24769496

  6. Identification of tropomyosin as major allergen of white squid (Loligo edulis) by two-dimensional immunoblotting and mass spectrometry.

    PubMed

    Yadzir, Zailatul Hani Mohamad; Misnan, Rosmilah; Murad, Shahnaz

    2012-01-01

    IgE-mediated allergic reaction to squid is one of the most frequent molluscan shellfish allergies. Previously, we have detected a 36 kDa protein as the major allergen of Loligo edulis (white squid) by immunoblotting using sera from patients with squid allergy. The aim of this present study was to further identify this major allergen using a proteomics approach. The major allergen was identified by a combination of two-dimensional electrophoresis (2-DE), immunoblotting, mass spectrometry and bioinformatics tools. The 2-DE gel fractionated the cooked white squid proteins to more than 50 different protein spots between 10 to 38 kDa and isoelectric point (pI) from 3.0 to 10.0. A highly reactive protein spot of a molecular mass of 36 kDa and pI of 4.55 was observed in all of the patients' serum samples tested. Mass spectrometry analysis led to identification of this allergen as tropomyosin. This finding can contribute to advancement in component-based diagnosis, management of squid allergic patients, to the development of immunotherapy and to the standardization of allergenic test products as tools in molecular allergology.

  7. Major grass pollen allergen Lol p 1 binds to diesel exhaust particles: implications for asthma and air pollution.

    PubMed

    Knox, R B; Suphioglu, C; Taylor, P; Desai, R; Watson, H C; Peng, J L; Bursill, L A

    1997-03-01

    Grass pollen allergens are known to be present in the atmosphere in a range of particle sizes from whole pollen grains (approx. 20 to 55 microns in diameter) to smaller size fractions < 2.5 microns (fine particles, PM25). These latter particles are within the respirable range and include allergen-containing starch granules released from within the grains into the atmosphere when grass pollen ruptures in rainfall and are associated with epidemics of thunderstorm asthma during the grass pollen season. The question arises whether grass pollen allergens can interact with other sources of fine particles, particularly those present during episodes of air pollution. We propose the hypothesis that free grass pollen allergen molecules, derived from dead or burst grains and dispersed in microdroplets of water in aerosols, can bind to fine particles in polluted air. We used diesel exhaust carbon particles (DECP) derived from the exhaust of a stationary diesel engine, natural highly purified Lol p 1, immunogold labelling with specific monoclonal antibodies and a high voltage transmission electron-microscopic imaging technique. DECP are visualized as small carbon spheres, each 30-60 nm in diameter, forming fractal aggregates about 1-2 microns in diameter. Here we test our hypothesis and show by in vitro experiments that the major grass pollen allergen, Lol p 1, binds to one defined class of fine particles, DECP. DECP are in the respirable size range, can bind to the major grass pollen allergen Lol p 1 under in vitro conditions and represent a possible mechanism by which allergens can become concentrated in polluted air and thus trigger attacks of asthma.

  8. Identification of tropomyosins as major allergens in antarctic krill and mantis shrimp and their amino acid sequence characteristics.

    PubMed

    Motoyama, Kanna; Suma, Yota; Ishizaki, Shoichiro; Nagashima, Yuji; Lu, Ying; Ushio, Hideki; Shiomi, Kazuo

    2008-01-01

    Tropomyosin represents a major allergen of decapod crustaceans such as shrimps and crabs, and its highly conserved amino acid sequence (>90% identity) is a molecular basis of the immunoglobulin E (IgE) cross-reactivity among decapods. At present, however, little information is available about allergens in edible crustaceans other than decapods. In this study, the major allergen in two species of edible crustaceans, Antarctic krill Euphausia superba and mantis shrimp Oratosquilla oratoria that are taxonomically distinct from decapods, was demonstrated to be tropomyosin by IgE-immunoblotting using patient sera. The cross-reactivity of the tropomyosins from both species with decapod tropomyosins was also confirmed by inhibition IgE immunoblotting. Sequences of the tropomyosins from both species were determined by complementary deoxyribonucleic acid cloning. The mantis shrimp tropomyosin has high sequence identity (>90% identity) with decapod tropomyosins, especially with fast-type tropomyosins. On the other hand, the Antarctic krill tropomyosin is characterized by diverse alterations in region 13-42, the amino acid sequence of which is highly conserved for decapod tropomyosins, and hence, it shares somewhat lower sequence identity (82.4-89.8% identity) with decapod tropomyosins than the mantis shrimp tropomyosin. Quantification by enzyme-linked immunosorbent assay revealed that Antarctic krill contains tropomyosin at almost the same level as decapods, suggesting that its allergenicity is equivalent to decapods. However, mantis shrimp was assumed to be substantially not allergenic because of the extremely low content of tropomyosin.

  9. Assessment of the allergenic potential of transgenic wheat (Triticum aestivum) with reduced levels of omega-5 gliadins, the major sensitizing allergen in wheat-dependent exercise-induced anaphylaxis

    USDA-ARS?s Scientific Manuscript database

    The omega-5 gliadins are the major sensitizing allergens in wheat-dependent exercise-induced anaphylaxis (WDEIA). In this study, two-dimensional immunoblot analysis was used to assess the allergenic potential of two transgenic wheat lines in which omega-5 gliadin genes were silenced by RNA interfe...

  10. Allergenicity of Hev b 13, a major esterase allergen in natural rubber latex (Hevea brasiliensis) allergy, does not only depend on its carbohydrate moiety.

    PubMed

    Rougé, Pierre; Culerrier, Raphaël; Campistron, Marion; Granier, Claude; Bienvenu, Françoise; Bienvenu, Jacques; Didier, Alain; Barre, Annick

    2010-01-01

    The three-dimensional model built for the major latex allergen Hev b 13 consists of the typical organization of plant esterases made of a central bundle of five parallel beta-strands surrounded by five alpha-helices associated to two shorter alpha-helical segments. Up to 12 sets of sequential IgE-binding peptides were identified in SPOT experiments along the amino acid sequence of Hev b 13. They correspond in fact to eight IgE-binding epitopic stretches exposed on the surface of the allergen. With the exception of epitope #5, all other epitopes contain charged residues. Epitope #8 contains the 3rd putative N-glycosylation site of Hev b 13 and should consist of a glycotope, whereas all other identified IgE-binding areas occur outside the two remaining putative N-glycosylation sites. Accordingly, the allergenicity of Hev b 13 does not primarily depends on its carbohydrate moiety. Copyright 2010 Elsevier Ltd. All rights reserved.

  11. Identification, cloning, and characterization of a major cat flea salivary allergen (Cte f 1).

    PubMed

    McDermott, M J; Weber, E; Hunter, S; Stedman, K E; Best, E; Frank, G R; Wang, R; Escudero, J; Kuner, J; McCall, C

    2000-05-01

    An 18 kDa protein isolated from saliva of the cat flea, Ctenocephalides felis, elicits a positive intradermal skin test (IDST) in 100 and 80% of experimental and clinical flea allergic dogs, respectively. Using solid-phase enzyme-linked immuno assay (ELISA), this protein detected IgE in 100 and 80% of experimental and clinical flea allergic dogs, respectively. A cDNA (pFSI) encoding a full-length Cte f 1 protein was isolated from a C. felis salivary gland cDNA library, using a combination of PCR and hybridization screening. This cDNA is 658 bp in length, and contains an open reading frame of 528 bp. The open reading frame encodes a protein of 176 amino acids, consisting of an 18 amino acid signal sequence and a 158 amino acid mature protein. The calculated molecular weight and pI of the mature protein are 18106 Da and 9.3, respectively. The protein, named Cte f 1, is the first novel major allergen described for canine flea allergy. Recombinant Cte f 1 (rCte f 1) was expressed in Escherichia coli, Pichia pastoris and baculovirus infected Trichoplusia ni cells. Approximately, 90% of the rCte f 1 expressed in E. coli accumulated in insoluble inclusion bodies, which could be refolded to a soluble mixture of disulfide isomers with partial IgE binding activity. Small quantities of an apparently correctly refolded form of rCte f 1, which had IgE binding activity equal to the native antigen, was isolated from the soluble fraction of E. coli cells. However, P. pastoris and baculovirus infected insect cells expressed and secreted a fully processed, correctly refolded and fully active form of rCte f 1. Mass spectrometry analysis of the active forms of rCte f 1confirmed that eight intact disulfide bonds were present, matching the number observed in the native allergen. The relative ability of rCte f 1 to bind IgE in the serum of flea allergic animals, produced in these three expression systems, matched that of the native allergen. Competition ELISA demonstrated that

  12. Cloning, expression, and purification of recombinant major mango allergen Man i 1 in Escherichia coli.

    PubMed

    Tsai, Wen-Che; Wu, Tzee-Chung; Chiang, Bor-Luen; Wen, Hsiao-Wei

    2017-02-01

    In recent years, the number of people around the world who suffer from fruit allergies has increased. Mango can induce anaphylaxis, and two major mango allergens have been identified - Man i 1 and Man i 2. Apart from their molecular weights and pI values, no other information about them is known. This work identifies the DNA and amino acid sequences of Man i 1 and constructs an expression system for recombinant Man i 1 (rMan i 1). Firstly, 3' and 5' RACE assays were used to identify the cDNA fragment of Man i 1. Subsequently, the full length of Man i 1 cDNA was inserted into a pET-21a(+) vector, and the inserted plasmid was transformed to Escherichia coli BL21 (DE3) to express rMan i 1. The conditions for the expression of rMan i 1, including IPTG concentration, induction temperature, and induction time, were optimized. The highest amount of soluble rMan i 1 was obtained after induction with 0.1 mM IPTG at 16 °C for 20 h. The His-tagged rMan i 1 was purified using Ni-NTA agarose and its identity was verified using an anti-histidine antibody and the serum of a mango-allergic person. Additionally, rMan i 1 was identified as glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and shared 86.2% identity in amino acid sequence of GAPDH from wheat. Finally, an E. coli expression system of rMan i 1 was established, with the potential to be used in immunotherapy against mango allergy or the development of assays for detecting the residues of mango allergens.

  13. Purification and characterization of parvalbumins, the major allergens in red stingray (Dasyatis akajei).

    PubMed

    Cai, Qiu-Feng; Liu, Guang-Ming; Li, Teng; Hara, Kenji; Wang, Xi-Chang; Su, Wen-Jin; Cao, Min-Jie

    2010-12-22

    Fish has received increasing attention because it induces IgE-mediated food allergy. Parvalbumin (PV) represents the major allergen of fish, and IgE cross-reactivity to PV in various teleost fish species has been shown, while little information is available about allergens in elasmobranch fish. In this study, two PV isoforms (named as PV-I and PV-II) from red stingray (Dasyatis akajei) were purified to homogeneity by a series of procedures including ammonium sulfate precipitation and column chromatographies of DEAE-Sepharose and Sephacryl S-200. Purified PVs revealed a single band on tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular masses of PV-I and PV-II were 12.29 and 11.95 kDa, respectively, as determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Western blot using antifrog PV monoclonal antibody (PARV-19) showed positive reactions to the two proteins, confirming that they were PVs, although their immunological reactivities were weaker than those of PV from silver carp. The N-terminal amino acid sequence of PV-I was determined, and comparison with PVs from other fish species showed low homology between teleost and elasmobranch fish. The isoelectric points of PV-I and PV-II were 5.4 and 5.0, respectively, as determined by two-dimensional electrophoresis (2-DE), suggesting that both isoforms belong to the α-group. IgE immunoblotting analysis showed that sera from fish-allergic patients reacted to both PV-I and PV-II from red stingray. Thermal stability revealed that PV-I easily formed oligomers than PV-II, which might contribute to the maintenance of its allerginicity during heat processing.

  14. Removal of cat major allergen (Fel d I) from futon (Japanese bedding) with a home washing machine.

    PubMed

    Hashimoto, M; Nigi, H; Sakaguchi, M; Inouye, S; Miyazawa, H; Watanabe, M; Mitsuseki, M; Yasueda, H; Nitta, H

    1994-06-01

    We evaluated the removal of a cat major allergen (Fel d I) from futons (Japanese bedding) with the use of a large-sized home washing machine. Before and after washing a futon that had been used in a home with a cat, a small amount of cotton was collected from the futon and Fel d I was extracted from the cotton. The levels of Fel d I were assayed by a sandwich enzyme-linked immunosorbent assay (ELISA). We found that washing reduced the Fel d I level in futons by more than 95%. In conclusion, washing of futons is an effective method for elimination of their cat allergens.

  15. Separation and characterization of nitrated variants of the major birch pollen allergen by CZE-ESI-μTOF MS.

    PubMed

    Gusenkov, Sergey; Ackaert, Chloé; Stutz, Hanno

    2013-09-01

    A CZE-ESI-TOF MS method has been optimized for the separation and identification of nitrated variants of the major birch pollen allergen from Betula verrucosa, isoform 1a (Bet v 1a). In-house nitration of recombinant Bet v 1a was done by peroxynitrite. As a BGE, 10 mmol/L ammonium bicarbonate with pH 7.50 provided best resolution. Nebulizer gas pressure and sheath liquid flow rate of 0.4 bar and 6 μL/min, respectively, maintained CZE selectivity and constituted stable electrospray conditions. A sheath liquid composition of 75% v/v methanol with 0.1% v/v formic acid in ultrapure water resulted in highest signal intensities. Alternatively, methanol could be replaced by 50% v/v isopropanol. Two modified allergen products derived from reaction mixtures that contained different amounts of the nitration reagent were compared by the elaborated CZE-ESI-TOF MS method. Up to twelve different Bet v 1a variants with one- to sixfold nitration could be distinguished. Several allergen fractions of equivalent nitration grade were resolved. Their different migration times indicate site-specific nitration with concomitant differences in pI and maybe also in hydrodynamic radius. The method allows for a characterization of in-house nitrated allergen samples that are intended for testing the postulated enhanced allergenicity of nitrated Bet v 1a variants. © 2013 The Authors. Electrophoresis published by WILEY-VCH Verlag GmbH & Co. KGaA.

  16. Separation and characterization of nitrated variants of the major birch pollen allergen by CZE-ESI-μTOF MS

    PubMed Central

    Gusenkov, Sergey; Ackaert, Chloé; Stutz, Hanno

    2013-01-01

    A CZE-ESI-TOF MS method has been optimized for the separation and identification of nitrated variants of the major birch pollen allergen from Betula verrucosa, isoform 1a (Bet v 1a). In-house nitration of recombinant Bet v 1a was done by peroxynitrite. As a BGE, 10 mmol/L ammonium bicarbonate with pH 7.50 provided best resolution. Nebulizer gas pressure and sheath liquid flow rate of 0.4 bar and 6 μL/min, respectively, maintained CZE selectivity and constituted stable electrospray conditions. A sheath liquid composition of 75% v/v methanol with 0.1% v/v formic acid in ultrapure water resulted in highest signal intensities. Alternatively, methanol could be replaced by 50% v/v isopropanol. Two modified allergen products derived from reaction mixtures that contained different amounts of the nitration reagent were compared by the elaborated CZE-ESI-TOF MS method. Up to twelve different Bet v 1a variants with one- to sixfold nitration could be distinguished. Several allergen fractions of equivalent nitration grade were resolved. Their different migration times indicate site-specific nitration with concomitant differences in pI and maybe also in hydrodynamic radius. The method allows for a characterization of in-house nitrated allergen samples that are intended for testing the postulated enhanced allergenicity of nitrated Bet v 1a variants. PMID:23857337

  17. Standardization of RP-HPLC methods for the detection of the major peanut allergens Ara h 1, Ara h 2 and Ara h 3.

    PubMed

    Singh, Harmit; Cantoria, Mary Jo; Malave, Poonam; Saputra, Denny; Maleki, Soheila

    2016-03-01

    Crude peanut extract (CPE) was analyzed for three major allergens (Ara h 1, h 2, and h 3) using a C12 and a C18 column at two wavelengths (280 and 220nm) and under different solvent conditions. HPLC profiles were compared for retention time, resolution, and peak heights. CPE samples were spiked with pure allergens to identify the peaks corresponding to allergens. The HPLC fractions of corresponding allergens were collected and freeze-dried in order to perform SDS-PAGE and immunoblotting tests. The best method was identified the one with a shorter retention time, better resolution, and greater peak height as compared with the other methods. In general, the peak heights were greater at 220nm than at 280nm. The major disadvantage of the C12 column was the need for two sets of conditions to identify the allergens as compared to the C18 column where all three allergens could be identified in one run.

  18. Subpollen particles are rich carriers of major short ragweed allergens and NADH dehydrogenases: quantitative proteomic and allergomic study.

    PubMed

    Smiljanic, K; Apostolovic, D; Trifunovic, S; Ognjenovic, J; Perusko, M; Mihajlovic, L; Burazer, L; van Hage, M; Cirkovic Velickovic, T

    2017-06-01

    Short ragweed (Ambrosia artemisiifolia) allergies affect more than 36 million people annually. Ragweed pollen grains release subpollen particles (SPP) of respirable size upon hydration or a change in air electrical conditions. The aim of this study was to characterize the proteomes and allergomes of short ragweed SPP and total pollen protein extract (TOT), and compare their effects with those of standard aqueous pollen protein extract (APE) using sera from short ragweed pollen-sensitized patients. Quantitative 2D gel-based and shotgun proteomics, 1D and 2D immunoblotting, and quantitative ELISA were applied. Novel SPP extraction and preparation protocols enabled appropriate sample preparation and further downstream analysis by quantitative proteomics. The SPP fraction contained the highest proportion (94%) of the allergome, with the largest quantities of the minor Amb a 4 and major Amb a 1 allergens, and as unique, NADH dehydrogenases. APE was the richest in Amb a 6, Amb a 5 and Amb a 3, and TOT fraction was the richest in the Amb a 8 allergens (89% and 83% of allergome, respectively). Allergenic potency correlated well among the three fractions tested, with 1D immunoblots demonstrating a slight predominance of IgE reactivity to SPP compared to TOT and APE. However, the strongest IgE binding in ELISA was noted against APE. New allergenic candidates, phosphoglycerate mutase and phosphoglucomutase, were identified in all the three pollen fractions. Enolase, UTP-glucose-1-phosphate uridylyltransferase and polygalacturonase were observed in SPP and TOT fractions as novel allergens of the short ragweed pollen, as previously described. We demonstrated that the complete major (Amb a 1 and 11) and almost all minor (Amb a 3, 4, 5, 6, 8 and 9) short ragweed pollen allergen repertoire as well as NADH oxidases are present in SPP, highlighting an important role for SPP in allergic sensitization to short ragweed. © 2016 John Wiley & Sons Ltd.

  19. Molecular characterization of a human immunoglobulin G4 antibody specific for the major birch pollen allergen, Bet v 1.

    PubMed

    Flicker, S; Steinberger, P; Eibensteiner, P B; Lebecque, S; Kraft, D; Valenta, R

    2008-02-01

    Allergen-specific IgG4 antibodies induced by specific immunotherapy are thought to represent a protective immune response. Objective Our aim was the molecular characterization of a human IgG4 antibody (BAB5) specific for the major birch pollen allergen Bet v 1 that was derived from an immunotherapy-treated patient. The cDNA coding for BAB5 was obtained by reverse transcriptase-PCR from the BAB5-producing cell line, compared with the germ line sequences and was expressed as a soluble antibody fragment in Escherichia coli. The epitope specificity and cross-reactivity of BAB5 were investigated with recombinant and synthetic Bet v 1 fragments and Bet v 1 homologous allergens from pollen. The ability of BAB5 to block allergic patients IgE was determined by competition experiments and sandwich ELISA. BAB5 is an affinity-matured Bet v 1-specific IgG4 antibody that reacts exclusively with Bet v 1 but not with Bet v 1-related allergens. Unlike an earlier-described monoclonal IgG1-blocking antibody, BAB1, which had been isolated from the same patient, BAB5 did not block allergic patients' IgE reactivity to Bet v 1. Our study demonstrates that not all allergen-specific IgG antibodies inhibit IgE recognition of allergens and can contribute to the success of immunotherapy. The epitope specificity and affinity of IgG antibodies but not their isotype are decisive for their protective activity.

  20. 27 CFR 4.32a - Voluntary disclosure of major food allergens.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... allergen means any of the following: (i) Milk, egg, fish (for example, bass, flounder, or cod), Crustacean..., or walnuts); (ii) In the case of Crustacean shellfish, it means the name of the species of Crustacean...

  1. Resolution and identification of major peanut allergens using a combination of fluorescence two-dimensional differential gel electrophoresis, western blotting and Q-TOF mass spectrometry.

    USDA-ARS?s Scientific Manuscript database

    Peanut allergy is triggered by several proteins known as allergens. The matching resolution and identification of major peanut allergens in 2D protein maps, was accomplished by the use of fluorescence two-dimensional differential gel electrophoresis (2D DIGE), Western blotting and quadrupole time-of...

  2. Monoclonal antibodies to the major Lolium perenne (rye grass) pollen allergen Lol p I (Rye I).

    PubMed

    Kahn, C R; Marsh, D G

    1986-12-01

    Thirteen monoclonal antibodies (MAbs) were produced against Lol p I (Rye I), the major Lolium perenne (rye grass) pollen allergen. Spleen cells from A/J and SJL mice immunized with highly purified Lol p I (Lol I) were allowed to fuse with cells from the non-secreting Sp2/0-Ag14 myeloma cell line. Each MAb was analyzed for antigenic specificity by radioimmunoassay (RIA) using 125I-Lol I. The epitope specificities of seven of the MAbs were examined by competitive binding against a labelled standard MAb for the Lol I antigen (Ag). The dissociation constant, Kd, of one MAb (No. 3.2) that was studied most extensively was determined by double Ab RIA to be 3.5 X 10(-6) L/M. This MAb recognized the related 27,000-30,000 Group I glycoproteins found in the pollens of nine other species of grass pollens tested, including weak binding to Bermuda grass Group I (Cyn d I), which by conventional analysis using polyclonal anti-Lol I serum shows no detectable binding. Monoclonal antibody No. 3.2 was coupled covalently to Sepharose 4B and used to prepare highly purified Lol I from a partially purified rye pollen extract. Finally, an RIA was developed which permitted the analysis of the Group I components in rye grass and nine other grass pollen species. The latter assay is likely to prove useful in the standardization of grass pollen extracts according to their Group I contents.

  3. Rubber elongation factor (REF), a major allergen component in Hevea brasiliensis latex has amyloid properties.

    PubMed

    Berthelot, Karine; Lecomte, Sophie; Estevez, Yannick; Coulary-Salin, Bénédicte; Bentaleb, Ahmed; Cullin, Christophe; Deffieux, Alain; Peruch, Frédéric

    2012-01-01

    REF (Hevb1) and SRPP (Hevb3) are two major components of Hevea brasiliensis latex, well known for their allergenic properties. They are obviously taking part in the biosynthesis of natural rubber, but their exact function is still unclear. They could be involved in defense/stress mechanisms after tapping or directly acting on the isoprenoid biosynthetic pathway. The structure of these two proteins is still not described. In this work, it was discovered that REF has amyloid properties, contrary to SRPP. We investigated their structure by CD, TEM, ATR-FTIR and WAXS and neatly showed the presence of β-sheet organized aggregates for REF, whereas SRPP mainly fold as a helical protein. Both proteins are highly hydrophobic but differ in their interaction with lipid monolayers used to mimic the monomembrane surrounding the rubber particles. Ellipsometry experiments showed that REF seems to penetrate deeply into the monolayer and SRPP only binds to the lipid surface. These results could therefore clarify the role of these two paralogous proteins in latex production, either in the coagulation of natural rubber or in stress-related responses. To our knowledge, this is the first report of an amyloid formed from a plant protein. This suggests also the presence of functional amyloid in the plant kingdom.

  4. Rubber Elongation Factor (REF), a Major Allergen Component in Hevea brasiliensis Latex Has Amyloid Properties

    PubMed Central

    Berthelot, Karine; Lecomte, Sophie; Estevez, Yannick; Coulary-Salin, Bénédicte; Bentaleb, Ahmed; Cullin, Christophe; Deffieux, Alain; Peruch, Frédéric

    2012-01-01

    REF (Hevb1) and SRPP (Hevb3) are two major components of Hevea brasiliensis latex, well known for their allergenic properties. They are obviously taking part in the biosynthesis of natural rubber, but their exact function is still unclear. They could be involved in defense/stress mechanisms after tapping or directly acting on the isoprenoid biosynthetic pathway. The structure of these two proteins is still not described. In this work, it was discovered that REF has amyloid properties, contrary to SRPP. We investigated their structure by CD, TEM, ATR-FTIR and WAXS and neatly showed the presence of β-sheet organized aggregates for REF, whereas SRPP mainly fold as a helical protein. Both proteins are highly hydrophobic but differ in their interaction with lipid monolayers used to mimic the monomembrane surrounding the rubber particles. Ellipsometry experiments showed that REF seems to penetrate deeply into the monolayer and SRPP only binds to the lipid surface. These results could therefore clarify the role of these two paralogous proteins in latex production, either in the coagulation of natural rubber or in stress-related responses. To our knowledge, this is the first report of an amyloid formed from a plant protein. This suggests also the presence of functional amyloid in the plant kingdom. PMID:23133547

  5. Molecular and immunological characterization of Tri a 36, a low molecular weight glutenin, as a novel major wheat food allergen.

    PubMed

    Baar, Alexandra; Pahr, Sandra; Constantin, Claudia; Scheiblhofer, Sandra; Thalhamer, Josef; Giavi, Stavroula; Papadopoulos, Nikolaos G; Ebner, Christof; Mari, Adriano; Vrtala, Susanne; Valenta, Rudolf

    2012-09-15

    Wheat is an essential element in our nutrition but one of the most important food allergen sources. Wheat allergic patients often suffer from severe gastrointestinal and systemic allergic reactions after wheat ingestion. In this study, we report the molecular and immunological characterization of a new major wheat food allergen, Tri a 36. The cDNA coding for a C-terminal fragment of Tri a 36 was isolated by screening a wheat seed cDNA expression library with serum IgE from wheat food-allergic patients. Tri a 36 is a 369-aa protein with a hydrophobic 25-aa N-terminal leader peptide. According to sequence comparison it belongs to the low m.w. glutenin subunits, which can be found in a variety of cereals. The mature allergen contains an N-terminal domain, a repetitive domain that is rich in glutamine and proline residues, and three C-terminal domains with eight cysteine residues contributing to intra- and intermolecular disulfide bonds. Recombinant Tri a 36 was expressed in Escherichia coli and purified as soluble protein. It reacted with IgE Abs of ∼80% of wheat food-allergic patients, showed IgE cross-reactivity with related allergens in rye, barley, oat, spelt, and rice, and induced specific and dose-dependent basophil activation. Even after extensive in vitro gastric and duodenal digestion, Tri a 36 released distinct IgE-reactive fragments and was highly resistant against boiling. Thus, recombinant Tri a 36 is a major wheat food allergen that can be used for the molecular diagnosis of, and for the development of specific immunotherapy strategies against, wheat food allergy.

  6. Mapping of IgE-binding regions on recombinant Cyn d 1, a major allergen from Bermuda Grass Pollen (BGP).

    PubMed

    Tiwari, Ruby; Bhalla, Prem L; Singh, Mohan B

    2009-02-02

    Bermuda grass (Cynodon dactylon; subfamily Chloridoideae) is an important source of seasonal aeroallergens in warm tropical and sub-tropical areas worldwide. Improved approaches to diagnosis and therapy of allergic diseases require a thorough understanding of the structure and epitopes on the allergen molecule that are crucial for the antigen-antibody interaction. This study describes the localization of the human IgE-binding regions of the major group 1 pollen allergen Cyn d 1 from Bermuda grass. A cDNA library was constructed from Bermuda grass pollen (BGP) using a Lambda gt11 expression vector. The gene encoding the Cyn d 1 allergen was isolated by screening the library with a mouse monoclonal antibody raised against grass group 1 allergen. In order to characterize the IgE epitopes on Cyn d 1, seven overlapping fragments and three deletion mutants were cloned and over-expressed in E. coli. The recombinant fragments and deletion mutants were evaluated for their comparative IgE reactivity with sera of non atopic individuals and grass pollen allergic patients by ELISA and a dot-blot assay. Analysis of IgE binding regions by overlapping fragments and deletion mutants identified two major allergenic regions corresponding to amino acids 120-170 and 224-244. Deletion of either or both regions led to a significant reduction in IgE binding, emphasizing the importance of the C-terminal region on Cyn d 1 in epitope-IgE interaction. Anti-Cyn d 1 IgE antibodies from allergic human sera recognize two epitopes located at the C-terminal end of the molecule. These data will enable the design of improved diagnostic and therapeutic approaches for BGP hypersensitivity.

  7. Mapping of IgE-binding regions on recombinant Cyn d 1, a major allergen from Bermuda Grass Pollen (BGP)

    PubMed Central

    Tiwari, Ruby; Bhalla, Prem L; Singh, Mohan B

    2009-01-01

    Background Bermuda grass (Cynodon dactylon; subfamily Chloridoideae) is an important source of seasonal aeroallergens in warm tropical and sub-tropical areas worldwide. Improved approaches to diagnosis and therapy of allergic diseases require a thorough understanding of the structure and epitopes on the allergen molecule that are crucial for the antigen-antibody interaction. This study describes the localization of the human IgE-binding regions of the major group 1 pollen allergen Cyn d 1 from Bermuda grass. Methods A cDNA library was constructed from Bermuda grass pollen (BGP) using a Lambda gt11 expression vector. The gene encoding the Cyn d 1 allergen was isolated by screening the library with a mouse monoclonal antibody raised against grass group 1 allergen. In order to characterize the IgE epitopes on Cyn d 1, seven overlapping fragments and three deletion mutants were cloned and over-expressed in E. coli. The recombinant fragments and deletion mutants were evaluated for their comparative IgE reactivity with sera of non atopic individuals and grass pollen allergic patients by ELISA and a dot-blot assay. Results Analysis of IgE binding regions by overlapping fragments and deletion mutants identified two major allergenic regions corresponding to amino acids 120–170 and 224–244. Deletion of either or both regions led to a significant reduction in IgE binding, emphasizing the importance of the C-terminal region on Cyn d 1 in epitope-IgE interaction. Conclusion Anti-Cyn d 1 IgE antibodies from allergic human sera recognize two epitopes located at the C-terminal end of the molecule. These data will enable the design of improved diagnostic and therapeutic approaches for BGP hypersensitivity. PMID:19187539

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

    PubMed

    Muñoz-García, Esther; Luengo-Sánchez, Olga; Haroun-Díaz, Elisa; Maroto, Aroa Sanz; Palacín, Arancha; Díaz-Perales, Araceli; de las Heras Gozalo, Manuel; Labrador-Horrillo, Moisés; Vivanco, Fernando; Cuesta-Herranz, Javier; Pastor-Vargas, Carlos

    2013-12-01

    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. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Lol p XI, a new major grass pollen allergen, is a member of a family of soybean trypsin inhibitor-related proteins.

    PubMed

    van Ree, R; Hoffman, D R; van Dijk, W; Brodard, V; Mahieu, K; Koeleman, C A; Grande, M; van Leeuwen, W A; Aalberse, R C

    1995-05-01

    Monoclonal antibodies were obtained against an unknown allergen from Lolium perenne grass pollen. The allergen had an apparent molecular mass of 18 kd on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Earlier immunoblotting studies had shown that carbohydrate-specific IgG antibodies recognize an antigen of similar size. We sought to characterize the allergen biochemically and immunologically. The amino acid sequence of the allergen was determined by automated Edman degradation, and its monosaccharide composition was determined by gas chromatographic analysis. A panel of 270 grass pollen-positive sera was assessed in a RAST with the purified allergen. Protease digestion (proteinase K) and chemical deglycosylation (trifluoromethane sulfonic acid) were used to distinguish between carbohydrate and peptide epitopes for IgE antibodies. The allergen was shown to be a glycoprotein with a molecular mass of 16 kd, of which 8% is carbohydrate. Its amino acid sequence shares 32% homology with soybean trypsin inhibitor (Kunitz) but lacks its active site. No homology was found with known grass pollen allergens, hence it was designated Lol p XI. A high degree of homology (44%) was found with a tree pollen allergen, Ole e I, the major allergen of olive pollen. More than 65% of grass pollen-positive sera had IgE against Lol p XI. IgE reactivity was demonstrated both with the carbohydrate moiety and the peptide backbone. Lol p XI is a new major grass pollen allergen carrying an IgE-binding carbohydrate determinant. Lol p XI is structurally related to the major allergen from olive pollen.

  10. Structural glycobiology of the major allergen of Artemisia vulgaris pollen, Art v 1: O-glycosylation influence on the protein dynamics and allergenicity.

    PubMed

    Pol-Fachin, Laercio; Verli, Hugo

    2012-06-01

    Art v 1 is the major allergen of mugwort (Artemisia vulgaris) pollen. It is formed by an N-terminal globular defensin-like part and a C-terminal proline-rich domain. As the structure and the dynamics of Art v 1 have been mostly described for its recombinant, non-glycosylated form, which does not occur in normal plant physiology, the present work intends to obtain a three-dimensional model for Art v 1 native O-glycosylation structure and to evaluate the influence of such glycans over the protein dynamics and allergenicity through molecular dynamics simulations in triplicates. Structural insights into the mutual recognition of Art v 1 protein and carbohydrate moieties recognition by antibodies were obtained, in which glycan chains remained close to the previously identified epitopes in the defensin-like domain, thus pointing to potential interferences with antibodies recognition. To our knowledge, this is the first structural report of an entire furanose-containing glycoprotein. As well, together with the previously determined NMR structures, the obtained results contribute in the comprehension of the effect of glycosylation over both proline-rich and defensin-like domains, providing an atomic representation of such alterations.

  11. Identification of Der p 23, a Peritrophin-like Protein, as a New Major Dermatophagoides pteronyssinus Allergen Associated with the Peritrophic Matrix of Mite Fecal Pellets

    PubMed Central

    Weghofer, Margit; Grote, Monika; Resch, Yvonne; Casset, Anne; Kneidinger, Michael; Kopec, Jolanta; Thomas, Wayne R.; Fernández-Caldas, Enrique; Kabesch, Michael; Ferrara, Rosetta; Mari, Adriano; Purohit, Ashok; Pauli, Gabrielle; Horak, Friedrich; Keller, Walter; Valent, Peter; Valenta, Rudolf; Vrtala, Susanne

    2015-01-01

    The house dust mite (HDM) Dermatophagoides pteronyssinus is one of most important allergen sources and a major elicitor of allergic asthma. We screened a D. pteronyssinus expression cDNA library with IgE Abs from HDM allergic patients. A cDNA coding for a new major allergen was isolated, which showed sequence homology to peritrophins, which contain chitin-binding domains and are part of the peritrophic matrix lining the gut of arthropods. The mature Der p 23 allergen was expressed in Escherichia coli as an 8-kDa protein without its hydrophobic leader sequence and purified to homogeneity. It reacted with IgE Abs from 74% of D. pteronyssinus allergic patients (n = 347) at levels comparable to the two major HDM allergens, Der p 1 and Der p 2. Thus, Der p 23 represents a new major D. pteronyssinus allergen. Furthermore, rDer p 23 exhibited high allergenic activity as demonstrated by upregulation of CD203c expression on basophils from D. pteronyssinus allergic patients. Immunogold electron microscopy localized the allergen in the peritrophic matrix lining the midgut of D. pteronyssinus as well as on the surface of the fecal pellets. Thus, we identified a new major D. pteronyssinus allergen as peritrophin-like protein. The high allergenic activity of Der p 23 and its frequent recognition as respiratory allergen may be explained by the fact that it becomes airborne and respirable through its association with mite feces. Der p 23 may be an essential component for diagnosis and specific immunotherapy of HDM allergy. PMID:23460742

  12. Identification of Der p 23, a peritrophin-like protein, as a new major Dermatophagoides pteronyssinus allergen associated with the peritrophic matrix of mite fecal pellets.

    PubMed

    Weghofer, Margit; Grote, Monika; Resch, Yvonne; Casset, Anne; Kneidinger, Michael; Kopec, Jolanta; Thomas, Wayne R; Fernández-Caldas, Enrique; Kabesch, Michael; Ferrara, Rosetta; Mari, Adriano; Purohit, Ashok; Pauli, Gabrielle; Horak, Friedrich; Keller, Walter; Valent, Peter; Valenta, Rudolf; Vrtala, Susanne

    2013-04-01

    The house dust mite (HDM) Dermatophagoides pteronyssinus is one of most important allergen sources and a major elicitor of allergic asthma. We screened a D. pteronyssinus expression cDNA library with IgE Abs from HDM allergic patients. A cDNA coding for a new major allergen was isolated, which showed sequence homology to peritrophins, which contain chitin-binding domains and are part of the peritrophic matrix lining the gut of arthropods. The mature Der p 23 allergen was expressed in Escherichia coli as an 8-kDa protein without its hydrophobic leader sequence and purified to homogeneity. It reacted with IgE Abs from 74% of D. pteronyssinus allergic patients (n = 347) at levels comparable to the two major HDM allergens, Der p 1 and Der p 2. Thus, Der p 23 represents a new major D. pteronyssinus allergen. Furthermore, rDer p 23 exhibited high allergenic activity as demonstrated by upregulation of CD203c expression on basophils from D. pteronyssinus allergic patients. Immunogold electron microscopy localized the allergen in the peritrophic matrix lining the midgut of D. pteronyssinus as well as on the surface of the fecal pellets. Thus, we identified a new major D. pteronyssinus allergen as peritrophin-like protein. The high allergenic activity of Der p 23 and its frequent recognition as respiratory allergen may be explained by the fact that it becomes airborne and respirable through its association with mite feces. Der p 23 may be an essential component for diagnosis and specific immunotherapy of HDM allergy.

  13. The major Alternaria alternata allergen, Alt a 1: A reliable and specific marker of fungal contamination in citrus fruits.

    PubMed

    Gabriel, M F; Uriel, N; Teifoori, F; Postigo, I; Suñén, E; Martínez, J

    2017-09-18

    The ubiquitously present spores of Alternaria alternata can spoil a wide variety of foodstuffs, including a variety of fruits belonging to the Citrus genus. The major allergenic protein of A. alternata, Alt a 1, is a species-specific molecular marker that has been strongly associated with allergenicity and phytopathogenicity of this fungal species. This study aimed to evaluate the potential of the detection of Alt a 1 as a reliable indicator of A. alternata contamination in citrus fruits. To accomplish this aim, sixty oranges were artificially infected with a spore suspension of A. alternata. Internal fruit material was collected at different incubation times (one, two and three weeks after the fungal inoculation) and used for both total RNA extraction and protein extraction. Alt a 1 detection was then performed by polymerase chain reaction (PCR) amplification using Alt a 1 specific primers and by enzyme-linked immunosorbent assay (ELISA). The experimental model presented in this work was effective to simulate the typical Alternaria black rot phenotype and its progression. Although both PCR and ELISA techniques have been successfully carried out for detecting Alt a 1 allergen in A. alternata infected oranges, the PCR method was found to be more sensitive than ELISA. Nevertheless, ELISA results were highly valuable to demonstrate that considerable amounts of Alt a 1 are produced during A. alternata fruit infection process, corroborating the recently proposed hypothesis that this protein plays a role in the pathogenicity and virulence of Alternaria species. Such evidence suggests that the detection of Alt a 1 by PCR-based assay may be used as a specific indicator of the presence of pathogenic and allergenic fungal species, A. alternata, in fruits. This knowledge can be employed to control the fungal infection and mitigate agricultural losses as well as human exposure to A. alternata allergens and toxins. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. An unfolded variant of the major peanut allergen Ara h 2 with decreased anaphylactic potential.

    PubMed

    Starkl, P; Felix, F; Krishnamurthy, D; Stremnitzer, C; Roth-Walter, F; Prickett, S R; Voskamp, A L; Willensdorfer, A; Szalai, K; Weichselbaumer, M; O'Hehir, R E; Jensen-Jarolim, E

    2012-12-01

    Peanut allergy causes severe type 1 hypersensitivity reactions and conventional immunotherapy against peanut allergy is associated with a high risk of anaphylaxis. Our current study reports proof of concept experiments on the safety of a stably denatured variant of the major peanut allergen Ara h 2 for immunotherapy. We determined the impact of structure loss of Ara h 2 on its IgE binding and basophil degranulation capacity, T cell reactivity as well as anaphylactic potential. The secondary structure of untreated and reduced/alkylated Ara h 2 variants was determined by circular dichroism spectroscopy. We addressed human patient IgE binding to Ara h 2 by ELISA and Western blot experiments. RBL-SX38 cells were used to test the degranulation induced by untreated and reduced/alkylated Ara h 2. We assessed the anaphylactic potential of Ara h 2 variants by challenge of sensitized BALB/c mice. T cell reactivity was investigated using human Ara h 2-specific T cell lines and splenocytes isolated from sensitized mice. Reduction/alkylation of Ara h 2 caused a decrease in IgE binding capacity, basophil degranulation and anaphylactic potential in vivo. However, the human T cell response to reduced/alkylated and untreated Ara h 2 was comparable. Mouse splenocytes showed higher metabolic activity upon stimulation with reduced/alkylated Ara h 2 and released similar IL-4, IL-13 and IFNγ levels upon treatment with either Ara h 2 variant. Reduced/alkylated Ara h 2 might be a safer alternative than native Ara h 2 for immunotherapeutic treatment of peanut allergic patients. © 2012 Blackwell Publishing Ltd.

  15. Purification, crystallization and initial crystallographic characterization of peanut major allergen Ara h 3

    SciTech Connect

    Jin, Tengchuan; Howard, Andrew; Zhang, Yu-Zhu

    2007-10-01

    The crystallization of peanut allergen Ara h 3 is reported. The peanut is a significant food source, but is responsible for many cases of anaphylaxis. The peanut 11S legumin-like seed storage protein Ara h 3 is one of the best characterized allergens. In this study, Ara h 3 was extracted from peanut kernels and purified by sequential anion-exchange, hydrophobic interaction and gel-filtration chromatography to very high purity to facilitate crystallization and structural studies. Well diffracting single crystals were obtained by the vapor-diffusion method. A molecular-replacement structural solution has been obtained and refinement of the structure is currently under way.

  16. Characterization of peach thaumatin-like proteins and their identification as major peach allergens.

    PubMed

    Palacín, A; Tordesillas, L; Gamboa, P; Sanchez-Monge, R; Cuesta-Herranz, J; Sanz, M L; Barber, D; Salcedo, G; Díaz-Perales, A

    2010-09-01

    Peach is the most important fruit related to food allergy in the Mediterranean area. Pru p 3, its lipid transfer protein, has been described as the principal allergen responsible for cross-reactivities with other foods and pollen and the severity of clinical symptoms. However, the involvement of other allergenic families cannot be ruled out. Thaumatin-like proteins (TLPs) have been described as food allergen in several fruits, such as apple, cherry, kiwi and banana, and pollen. To identify members of the TLP family in peach fruit and to characterize putative allergens. Through two-dimensional (2D) electrophoresis of peach extract and immunodetections with a pool of peach-allergic patients, IgE-binding spots were identified and the corresponding proteins purified and characterized as allergens by in vitro and in vivo assays. Three isoforms, belonging to the TLP family, were purified by different chromatographic systems and characterized by N-terminal amino acid sequences, molecular weight determination (MALDI) and enzymatic activity analysis (beta-1,3-gluconase test and inhibition growth of fungi). In the same way, their IgE-binding capacity and allergenic activity were tested by ELISA assays, basophil activation tests and skin prick tests (SPT). Two peach-TLPs, Pru p 2.0101 and Pru p 2.0201, were identified as IgE-binding spots by 2D electrophoresis. Another peach-TLP, Pru p 2.0301, was cloned and produced as recombinant protein in a yeast system. The three isoforms were purified and characterized as TLPs by immunoblotting with anti-chestnut TLP antibodies and anti-plant N-asparagine complex glycan (anti-cross-reactive carbohydrate determinant). All of them showed beta-1,3-glucanase activity and inhibition of fungal growth. The three TLPs were recognized by around 50% of the sera from 31 patients analysed in ELISA experiments. All three gave a positive response to an SPT and/or in basophil activation experiments. Three isoforms, belonging to the TLP family, were

  17. Evaluation of different glycoforms of honeybee venom major allergen phospholipase A2 (Api m 1) produced in insect cells.

    PubMed

    Blank, Simon; Michel, Yvonne; Seismann, Henning; Plum, Melanie; Greunke, Kerstin; Grunwald, Thomas; Bredehorst, Reinhard; Ollert, Markus; Braren, Ingke; Spillner, Edzard

    2011-04-01

    Allergic reactions to hymenoptera stings are one of the major reasons for IgE-mediated anaphylaxis. However, proper diagnosis using venom extracts is severely affected by molecular cross-reactivity. In this study recombinant honeybee venom major allergen phospholipase A2 (Api m 1) was produced for the first time in insect cells. Using baculovirus infection of different insect cell lines allergen versions providing a varying degree of cross-reactive carbohydrate determinants as well as a non glycosylated variant could be obtained as secreted soluble proteins in high yields. The resulting molecules were analyzed for their glycosylation and proved to show advantageous properties regarding cross-reactivity in sIgE-based assays. Additionally, in contrast to the enzymatically active native protein the inactivated allergen did not induce IgE-independent effector cell activation. Thus, insect cell-derived recombinant Api m 1 with defined CCD phenotypes might provide further insights into hymenoptera venom IgE reactivities and contribute to an improved diagnosis of hymenoptera venom allergy.

  18. Cloning and sequencing of Lol pI, the major allergenic protein of rye-grass pollen.

    PubMed

    Griffith, I J; Smith, P M; Pollock, J; Theerakulpisut, P; Avjioglu, A; Davies, S; Hough, T; Singh, M B; Simpson, R J; Ward, L D

    1991-02-25

    We have isolated a full length cDNA clone encoding the major glycoprotein allergen Lol pI. The clone was selected using a combination of immunological screening of a cDNA expression library and PCR amplification of Lol pI-specific transcripts. Lol pI expressed in bacteria as a fusion protein shows recognition by specific IgE antibodies present in sera of grass pollen-allergic subjects. Northern analysis has shown that the Lol pI transcripts are expressed only in pollen of rye-grass. Molecular cloning of Lol pI provides a molecular genetic approach to study the structure-function relationship of allergens.

  19. Crystallization and preliminary X-ray analysis of the major peanut allergen Ara h 1 core region

    PubMed Central

    Cabanos, Cerrone; Urabe, Hiroyuki; Masuda, Taro; Tandang-Silvas, Mary Rose; Utsumi, Shigeru; Mikami, Bunzo; Maruyama, Nobuyuki

    2010-01-01

    Peanuts contain some of the most potent food allergens known to date. Ara h 1 is one of the three major peanut allergens. As a first step towards three-dimensional structure elucidation, recombinant Ara h 1 core region was cloned, expressed in Escherichia coli and purified to homogeneity. Crystals were obtained using 0.1 M sodium citrate pH 5.6, 0.1 M NaCl, 15% PEG 400 as precipitant. The crystals diffracted to 2.25 Å resolution using synchrotron radiation and belonged to the monoclinic space group C2, with unit-cell parameters a = 156.521, b = 88.991, c = 158.971 Å, β = 107.144°. Data were collected at the BL-38B1 station of SPring-8 (Hyogo, Japan). PMID:20823529

  20. Molecular cloning, expression and immunological characterisation of Pas n 1, the major allergen of Bahia grass Paspalum notatum pollen.

    PubMed

    Davies, Janet M; Mittag, Diana; Dang, Thanh D; Symons, Karen; Voskamp, Astrid; Rolland, Jennifer M; O'Hehir, Robyn E

    2008-12-01

    Bahia grass, Paspalum notatum, is a clinically important subtropical grass with a prolonged pollination season from spring to autumn. We aimed to clone and characterise the major Bahia grass pollen allergen, Pas n 1. Grass pollen-allergic patients presenting to a tertiary hospital allergy clinic were tested for IgE reactivity with Bahia grass pollen extract by skin prick testing, ImmunoCAP, ELISA and immunoblotting. Using primers deduced from the N-terminal peptide sequence of a group 1 allergen of Bahia grass pollen extract separated by two-dimensional gel electrophoresis, the complete Pas n 1 cDNA was obtained by rapid amplification of cDNA ends and cloned. Biological relevance of recombinant Pas n 1 expressed in Escherichia coli was assessed by serum IgE reactivity and basophil activation. Twenty-nine of 34 (85%) consecutive patients presenting with grass pollen allergy were skin prick test positive to Bahia grass pollen. The Pas n 1 cDNA has sequence homology with the beta-expansin 1 glycoprotein family and is more closely related to the maize pollen group 1 allergen (85% identity) than to ryegrass Lol p 1 or Timothy grass Phl p 1 (64 and 66% identity, respectively). rPas n 1 reacted with serum IgE in 47 of 55 (85%) Bahia grass pollen-allergic patients, activated basophils and inhibited serum IgE reactivity with the 29 kDa band of Bahia grass pollen extract. In conclusion the cDNA for the major group 1 allergen of the subtropical Bahia grass pollen, Pas n 1, was identified and cloned. rPas n 1 is immunologically active and is a valuable reagent for diagnosis and specific immunotherapy of grass pollen allergy.

  1. The amino acid sequence of Ole e I, the major allergen from olive tree (Olea europaea) pollen.

    PubMed

    Villalba, M; Batanero, E; López-Otín, C; Sánchez, L M; Monsalve, R I; González de la Peña, M A; Lahoz, C; Rodríguez, R

    1993-09-15

    The complete primary structure of the major allergen from Olea europaea (olive tree) pollen, Ole e I (IUIS nomenclature), has been determined. The amino acid sequence was established by automated Edman degradation of the reduced and alkylated molecule as well as of selected fragments obtained by proteolytic digestions. Ole e I contains a single polypeptide chain of 145 amino acid residues with a calculated molecular mass of 16331 Da. No free sulfhydryl groups have been detected in the native protein. The molecule contains a putative glycosylation site. A high degree of microheterogeneity has been observed, mainly centered in the first 33% of the molecule. Comparison of Ole e I sequence with protein sequence databases showed no similarity with other known allergens. However, it has a 36% and 38% sequence identity with the putative polypeptide structures, deduced, respectively, from nucleotide sequences of genes isolated from tomato anthers and corn pollen, which have been suggested to be involved in the growing of the pollen tube. Therefore, the olive tree allergen may be a constitutive protein of the pollen involved in reproductive functions.

  2. Heat processing of peanut seed enhances the sensitization potential of the major peanut allergen Ara h 6

    PubMed Central

    Guillon, Blanche; Bernard, Hervé; Drumare, Marie‐Françoise; Hazebrouck, Stéphane

    2016-01-01

    1 Scope Processing of food has been shown to impact IgE binding and functionality of food allergens. In the present study, we investigated the impact of heat processing on the sensitization capacity of Ara h 6, a major peanut allergen and one of the most potent elicitors of the allergic reaction. 2 Methods and results Peanut extracts obtained from raw or heat‐processed peanut and some fractions thereof were biochemically and immunochemically characterized. These extracts/fractions, purified Ara h 6, or recombinant Ara h 6 including Ara h 6 mutants lacking disulfide bridges were used in in vitro digestion tests and mouse models of experimental sensitization. Peanut roasting led to the formation of complexes of high molecular weight, notably between Ara h 6 and Ara h 1, which supported the induction of IgE specific to native Ara h 6. On the contrary, a fraction containing free monomeric 2S albumins or purified native Ara h 6 displayed no intrinsic allergenicity. In addition to complex formation, heat denaturation and/or partial destabilization enhanced Ara h 6 immunogenicity and increased its sensitivity to digestion. 3 Conclusion These results suggest that sensitization potency and IgE binding capacity can be supported by different structures, modified and/or produced during food processing in interaction with other food constituents. PMID:27374416

  3. Heat processing of peanut seed enhances the sensitization potential of the major peanut allergen Ara h 6.

    PubMed

    Guillon, Blanche; Bernard, Hervé; Drumare, Marie-Françoise; Hazebrouck, Stéphane; Adel-Patient, Karine

    2016-12-01

    Processing of food has been shown to impact IgE binding and functionality of food allergens. In the present study, we investigated the impact of heat processing on the sensitization capacity of Ara h 6, a major peanut allergen and one of the most potent elicitors of the allergic reaction. Peanut extracts obtained from raw or heat-processed peanut and some fractions thereof were biochemically and immunochemically characterized. These extracts/fractions, purified Ara h 6, or recombinant Ara h 6 including Ara h 6 mutants lacking disulfide bridges were used in in vitro digestion tests and mouse models of experimental sensitization. Peanut roasting led to the formation of complexes of high molecular weight, notably between Ara h 6 and Ara h 1, which supported the induction of IgE specific to native Ara h 6. On the contrary, a fraction containing free monomeric 2S albumins or purified native Ara h 6 displayed no intrinsic allergenicity. In addition to complex formation, heat denaturation and/or partial destabilization enhanced Ara h 6 immunogenicity and increased its sensitivity to digestion. These results suggest that sensitization potency and IgE binding capacity can be supported by different structures, modified and/or produced during food processing in interaction with other food constituents. © 2016 The Authors. Molecular Nutrition & Food Research published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Comparison of six commercial ELISA kits for their specificity and sensitivity in detecting different major peanut allergens.

    PubMed

    Jayasena, Shyamali; Smits, Mieke; Fiechter, Daniëlle; de Jong, Aard; Nordlee, Julie; Baumert, Joe; Taylor, Steve L; Pieters, Raymond H; Koppelman, Stef J

    2015-02-18

    Six commercial peanut enzyme-linked immunosorbent assay kits were assessed for their ability to recover peanut from the standard reference material 2387 peanut butter and also for their specificity in detecting four major peanut allergens, Ara h 1, Ara h 2, Ara h 3, and Ara h 6. The percentage recovery of peanut from peanut butter differed across different kits as well as at different sample concentrations. The highest recovery was observed with the Romer and R-Biopharm kits, while four other kits were found to underestimate the protein content of the reference peanut butter samples. Five of the kits were most sensitive in detecting Ara h 3 followed by Ara h 1, while hardly recognizing Ara h 2 and Ara h 6. The other kit showed the highest sensitivity to Ara h 2 and Ara h 6, while Ara h 1 and Ara h 3 were poorly recognized. Although Ara h 2 and Ara h 6 are known to be heat stable and more potent allergens, antisera specific to any of these four peanut proteins/allergens may serve as good markers for the detection of peanut residues.

  5. Genetic engineering of trimers of hypoallergenic fragments of the major birch pollen allergen, Bet v 1, for allergy vaccination.

    PubMed

    Vrtala, Susanne; Fohr, Monika; Campana, Raffaela; Baumgartner, Christian; Valent, Peter; Valenta, Rudolf

    2011-03-03

    An immunotherapy trial performed in allergic patients with hypoallergenic recombinant fragments, comprising aa 1-74 and 75-160 of the major birch pollen allergen, Bet v 1, has indicated that the induction of allergen-specific IgG responses may be an important mechanism of this treatment. To investigate whether the immunogenicity of the rBet v 1 fragments can be increased, recombinant trimers of the fragments were produced. For this purpose, DNA trimers of rBet v 1 aa 1-74 as well as of rBet v 1 aa 75-160 were subcloned into expression plasmid pET 17b, expressed in Escherichia coli and purified. The fragments as well as the fragment trimers showed a reduced IgE-binding capacity and allergenic activity compared to rBet v 1 wildtype when tested in allergic patients. Both rBet v 1 aa 75-160 monomer and trimer induced high titers of allergen-specific IgG1 Abs in mice. Interestingly, rBet v 1 aa 1-74 trimer induced a much higher IgG(1) response to rBet v 1 than rBet v 1 aa 1-74 monomer. Consequently, IgG Abs induced with the rBet v 1 aa 1-74 trimer inhibited birch pollen allergic patients' IgE-binding 10-fold more efficiently than IgG Abs induced with the monomer. Our data show that the immunogenicity of allergy vaccines can be increased by oligomerization.

  6. Solution Structure, Copper Binding and Backbone Dynamics of Recombinant Ber e 1–The Major Allergen from Brazil Nut

    PubMed Central

    Rundqvist, Louise; Tengel, Tobias; Zdunek, Janusz; Björn, Erik; Schleucher, Jürgen; Alcocer, Marcos J. C.; Larsson, Göran

    2012-01-01

    Background The 2S albumin Ber e 1 is the major allergen in Brazil nuts. Previous findings indicated that the protein alone does not cause an allergenic response in mice, but the addition of components from a Brazil nut lipid fraction were required. Structural details of Ber e 1 may contribute to the understanding of the allergenic properties of the protein and its potential interaction partners. Methodology/Principal Findings The solution structure of recombinant Ber e 1 was solved using NMR spectroscopy and measurements of the protein back bone dynamics at a residue-specific level were extracted using 15N-spin relaxation. A hydrophobic cavity was identified in the structure of Ber e 1. Using the paramagnetic relaxation enhancement property of Cu2+ in conjunction with NMR, it was shown that Ber e 1 is able to specifically interact with the divalent copper ion and the binding site was modeled into the structure. The IgE binding region as well as the copper binding site show increased dynamics on both fast ps-ns timescale as well as slower µs-ms timescale. Conclusions/Significance The overall fold of Ber e 1 is similar to other 2S albumins, but the hydrophobic cavity resembles that of a homologous non-specific lipid transfer protein. Ber e 1 is the first 2S albumin shown to interact with Cu2+ ions. This Cu2+ binding has minimal effect on the electrostatic potential on the surface of the protein, but the charge distribution within the hydrophobic cavity is significantly altered. As the hydrophobic cavity is likely to be involved in a putative lipid interaction the Cu2+ can in turn affect the interaction that is essential to provoke an allergenic response. PMID:23056307

  7. IgE-Binding Epitope Mapping and Tissue Localization of the Major American Cockroach Allergen Per a 2

    PubMed Central

    Lee, Mey-Fann; Chang, Chia-Wei; Song, Pei-Pong; Hwang, Guang-Yuh; Lin, Shyh-Jye

    2015-01-01

    Purpose Cockroaches are the second leading allergen in Taiwan. Sensitization to Per a 2, the major American cockroach allergen, correlates with clinical severity among patients with airway allergy, but there is limited information on IgE epitopes and tissue localization of Per a 2. This study aimed to identify Per a 2 linear IgE-binding epitopes and its distribution in the body of a cockroach. Methods The cDNA of Per a 2 was used as a template and combined with oligonucleotide primers specific to the target areas with appropriate restriction enzyme sites. Eleven overlapping fragments of Per a 2 covering the whole allergen molecule, except 20 residues of signal peptide, were generated by PCR. Mature Per a 2 and overlapping deletion mutants were affinity-purified and assayed for IgE reactivity by immunoblotting. Three synthetic peptides comprising the B cell epitopes were evaluated by direct binding ELISA. Rabbit anti-Per a 2 antibody was used for immunohistochemistry. Results Human linear IgE-binding epitopes of Per a 2 were located at the amino acid sequences 57-86, 200-211, and 299-309. There was positive IgE binding to 10 tested Per a 2-allergic sera in 3 synthetic peptides, but none in the controls. Immunostaining revealed that Per a 2 was localized partly in the mouth and midgut of the cockroach, with the most intense staining observed in the hindgut, suggesting that the Per a 2 allergen might be excreted through the feces. Conclusions Information on the IgE-binding epitope of Per a 2 may be used for designing more specific diagnostic and therapeutic approaches to cockroach allergy. PMID:25749772

  8. Identification of a new natural Ara h 6 isoform and of its proteolytic product as major allergens in peanut.

    PubMed

    Bernard, H; Mondoulet, L; Drumare, M F; Paty, E; Scheinmann, P; Thaï, R; Wal, J M

    2007-11-14

    Numerous food allergens of plant origin belong to the 2S albumin family, including peanut Ara h 2. In addition to Ara h 2, several other conglutins related to 2S albumins are present in peanut seeds. We evaluated the allergenicity of different peanut conglutins as compared with Ara h 2. Several conglutins were isolated from the kernel, i.e. Ara h 2, a new isoform of Ara h 6 and its derived product, which is likely to be naturally formed during seed processing. Enzyme allergosorbent tests performed on sera of peanut allergic patients showed that more than 94% of 47 analyzed patients had positive IgE responses to Ara h 6 isoform and to its degradation product. Skin prick tests with the new isoform of Ara h 6 led to a positive response in seven out of the eight tested patients. Both enzyme allergosorbent tests and skin prick tests showed that the reactivity of Ara h 6 was similar to, or even higher than, that of Ara h 2, suggesting that the present isoform of Ara h 6 is as allergenic as Ara h 2. In addition the IgE response to the plant processed (i.e., hydrolyzed) Ara h 6 new isoform is equivalent to the IgE response to the native isoform. The IgE immunoreactivity is mostly abrogated by chemical reduction and denaturation of Ara h 6 isoforms, which underlined the importance of tertiary structure in Ara h 6 immunoreactivity. These results, and particularly the high correlation between anti-Ara h 2 and anti-Ara h 6 IgE responses, emphasise the major role of 2S albumins in peanut allergenicity.

  9. Molecular modelling of the major peanut allergen Ara h 1 and other homotrimeric allergens of the cupin superfamily: a structural basis for their IgE-binding cross-reactivity.

    PubMed

    Barre, Annick; Borges, Jean-Philippe; Rougé, Pierre

    2005-06-01

    Three-dimensional models of the major vicilin allergens from peanut (Ara h 1), lentil (Len c 1) and pea (Pis s 1), were built by homology-based modelling from the X-ray coordinates of the structurally closely related soybean beta-conglycinin. All the allergen monomers exhibit the typical cupin motif made of two modules related by a pseudo-dyad axis. Each module consists of a beta-barrel core domain associated to a loop domain which mainly contains alpha-helices. The three cupin motifs are assumed to be arranged in a homotrimeric structure similar to that observed in beta-conglycinin, phaseolin or canavalin. Most of the sequential B-cell epitopes characterized on the C-terminus of the Ara h 1 allergen are well conserved in both Len c 1 and Pis s 1 allergens. They occupy very comparable areas on the molecular surface of the allergens and exhibit a similar three-dimensional conformation. This antigenic community readily accounts for the IgE-binding cross-reactivity commonly observed between the vicilin allergens from edible legume seeds. The clinical implication of this cross-reactivity is addressed for a definite diagnosis of legume seed allergy.

  10. Single recombinant and purified major allergens and peptides: How they are made and how they change allergy diagnosis and treatment.

    PubMed

    Curin, Mirela; Garib, Viktoriya; Valenta, Rudolf

    2017-09-01

    To review the current knowledge regarding recombinant and purified allergens and allergen-derived peptides. PubMed, homepages relevant to the topic, and the National Institutes of Health clinical trial database were searched. The literature was screened for studies describing purified and recombinant allergens and allergen-derived peptides. Studies relevant to the topic were included in this review. Advantages and drawbacks of pure and defined recombinant allergens and peptides over allergen extracts in the context of allergy research, diagnosis, and allergen immunotherapy are discussed. We describe how these molecules are manufactured, which products are currently available on the market, and what the regulative issues are. We furthermore provide an overview of clinical studies with vaccines based on recombinant allergens and synthetic peptides. The possibility of prophylactic vaccination based on recombinant fusion proteins consisting of viral carrier proteins and allergen-derived peptides without allergenic activity are also discussed. During the last 25 years more than several hundred allergen sequences were determined, which led to a production of recombinant allergens that mimic biochemically and immunologically their natural counterparts. Especially in Europe, recombinant allergens are increasingly replacing allergen extracts in diagnosis of allergy. Despite many challenges, such as high cost of clinical trials and regulative issues, allergy vaccines based on recombinant allergens and peptides are being developed and will likely soon be available on the market. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  11. Immunological cross-reactivity of the major allergen from perennial ryegrass (Lolium perenne), Lol p I, and the cysteine proteinase, bromelain.

    PubMed

    Pike, R N; Bagarozzi, D; Travis, J

    1997-04-01

    Antibodies prepared in rabbits against the major allergen from ryegrass (Lolium perenne), Lol p I, cross-reacted with the cysteine proteinase bromelain from pineapple and vice versa. Deglycosylation of the proteins showed that the cross-reaction was based on recognition of the carbohydrate moiety of the allergen, but for bromelain the cross-reaction was most likely due to a combination of factors. The results indicate that the carbohydrate residues from these allergens play an important role in cross-reactions found between them and possibly those from other species.

  12. Pectin methylesterases of pollen tissue, a major allergen in olive tree.

    PubMed

    Salamanca, Guillermo; Rodríguez, Rosalía; Quiralte, Joaquín; Moreno, Carmen; Pascual, Cristina Y; Barber, Domingo; Villalba, Mayte

    2010-07-01

    Olive tree (Olea europaea) pollen is a main cause of allergy in Mediterranean areas and North America. A novel allergen, Ole e 11, has been detected by proteomic techniques. Protein bands binding IgE from allergic sera were excised from a 2D electrophoresis gel and analysed by Edman degradation and MALDI-TOF MS. Four peptides were sequenced and used for designing primers to clone the cDNA codifying the protein. Ole e 11 consists of a 342 amino acid length polypeptide with a molecular mass of 37.4 kDa and a pI of 7.8. The allergen was identified as a pectin methylesterase and showed low identity with other members of this family from foods such as those from carrot (23%), orange (25%) and tomato (24%), and higher identity with those from Arabidopsis thaliana (57%) and Salsola kali (54%) pollen. The protein was overproduced in Pichia pastoris, purified, and characterized as an active enzyme. CD analysis rendered 3%alpha-helix, 50%beta-sheet and 27%beta-turns for its secondary structure, which is in agreement with other pectin methylesterase structures. The recombinant protein was demonstrated to be immunologically equivalent to the natural form by immunoblotting, indirect ELISA and inhibition experiments, using polyclonal antiserum and sera from olive pollen allergic patients. The prevalence fluctuated between 55.9% and 75.6% in three different allergic populations. The availability of this new olive pollen allergen could improve the component-resolved diagnosis. Its allergenic relevance is stepped up by the biotechnological use of these enzymes to improve organoleptic properties in processing foods and further confirms the need to include it in an accurate diagnosis.

  13. Crystallization and preliminary diffraction data of a major pollen allergen. Crystal growth separates a low molecular weight form with elevated biological activity.

    PubMed

    Bufe, A; Betzel, C; Schramm, G; Petersen, A; Becker, W M; Schlaak, M; Perbandt, M; Dauter, Z; Weber, W

    1996-11-01

    Group V major allergen Phl p 5b of timothy grass pollen induces allergic rhinitis and bronchial asthma in 90% of grass pollen-allergic patients. In addition to its allergenicity ribonuclease activity has recently been attributed to this 29-kDa protein. The allergen was expressed in Escherichia coli and subsequently purified. Spontaneous conversion of these preparations to a mixture of various forms with molecular sizes between 10 and 29 kDa was consistently observed. Surprisingly, crystals could be grown from this heterogenous preparation. Single crystals, redissolved and analyzed by SDS-polyacrylamide gel electrophoresis and immunoblot, yielded one distinct low molecular weight protein, which was identified by amino acid sequencing as the C-terminal 13-kDa portion of the allergen. Histamine release assays with single crystal solutions using basophils of an allergic patient demonstrated allergenicity comparable with that of the holo-allergen. By contrast, RNase activity of the crystallized C-terminal form was 23 times higher than that of the full-length parent allergen. Crystals were used to collect preliminary diffraction data; the space group was evaluated to I4122 with cell dimensions of a = 87.7 A, b = 87.7 A, and c = 59.6 A. We conclude that preferential crystal growth of the 13-kDa form is indicative of a compact conformation of this particular C-terminal portion of the allergen. Thus, we show here that protein crystallization is not only a prerequisite for structural analyses, but it also can provide a unique separation technique to localize the functional domain of a major allergen.

  14. Insect Sting Reactions and Specific IgE to Venom and Major Allergens in a General Population.

    PubMed

    Mosbech, Holger; Tang, Line; Linneberg, Allan

    2016-01-01

    Insect sting reactions are frequently reported, but population studies documenting the frequency and the relation to IgE-sensitization and serum tryptase are scarce. Questionnaire data and results from measurements of specific IgE against venom, major allergens and cross-reacting carbohydrate determinants (CCDs) were collected from 2,090 adult participants in a cross-sectional survey. 13% of the population reported symptoms of sting reactions and about half were systemic in nature. In all, 15% were sensitized to venom but only 31% of these had reacted to stings and only 38% of those with reactions had IgE to venom. In addition, 12% with IgE to venom were double-sensitized (DS), i.e. to both bee and wasp venom. Among DS IgE to major venom allergens, rApi m 1, rVes v 1 and rVes v 5 were negative and of no help in 31%, but 59% could be identified as likely sensitized to bee or wasp. IgE to CCDs occurred in only 0.7%, but 80% of these were DS. Finally, 36% with IgE to CCDs had had symptoms, mostly local. Serum tryptase was not associated with a history of sting reactions. In a temperate climate, self-reported insect sting reactions and sensitization to venom are frequent, but in most cases, these are not seen in the same individual. In DS individuals, measurements of IgE to major allergens can be helpful in some but not all cases and additional analyses are needed. IgE to CCDs may have some clinical relevance. © 2016 S. Karger AG, Basel.

  15. New tree nut allergens

    USDA-ARS?s Scientific Manuscript database

    The 7S vicilin and 11S legumin seed storage globulins belong to the cupin protein superfamily and are major food allergens in many of the “big eight” food allergen groups. Korean pine vicilin and pecan vicilin are thus predicted to be food allergens. Recombinant vicilins were expressed in E. coli an...

  16. Trypsin resistance of the major peanut allergen Ara h 6 and allergenicity of the digestion products are abolished after selective disruption of disulfide bonds.

    PubMed

    Hazebrouck, Stéphane; Guillon, Blanche; Drumare, Marie-Françoise; Paty, Evelyne; Wal, Jean-Michel; Bernard, Hervé

    2012-04-01

    2S-albumins Ara h 2 and Ara h 6 are the most widely recognized and potent allergens for peanut-allergic patients. These allergens are particularly resistant to proteolysis and the digestion products generally retain significant allergenicity. Five disulfide bridges (DB) stabilize Ara h 6 overall structure and their influence on the trypsin resistance and on the allergenicity of the digestion products was investigated. Progressive disruption of each DB was performed by site-directed mutagenesis. Successful refolding of Ara h 6 variants was confirmed by circular dichroism. Trypsin resistance, IgE-binding capacity and allergenic potency, as assessed by in vitro mediator release assay with sera from peanut-allergic patients, was not affected by the deletion of the C-terminal DB at Cys(84) -Cys(124) . Additional disruption of DB at Cys(14) -Cys(71) or at Cys(73) -Cys(115) rendered Arg(16/20) or Arg(114) susceptible to trypsinolysis, respectively, but affected principally the IgE-binding capacity of Ara h 6. DB disruption at Cys(26) -Cys(58) or at Cys(59) -Cys(107) led to an extensive proteolytic degradation and a complete loss of allergenic potency of the digestion products. Selective disruption of the DB stabilizing the protease-resistant core of Ara h 6 eliminated the IgE-binding capacity of the trypsin-degradation products and their ability to trigger mast cell degranulation. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Cloning and characterization of an 11S legumin, Car i 4, a major allergen in pecan.

    PubMed

    Sharma, Girdhari M; Irsigler, Andre; Dhanarajan, Pushparani; Ayuso, Rosalia; Bardina, Luda; Sampson, Hugh A; Roux, Kenneth H; Sathe, Shridhar K

    2011-09-14

    Among tree nut allergens, pecan allergens remain to be identified and characterized. The objective was to demonstrate the IgE-binding ability of pecan 11S legumin and characterize its sequential IgE-binding epitopes. The 11S legumin gene was amplified from a pecan cDNA library and expressed as a fusion protein in Escherichia coli. The native 11S legumin in pecan extract was identified by mass spectrometry/mass spectrometry (MS/MS). Sequential epitopes were determined by probing the overlapping peptides with three serum pools prepared from different patients' sera. A three-dimensional model was generated using almond legumin as a template and compared with known sequential epitopes on other allergenic tree nut homologues. Of 28 patients tested by dot blot, 16 (57%) bound to 11S legumin, designated Car i 4. MS/MS sequencing of native 11S legumin identified 33 kDa acidic and 20-22 kDa basic subunits. Both pecan and walnut seed protein extracts inhibited IgE binding to recombinant Car i 4, suggesting cross-reactivity with Jug r 4. Sequential epitope mapping results of Car i 4 revealed weak, moderate, and strong reactivity of serum pools against 10, 5, and 4 peptides, respectively. Seven peptides were recognized by all three serum pools, of which two were strongly reactive. The strongly reactive peptides were located in three discrete regions of the Car i 4 acidic subunit sequence (residues 118-132, 208-219, and 238-249). Homology modeling of Car i 4 revealed significant overlapping regions shared in common with other tree nut legumins.

  18. Purification, crystallization and initial crystallographic characterization of peanut major allergen Ara h 3

    PubMed Central

    Jin, Tengchuan; Howard, Andrew; Zhang, Yu-Zhu

    2007-01-01

    The peanut is a significant food source, but is responsible for many cases of anaphylaxis. The peanut 11S legumin-like seed storage protein Ara h 3 is one of the best characterized allergens. In this study, Ara h 3 was extracted from peanut kernels and purified by sequential anion-exchange, hydrophobic interaction and gel-filtration chromatography to very high purity to facilitate crystallization and structural studies. Well diffracting single crystals were obtained by the vapor-diffusion method. A molecular-replacement structural solution has been obtained and refinement of the structure is currently under way. PMID:17909286

  19. Isolation of cDNA encoding a newly identified major allergenic protein of rye-grass pollen: intracellular targeting to the amyloplast.

    PubMed Central

    Singh, M B; Hough, T; Theerakulpisut, P; Avjioglu, A; Davies, S; Smith, P M; Taylor, P; Simpson, R J; Ward, L D; McCluskey, J

    1991-01-01

    We have identified a major allergenic protein from rye-grass pollen, tentatively designated Lol pIb of 31kDa and with pI 9.0. A cDNA clone encoding Lol pIb has been isolated, sequenced, and characterized. Lol pIb is located mainly in the starch granules. This is a distinct allergen from Lol pI, which is located in the cytosol. Lol pIb is synthesized in pollen as a pre-allergen with a transit peptide targeting the allergen to amyloplasts. Epitope mapping of the fusion protein localized the IgE binding determinant in the C-terminal domain. Images PMID:1671715

  20. Seasonal levels of the major American cockroach allergen per a 9 (arginine kinase) in Bangkok and their relevance for disease severity.

    PubMed

    Tungtrongchitr, Anchalee; Sookrung, Nitat; Indrawattana, Nitaya; Sae-Lim, Jenjira; Puduang, Somchai; Phonrat, Benjaluk; Wanachiwanawin, Darawan; Chaicumpa, Wanpen

    2009-03-01

    Monitoring the levels of cockroach (CR) allergen in the environment has medical relevance as a clear dose response relationship between CR allergen exposure, sensitization and hospitalization has been reported. In this study, a cross-sectional survey of the levels of a major American cockroach (Periplaneta americana) allergen, i.e. Per a 9 (arginine kinase) in dust samples collected in various seasons throughout the year 2007 from 76 houses of CR allergic Thai patients in the Bangkok metropolitan area were determined. A monoclonal antibody-polyclonal antibody (MAb-PAb) based-sandwich ELISA was used. The MAb was specific to Per a 9 and the PAb was raised in a rabbit against the crude extract of P. americana. The detection limit of the assay was 122 pg of the allergen or 0.024 microg per gram of fine dust powder. The concentrations of Per a 9 were found to be highest during the winter months and lowest in summer. The levels of this CR allergen had a direct correlation with disease exacerbation; i.e. the majority of the CR allergic patients had their most severe clinical manifestations during winter. Moreover, the CR allergen levels were found to be higher in wood based-houses than in concrete houses.

  1. Ani s 11-Like Protein Is a Pepsin- and Heat-Resistant Major Allergen of Anisakis spp. and a Valuable Tool for Anisakis Allergy Component-Resolved Diagnosis.

    PubMed

    Carballeda-Sangiao, Noelia; Rodríguez-Mahillo, Ana I; Careche, Mercedes; Navas, Alfonso; Caballero, Teresa; Dominguez-Ortega, Javier; Jurado-Palomo, Jesús; González-Muñoz, Miguel

    2016-01-01

    Anisakis simplex is a fish parasite responsible for gastrointestinal and allergic symptoms in humans. The Ani s 11-like protein has been proposed as an Anisakis allergen because its primary structure is similar to that of Ani s 11. The aims of this work were to analyse the frequency of detection of the Ani s 11-like protein and assess its diagnostic value. rAni s 11-like protein, rAni s 5 and rAni s 4 were expressed in Escherichia coli and rAni s 1 was produced in Pichia pastoris. Recombinant allergen detection patterns in 37 Anisakis-sensitised patients were determined. The stability to pepsin digestion and heat treatment of rAni s 11-like protein was also analysed by IgE immunoblotting. Ani s 11-like protein is a major allergen detected by 78% of Anisakis-allergic patients, and 13.5% of patients detect only the rAni s 11-like allergen. This allergen is heat stable because it retains its capability of binding IgE after boiling for 30 min and it is resistant to pepsin digestion for 120 min. These data indicate that the Ani s 11-like protein is a pepsin- and heat-resistant major allergen (Ani s 11.0201) of Anisakis spp. and a valuable tool for Anisakis allergy component-resolved diagnosis. © 2016 S. Karger AG, Basel.

  2. Identification of T-cell epitopes of Lol p 9, a major allergen of ryegrass (Lolium perenne) pollen.

    PubMed

    Blaher, B; Suphioglu, C; Knox, R B; Singh, M B; McCluskey, J; Rolland, J M

    1996-07-01

    T-cell recognition of Lol p 9, a major allergen of ryegrass pollen, was investigated by using a T-cell line and T-cell clones generated from the peripheral blood of an atopic donor. The T-cell line reacted with purified Lol p 9, as well as with crude ryegrass pollen extract, but failed to cross-react with Bermuda grass pollen extract. All of six T-cell clones generated from this line proliferated in response to Lol p 9. Epitope mapping was carried out with a panel of 34 overlapping synthetic peptides, which spanned the entire sequence of the Lol p 9 12R isoform. The T-cell line responded to two of the peptides, Lol p 9 (105-116) and Lol p 9 (193-204), whereas reactivity with one or other of these peptides was shown by five T-cell clones. These two peptides contained sequences consistent with motifs previously reported for major histocompatibility complex class II-restricted peptides. HLA antibody blocking studies showed that presentation of peptide Lol p 9 (105-116) to one T-cell clone was HLA-DR-restricted; this clone expressed a T helper cell phenotype (CD3+, CD4+) and the T-cell receptor alpha beta. The identification of immunodominant T-cell epitope(s) on allergens is essential for devising safer and more effective immunotherapy strategies, which can interrupt the chain of events leading to allergic disease.

  3. Cloning and expression of Ole e I, the major allergen from olive tree pollen. Polymorphism analysis and tissue specificity.

    PubMed

    Villalba, M; Batanero, E; Monsalve, R I; González de la Peña, M A; Lahoz, C; Rodríguez, R

    1994-05-27

    Ole e I, the major allergen from the olive tree (Olea europaea), is one of the main causes of allergy in Mediterranean countries and some areas of North America. The cloning and sequencing of several cDNAs coding for the olive allergen have been achieved. cDNA has been synthesized from total pollen RNA and amplified by using the polymerase chain reaction. The nucleotide sequence data demonstrate the existence of microheterogeneities in at least 37 positions out of the 145 amino acids of Ole e I, thus explaining the high degree of polymorphism exhibited by the natural protein. One of the sequenced cDNAs encoding a full-length isoform was inserted into the plasmid vector pGEX-2T and overexpressed. The recombinant Ole e I has been produced in Escherichia coli as a fusion protein with glutathione S-transferase of Schistosoma japonicum. This chimeric protein was purified by affinity chromatography on a glutathione-Sepharose 4B column and digested with thrombin to release the recombinant allergen. Both the fusion protein and the recombinant Ole e I were recognized in Western blot analysis by rabbit polyclonal and mouse monoclonal antisera raised against native Ole e I as well as by the IgE of olive pollen-sensitive human sera. This indicates that the recombinant production of individual isoforms may be useful for the improvement of reagents to be used in diagnosis and therapy of IgE-mediated disorders. In addition, Ole e I mRNA has been observed to be pollen-specific as shown in a Northern blot analysis.

  4. Vaccines for birch pollen allergy based on genetically engineered hypoallergenic derivatives of the major birch pollen allergen, Bet v 1.

    PubMed

    Mahler, V; Vrtala, S; Kuss, O; Diepgen, T L; Suck, R; Cromwell, O; Fiebig, H; Hartl, A; Thalhamer, J; Schuler, G; Kraft, D; Valenta, R

    2004-01-01

    We have recently engineered recombinant derivatives of the major birch pollen allergen Bet v 1 (rBet v 1 fragments and trimer) with strongly reduced allergenic activity. The aim of this study was the in vivo characterization of potential allergy vaccines based on Al(OH)3-adsorbed genetically modified rBet v 1 derivatives in mice. BALB/c mice were immunized either with courses of nine injections of increasing doses of Al(OH)3-adsorbed rBet v 1 wild-type, rBet v 1 fragments, rBet v 1 trimer or Al(OH)3 alone in weekly intervals or with three high-dose injections applied in intervals of 3 weeks. Humoral immune responses to rBet v 1 wild-type and homologous plant allergens were measured by ELISA and Western blotting, and the ability of mouse antibodies to inhibit the binding of allergic patients IgE to Bet v 1 was studied by ELISA competition experiments. In both schemes, hypoallergenic rBet v 1 derivatives induced low IgE but high IgG1 responses against rBet v 1 wild-type. The IgG1 antibodies induced by genetically modified rBet v 1 derivatives cross-reacted with natural Bet v 1 and its homologues from alder (Aln g 1) as well as hazel (Cor a 1) and strongly inhibited the binding of birch pollen allergic patients' IgE to Bet v 1 wild-type. Genetically modified hypoallergenic rBet v 1 derivatives induce blocking antibodies in vivo. Their safety and efficacy for the treatment of birch pollen and associated plant allergies can now be evaluated in clinical immunotherapy studies.

  5. Stability of the major allergen Brazil nut 2S albumin (Ber e 1) to physiologically relevant in vitro gastrointestinal digestion.

    PubMed

    Moreno, F Javier; Mellon, Fred A; Wickham, Martin S J; Bottrill, Andrew R; Mills, E N Clare

    2005-01-01

    The major 2S albumin allergen from Brazil nuts, Ber e 1, was subjected to gastrointestinal digestion using a physiologically relevant in vitro model system either before or after heating (100 degrees C for 20 min). Whilst the albumin was cleaved into peptides, these were held together in a much larger structure even when digested by using a simulated phase 1 (gastric) followed by a phase 2 (duodenal) digestion system. Neither prior heating of Ber e 1 nor the presence of the physiological surfactant phosphatidylcholine affected the pattern of proteolysis. After 2 h of gastric digestion, approximately 25% of the allergen remained intact, approximately 50% corresponded to a large fragment of M(r) 6400, and the remainder comprised smaller peptides. During duodenal digestion, residual intact 2S albumin disappeared quickly, but a modified form of the 'large fragment' remained, even after 2 h of digestion, with a mass of approximately 5000 Da. The 'large fragment' comprised several smaller peptides that were identified, by using different MS techniques, as deriving from the large subunit. In particular, sequences corresponding to the hypervariable region (Q37-M47) and to another peptide (P42-P69), spanning the main immunoglobulin E epitope region of 2S albumin allergens, were found to be largely intact following phase 1 (gastric) digestion. They also contained previously identified putative T-cell epitopes. These findings indicate that the characteristic conserved skeleton of cysteine residues of 2S albumin family and, particularly, the intrachain disulphide bond pattern of the large subunit, play a critical role in holding the core protein structure together even after extensive proteolysis, and the resulting structures still contain potentially active B- and T-cell epitopes.

  6. Flavonoid analysis of buckwheat sprouts.

    PubMed

    Nam, Tae-Gyu; Lee, Sun Mi; Park, Ji-Hae; Kim, Dae-Ok; Baek, Nam-in; Eom, Seok Hyun

    2015-03-01

    It is known that common buckwheat sprouts contain several flavonoids, including orientin, isoorientin, vitexin, isovitexin, rutin, and quercetrin, whereas tartary buckwheat sprouts contain only rutin. In this study, we evaluated flavonoids present in buckwheat sprouts and identified a previously unreported flavonoid. Simultaneous detection by HPLC was used to separate rutin and a compound that was not separated in previous studies. We used a novel HPLC elution gradient method to successfully separate rutin and the previously unidentified compound, for which we performed structural analysis. The identification of six flavonoids by HPLC was confirmed using HPLC-ESI-MS/MS analysis. The newly identified compound, [M+H](+) =611.17, was identified by NMR as the rutin epimer quercetin-3-O-robinobioside. Unlike common buckwheat sprout, tartary buckwheat sprout contained rutin as a main flavonoid, whereas other flavonoids appeared only in trace amounts or were not detected. Quercetin-3-O-robinobioside was not detected in tartary buckwheat sprout. Copyright © 2014 Elsevier Ltd. All rights reserved.

  7. Seven different genes encode a diverse mixture of isoforms of Bet v 1, the major birch pollen allergen.

    PubMed

    Schenk, Martijn F; Gilissen, Ludovicus Jwj; Esselink, Gerhard D; Smulders, Marinus Jm

    2006-07-04

    Pollen of the European white birch (Betula pendula, syn. B. verrucosa) is an important cause of hay fever. The main allergen is Bet v 1, member of the pathogenesis-related class 10 (PR-10) multigene family. To establish the number of PR-10/Bet v 1 genes and the isoform diversity within a single tree, PCR amplification, cloning and sequencing of PR-10 genes was performed on two diploid B. pendula cultivars and one interspecific tetraploid Betula hybrid. Sequences were attributed to putative genes based on sequence identity and intron length. Information on transcription was derived by comparison with homologous cDNA sequences available in GenBank/EMBL/DDJB. PCR-cloning of multigene families is accompanied by a high risk for the occurrence of PCR recombination artifacts. We screened for and excluded these artifacts, and also detected putative artifact sequences among database sequences. Forty-four different PR-10 sequences were recovered from B. pendula and assigned to thirteen putative genes. Sequence homology suggests that three genes were transcribed in somatic tissue and seven genes in pollen. The transcription of three other genes remains unknown. In total, fourteen different Bet v 1-type isoforms were identified in the three cultivars, of which nine isoforms were entirely new. Isoforms with high and low IgE-reactivity are encoded by different genes and one birch pollen grain has the genetic background to produce a mixture of isoforms with varying IgE-reactivity. Allergen diversity is even higher in the interspecific tetraploid hybrid, consistent with the presence of two genomes. Isoforms of the major birch allergen Bet v 1 are encoded by multiple genes, and we propose to name them accordingly. The present characterization of the Bet v 1 genes provides a framework for the screening of specific Bet v 1 genes among other B. pendula cultivars or Betula species, and for future breeding for trees with a reduced allergenicity. Investigations towards sensitization and

  8. Rapid production of the major birch pollen allergen Bet v 1 in Nicotiana benthamiana plants and its immunological in vitro and in vivo characterization.

    PubMed

    Krebitz, M; Wiedermann, U; Essl, D; Steinkellner, H; Wagner, B; Turpen, T H; Ebner, C; Scheiner, O; Breiteneder, H

    2000-07-01

    Type I allergies are immunological disorders that afflict a quarter of the world's population. Improved diagnosis of allergic diseases and the formulation of new therapeutic approaches are based on the use of recombinant allergens. We describe here for the first time the application of a rapid plant-based expression system for a plant-derived allergen and its immunological characterization. We expressed our model allergen Bet v 1, the major birch pollen allergen, in the tobacco-related species Nicotiana benthamiana using a tobacco mosaic virus vector. Two weeks postinoculation, plants infected with recombinant viral RNA containing the Bet v 1 coding sequence accumulated the allergen to levels of 200 microg/g leaf material. Total nonpurified protein extracts from plants were used for immunological characterizations. IgE immunoblots and ELISA (enzyme-linked immunoassay) inhibition assays showed comparable IgE binding properties for tobacco recombinant (r) Bet v 1 and natural (n) Bet v 1, suggesting that the B cell epitopes were preserved when the allergen was expressed in N. benthamiana plants. Using a murine model of type I allergy, mice immunized with crude leaf extracts containing Bet v 1 with purified rBet v 1 produced in E. coli or with birch pollen extract generated comparable allergen-specific IgE and IgG1 antibody responses and positive type I skin test reactions. These results demonstrate that nonpurified Bet v 1 overexpressed in N. benthamina has the same immunogenicity as purified Bet v 1 produced in E. coli or nBet v 1. We therefore conclude that this plant expression system offers a viable alternative to fermentation-based production of allergens in bacteria or yeasts. In addition, there may be a broad utility of this system for the development of new and low-cost vaccination strategies against allergy.

  9. The major royal jelly proteins 8 and 9 (Api m 11) are glycosylated components of Apis mellifera venom with allergenic potential beyond carbohydrate-based reactivity.

    PubMed

    Blank, S; Bantleon, F I; McIntyre, M; Ollert, M; Spillner, E

    2012-06-01

    As hymenoptera venoms are one of the allergen sources causing the highest incidence of anaphylaxis and sometimes fatal consequences, the detailed characterization of all venom allergens is imperative for design of component-resolved diagnostic approaches and improved intervention strategies. Our aim was the immunochemical characterization of major royal jelly proteins (MRJP) 8 and 9, both components identified in honeybee venom (HBV) and putative allergens. Both MRJPs were recombinantly produced as soluble differentially glycosylated proteins providing a defined degree of reactivity to cross-reactive carbohydrate determinants (CCD) in insect cells. Allergen-specific IgE(sIgE) reactivity of HBV-allergic patients was analysed by ELISA and immunoblotting. MRJP8 and MRJP9 were identified as venom components by MS-based proteomic analyses. In a population of 47 HBV-allergic patients, reactivities with CCD-carrying MRJPs were in the range of 56% (61%), underlining the contribution of CCDs to allergen-binding. Beyond CCD-reactivity, 15% of patients showed sIgE reactivity with MRJP8 and 34% with MRJP9 respectively. These reactivities roughly in the range of Api m 2 render the MRJPs minor, but important allergens. The glycosylated MRJP8 and MRJP9 of HBV have IgE-sensitizing potential in HBV-allergic patients beyond CCD reactivity and have to be considered as allergens, which might be potentially important for a fraction of venom allergic patients. They are valuable tools to elucidate individual component-resolved reactivity profiles of venom allergic patients and to provide insights into the role of particular venom components. Due to their allergenic properties, MRJP8 and MRJP9 were designated as isoallergens Api m 11.0101 and Api m 11.0201 respectively. © 2012 Blackwell Publishing Ltd.

  10. Crystal structure of Sol I 2: a major allergen from fire ant venom.

    PubMed

    Borer, Aline S; Wassmann, Paul; Schmidt, Margit; Hoffman, Donald R; Zhou, Jing-Jiang; Wright, Christine; Schirmer, Tilman; Marković-Housley, Zora

    2012-01-27

    Sol i 2 is a potent allergen from the venom of red imported fire ant, which contains allergens Sol i 1, Sol i 2, Sol i 3, and Sol i 4 that are known to be powerful triggers of anaphylaxis. Sol i 2 causes IgE antibody production in about one-third of individuals stung by fire ants. Baculovirus recombinant dimeric Sol i 2 was crystallized as a native and selenomethionyl-derivatized protein, and its structure has been determined by single-wavelength anomalous dispersion at 2.6 Å resolution. The overall fold of each subunit consists of five helices that enclose a central hydrophobic cavity. The structure is stabilized by three intramolecular disulfide bridges and one intermolecular disulfide bridge. The nearest structural homologue is the sequence-unrelated odorant binding protein and pheromone binding protein LUSH of the fruit fly Drosophila, which may suggest a similar biological function. To test this hypothesis, we measured the reversible binding of various pheromones, plant odorants, and other ligands to Sol i 2 by the changes in N-phenyl-1-naphthylamine fluorescence emission upon binding of ligands that compete with N-phenyl-1-naphthylamine. The highest binding affinity was observed for hydrophobic ligands such as aphid alarm pheromone (E)-β-farnesene, analogs of ant alarm pheromones, and plant volatiles decane, undecane, and β-caryophyllene. Conceivably, Sol i 2 may play a role in capturing and/or transporting small hydrophobic ligands such as pheromones, odors, fatty acids, or short-living hydrophobic primers. Molecular surface analysis, in combination with sequence alignment, can explain the serological cross-reactivity observed between some ant species. Copyright © 2011 Elsevier Ltd. All rights reserved.

  11. NMR solution structure of Ole e 6, a major allergen from olive tree pollen.

    PubMed

    Treviño, Miguel Angel; García-Mayoral, María Flor; Barral, Patricia; Villalba, Mayte; Santoro, Jorge; Rico, Manuel; Rodríguez, Rosalía; Bruix, Marta

    2004-09-10

    Ole e 6 is a pollen protein from the olive tree (Olea europaea) that exhibits allergenic activity with a high prevalence among olive-allergic individuals. The three-dimensional structure of Ole e 6 has been determined in solution by NMR methods. This is the first experimentally determined structure of an olive tree pollen allergen. The structure of this 50-residue protein is based on 486 upper limit distance constraints derived from nuclear Overhauser effects and 24 torsion angle restraints. The global fold of Ole e 6 consists of two nearly antiparallel alpha-helices, spanning residues 3-19 and 23-33, that are connected by a short loop and followed by a long, unstructured C-terminal tail. Viewed edge-on, the structured N terminus has a dumbbell-like shape with the two helices on the outside and with the hydrophobic core, mainly composed of 3 aromatic and 6 cysteine residues, on the inside. All the aromatic rings lie on top of and pack against the three disulfide bonds. The lack of thermal unfolding, even at 85 degrees C, indicates a high conformational stability. Based on the analysis of the molecular surface, we propose five plausible epitopes for IgE recognition. The results presented here provide the structural foundation for future experiments to verify the antigenicity of the proposed epitopes, as well as to design novel hypoallergenic forms of the protein suitable for diagnosis and treatment of type-I allergies. In addition, three-dimensional structure features of Ole e 6 are discussed to provide a basis for future functional studies.

  12. Cloning and characterization of 2S albumin, Car i 1, a major allergen in pecan.

    PubMed

    Sharma, Girdhari M; Irsigler, Andre; Dhanarajan, Pushparani; Ayuso, Rosalia; Bardina, Luda; Sampson, Hugh A; Roux, Kenneth H; Sathe, Shridhar K

    2011-04-27

    Although pecans are associated with IgE-mediated food allergies, the allergens responsible remain to be identified and characterized. The 2S albumin gene was amplified from the pecan cDNA library. Dot-blots were used to screen the recombinant protein with pecan allergic patients' serum. The affinity purified native protein was analyzed by Edman sequencing and mass spectrometry/mass spectrometry (MS/MS) analysis. Cross-reactivity with walnut was determined by inhibition enzyme-linked immunosorbent assay (ELISA). Sequential epitopes were determined by probing the overlapping peptides with three different patients' serum pool. The 3-dimensional homology model was generated, and the locations of the pecan epitopes were compared with those of known sequential epitopes on other allergenic tree nut homologues. Of 28 patients tested by dot-blot, 22 (79%) bound to 2S albumin, designated as Car i 1. Edman sequencing and the MS/MS sequencing of native 2S albumin confirmed the identity of recombinant (r) Car i 1. Both pecan and walnut protein extracts inhibited the IgE-binding to rCar i 1. Sequential epitope mapping indicated weak, moderate, and strong reactivity against 12, 7, and 5 peptides, respectively. Of the 11 peptides recognized by all serum pools, 5 peptides were strongly reactive and located in 3 discrete regions of the Car i 1 (amino acids 43-57, 67-78, and 106-120). Three-dimensional modeling revealed IgE-reactive epitopes to be solvent accessible and share significant homology with other tree nuts providing a possible basis for previously observed cross-reactivity.

  13. A rapid and efficient two-step gel electrophoresis method for the purification of major rye grass pollen allergens.

    PubMed

    Levy, D; Davies, J; O'Hehir, R; Suphioglu, C

    2001-06-01

    Purified proteins are mandatory for molecular, immunological and cellular studies. However, purification of proteins from complex mixtures requires specialised chromatography methods (i.e., gel filtration, ion exchange, etc.) using fast protein liquid chromatography (FPLC) or high-performance liquid chromatography (HPLC) systems. Such systems are expensive and certain proteins require two or more different steps for sufficient purity and generally result in low recovery. The aim of this study was to develop a rapid, inexpensive and efficient gel-electrophoresis-based protein purification method using basic and readily available laboratory equipment. We have used crude rye grass pollen extract to purify the major allergens Lol p 1 and Lol p 5 as the model protein candidates. Total proteins were resolved on large primary gel and Coomassie Brilliant Blue (CBB)-stained Lol p 1/5 allergens were excised and purified on a secondary "mini"-gel. Purified proteins were extracted from unstained separating gels and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analyses. Silver-stained SDS-PAGE gels resolved pure proteins (i.e., 875 microg of Lol p 1 recovered from a 8 mg crude starting material) while immunoblot analysis confirmed immunological reactivity of the purified proteins. Such a purification method is rapid, inexpensive, and efficient in generating proteins of sufficient purity for use in monoclonal antibody (mAb) production, protein sequencing and general molecular, immunological, and cellular studies.

  14. Reduction of major peanut allergens Ara h 1 and Ara h 2, in roasted peanuts by ultrasound assisted enzymatic treatment.

    PubMed

    Li, Hao; Yu, Jianmei; Ahmedna, Mohamed; Goktepe, Ipek

    2013-11-15

    This study investigated the effects of ultrasound, enzyme concentration and enzyme treatment time on soluble protein and major allergenic proteins (Ara h 1 and Ara h 2) of roasted peanut kernels. A 3-factor, five-level orthogonal experimental design was implemented with various ultrasonication times, concentrations of trypsin or α-chymotrypsin and treatment times. The total soluble proteins were determined by the Bicinchoninic acid (BCA) method, Ara h 1 and Ara h 2 were evaluated by SDS-PAGE and sandwich ELISA. The IgE-binding of peanut extracts was analysed by a competitive inhibition ELISA. Results indicate that ultrasound treatment, followed by protease digestion of peanuts, significantly increased the solubility of peanut protein and decreased the concentrations of Ara h 1 and Ara h 2. The sequential treatment of peanuts by ultrasonication-trypsin-alpha chymotrypsin, resulted in maximum reductions of Ara h 1/Ara h 2, and lowest IgE-binding. This study provides an approach to significantly reduce allergenic proteins in peanut product. Published by Elsevier Ltd.

  15. High-level expression and purification of the major house dust mite allergen Der p 2 in Escherichia coli.

    PubMed

    Cao, Tuo; Zhang, Zhang; Liu, Zhi-Gao; Dou, Xia; Zhang, Jie; Zhang, Wei; Wu, Bo; Yu, Zhen-Dong; Wei, Zhun; Yu, Bo

    2016-05-01

    Der p 2, a major allergen derived from the house dust mite Dermatophagoides pteronyssinus, is one of the most clinically relevant allergens worldwide. Recombinant Der p 2 (rDer p 2) is useful in clinical diagnosis and disease-specific immunotherapy. However, previous studies showed that Der p 2 can only be expressed in Escherichia coli (E. coli) cells as inclusion bodies, thus protein refolding is required to obtain functional products. Here we report a new method to produce biologically active Der p 2 protein in E. coli. N-terminal hexahistidine- and trigger factor (TF)-tagged Der p 2 was expressed in soluble form in E. coli and purified using a combination of chromatography processes. This procedure produced milligram-level high purity Der p 2 per liter of bacterial culture. Moreover, far-UV region circular dichroism (CD) analysis and serum specific IgE reactivity test demonstrated that the secondary structure and IgE reactivity properties of rDer p 2 produced in our study were almost identical to those of natural Der p 2 (nDer p 2). In conclusion, the method developed in this work provides a useful tool for the production of immunologically active recombinant Der p 2 for clinical applications.

  16. Screening and identification of mimotopes of the major shrimp allergen tropomyosin using one-bead-one-compound peptide libraries

    PubMed Central

    Leung, Nicki YH; Wai, Christine YY; Ho, Marco HK; Liu, Ruiwu; Lam, Kit S; Wang, Jin Jun; Shu, Shang An; Chu, Ka Hou; Leung, Patrick SC

    2017-01-01

    The one-bead-one-compound (OBOC) combinatorial peptide library is a powerful tool to identify ligand and receptor interactions. Here, we applied the OBOC library technology to identify mimotopes specific to the immunoglobulin E (IgE) epitopes of the major shellfish allergen tropomyosin. OBOC peptide libraries with 8–12 amino acid residues were screened with serum samples from patients with shellfish allergy for IgE mimotopes of tropomyosin. Twenty-five mimotopes were identified from the screening and their binding reactivity to tropomyosin-specific IgE was confirmed by peptide ELISA. These mimotopes could be divided into seven clusters based on sequence homology, and epitope mapping by EpiSearch of the clustered mimotopes was performed to characterize and confirm the validity of mimotopes. Five out of six of the predicted epitopes were found to overlap with previously identified epitopes of tropomyosin. To further confirm the mimicry potential of mimotopes, BALB/c mice were immunized with mimotopes conjugated to keyhole limpet hemocyanin and assayed for their capacity to induce tropomyosin-specific antibodies. BALB/c mice that received mimotope immunization were found to have an elevated level of tropomyosin-specific immunoglobulin G, but not mice that received an irrelevant mimotope. This study pioneers the successful application of the OBOC libraries using whole sera to screen and identify multiple shrimp allergen mimotopes and validates their mimicry potential using in vitro, in vivo, and in silico methods. PMID:26364917

  17. Use of on-line stop-flow heart-cutting two-dimensional high performance liquid chromatography for simultaneous determination of 12 major constituents in tartary buckwheat (Fagopyrum tataricum Gaertn).

    PubMed

    Ren, Qiang; Wu, Caisheng; Zhang, Jinlan

    2013-08-23

    The use of two-dimensional liquid chromatography (2D-LC) for quantification studies presents challenges with respect to repeatability, precision, and robustness. The present study used an on-line stop-flow heart-cutting 2D-LC system to determine 12 chemical constituents in tartary buckwheat. A combination of various stationary phases was developed and bridged using two switch valves as the interface. Hydrophilic interaction chromatography was chosen for separation in the first dimension ((1)D), and mixed mode stationary phases (an amide polar-embedded phase and alkyl-phenyl phase) were used in parallel for separation in the second dimension ((2)D). The mobile phase comprised acetonitrile and water containing 0.03% aqueous phosphoric acid. The sample was separated into two fractions on the (1)D column (HILIC-10 column) using 5% acetonitrile. One fraction, mainly comprising flavonoids, was directly eluted onto the head of (2)D column (Polar Advantage II column) and further separated using a linear gradient of 11-23% acetonitrile. The second fraction, containing phenylpropanoid glycosides, was trapped on the (1)D column. This retained fraction was back-flushed onto the (2)D column (Phenyl-1 column) and separated using a linear gradient of 35-43% acetonitrile. An on-line stop-flow heart-cutting 2D-LC system was successfully developed with column switching and back-flush. This 2D-LC system was validated and was able to simultaneously determine 12 major components in tartary buckwheat: seven flavonoids, four phenylpropanoid glycosides, and N-trans-feruloyltyramine. The system showed good performance with respect to linearity (r>0.996), repeatability (RSD, relative standard deviation<3.4%), intra-day and inter-day precision (RSD<4.6%), recovery (91.2-108%), limit of detection (LOD) (0.05-0.21μg/mL), and limit of quantification (LOQ) (0.10-0.41μg/mL). The on-line stop-flow heart-cutting 2D-LC system offers a potential approach to analyze compounds, which have similar

  18. Major house dust mite allergens Dermatophagoides pteronyssinus 1 and Dermatophagoides farinae 1 degrade and inactivate lung surfactant proteins A and D.

    PubMed

    Deb, Roona; Shakib, Farouk; Reid, Kenneth; Clark, Howard

    2007-12-21

    Lung surfactant proteins (SP) A and D are calcium-dependent carbohydrate-binding proteins. In addition to playing multiple roles in innate immune defense such as bacterial aggregation and modulation of leukocyte function, SP-A and SP-D have also been implicated in the allergic response. They interact with a wide range of inhaled allergens, competing with their binding to cell-sequestered IgE resulting in inhibition of mast cell degranulation, and exogenous administration of SP-A and SP-D diminishes allergic hypersensitivity in vivo. House dust mite allergens are a major cause of allergic asthma in the western world, and here we confirm the interaction of SP-A and SP-D with two major mite allergens, Dermatophagoides pteronyssinus 1 and Dermatophagoides farinae 1, and show that the cysteine protease activity of these allergens results in the degradation of SP-A and SP-D under physiological conditions, with multiple sites of cleavage. A recombinant fragment of SP-D that is effective in diminishing allergic hypersensitivity in mouse models of dust mite allergy was more susceptible to degradation than the native full-length protein. Degradation was enhanced in the absence of calcium, with different sites of cleavage, indicating that the calcium associated with SP-A and SP-D influences accessibility to the allergens. Degradation of SP-A and SP-D was associated with diminished binding to carbohydrates and to D. pteronyssinus 1 itself and diminished capacity to agglutinate bacteria. Thus, the degradation and consequent inactivation of SP-A and SP-D may be a novel mechanism to account for the potent allergenicity of these common dust mite allergens.

  19. Ancient whole grain gluten-free buckwheat snacks

    USDA-ARS?s Scientific Manuscript database

    Four kinds of whole grain Buckwheat snacks (gluten-free, low in fat and salt) were evaluated. The snacks were Buckwheat, Buckwheat-Turmeric, Buckwheat-Pepper and Buckwheat-Ginger. Turmeric and Ginger are very common ingredients of Asian cuisines. Turmeric and ginger have been reported to have heal...

  20. Effect of roasting on the allergenicity of major peanut allergens Ara h 1 and Ara h 2/6: the necessity of degranulation assays.

    PubMed

    Vissers, Y M; Iwan, M; Adel-Patient, K; Stahl Skov, P; Rigby, N M; Johnson, P E; Mandrup Müller, P; Przybylski-Nicaise, L; Schaap, M; Ruinemans-Koerts, J; Jansen, A P H; Mills, E N C; Savelkoul, H F J; Wichers, H J

    2011-11-01

    Peanuts are often consumed after roasting, a process that alters the three-dimensional structure of allergens and leads to Maillard modification. Such changes are likely to affect their allergenicity. We aimed to establish the effect of thermal treatment mimicking the roasting process on the allergenicity of Ara h 1 and a mix of 2S albumins from peanut (Ara h 2/6). Ara h 1 and Ara h 2/6 were purified from raw peanuts and heated in a dry form for 20 min at 145°C in the presence (R+g) or absence (R-g) of glucose, and soluble proteins were then extracted. Sera obtained from 12 well-characterized peanut-allergic patients were used to assess the IgE binding and degranulation capacities of the allergens. Extensive heating at low moisture resulted in the hydrolysis of both Ara h 1 and Ara h 2/6. However, in contrast to Ara h 2/6, soluble R+g Ara h 1 formed large aggregates. Although the IgE-binding capacity of R+g and R-g Ara h 1 was decreased 9000- and 3.6-fold, respectively, compared with native Ara h 1, their capacity to elicit mediator release was increased. Conversely, both the IgE-binding capacity and the degranulation capacity of R-g Ara h 2/6 were 600-700-fold lower compared with the native form, although the presence of glucose during heating significantly moderated these losses. Extensive heating reduced the degranulation capacity of Ara h 2/6 but significantly increased the degranulation capacity of Ara h 1. This observation can have important ramifications for component-resolved approaches for diagnosis and demonstrates the importance of investigating the degranulation capacity in addition to IgE reactivity when assessing the effects of food processing on the allergenicity of proteins. © 2011 Blackwell Publishing Ltd.

  1. The Calcium Goes Meow: Effects of Ions and Glycosylation on Fel d 1, the Major Cat Allergen.

    PubMed

    Ligabue-Braun, Rodrigo; Sachett, Liana Guimarães; Pol-Fachin, Laércio; Verli, Hugo

    2015-01-01

    The major cat allergen, Fel d 1, is a structurally complex protein with two N-glycosylation sites that may be filled by different glycoforms. In addition, the protein contains three putative Ca2+ binding sites. Since the impact of these Fel d 1 structure modifications on the protein dynamics, physiology and pathology are not well established, the present work employed computational biology techniques to tackle these issues. While conformational effects brought upon by glycosylation were identified, potentially involved in cavity volume regulation, our results indicate that only the central Ca2+ ion remains coordinated to Fel d 1 in biological solutions, impairing its proposed role in modulating phospholipase A2 activity. As these results increase our understanding of Fel d 1 structural biology, they may offer new support for understanding its physiological role and impact into cat-promoted allergy.

  2. Epitope mapping of the Dermatophagoides pteronyssinus house dust mite major allergen Der p II using overlapping synthetic peptides.

    PubMed

    van 't Hof, W; Driedijk, P C; van den Berg, M; Beck-Sickinger, A G; Jung, G; Aalberse, R C

    1991-11-01

    Fourteen synthetic peptides of 15 amino acid residues length, overlapping by five residues and spanning the entire sequence of the major allergen Der p II from the house dust mite Dermatophagoides pteronyssinus were synthesized. These peptides were coupled to CNBr-activated Sepharose-4B and used as solid-phase antigens in epitope mapping studies using human IgE antisera. These antibodies bound predominantly to the peptide comprising residues 65-78, the binding of which was inhibited by native Der p II. In addition these antisera bound, to a lesser extent, to the peptide that comprised residues 1-15, which binding was not inhibited by native Der p II. Thus, we found one sequential epitope for a number of IgE sera.

  3. The Calcium Goes Meow: Effects of Ions and Glycosylation on Fel d 1, the Major Cat Allergen

    PubMed Central

    Pol-Fachin, Laércio; Verli, Hugo

    2015-01-01

    The major cat allergen, Fel d 1, is a structurally complex protein with two N-glycosylation sites that may be filled by different glycoforms. In addition, the protein contains three putative Ca2+ binding sites. Since the impact of these Fel d 1 structure modifications on the protein dynamics, physiology and pathology are not well established, the present work employed computational biology techniques to tackle these issues. While conformational effects brought upon by glycosylation were identified, potentially involved in cavity volume regulation, our results indicate that only the central Ca2+ ion remains coordinated to Fel d 1 in biological solutions, impairing its proposed role in modulating phospholipase A2 activity. As these results increase our understanding of Fel d 1 structural biology, they may offer new support for understanding its physiological role and impact into cat-promoted allergy. PMID:26134118

  4. Immune-reactivity of recombinant isoforms of the major house dust mite allergen Der p 2.

    PubMed

    Hakkaart, G A; Chapman, M D; Aalberse, R C; van Ree, R

    1998-02-01

    Recombinant Der p 2, expressed in yeast, lacked reactivity with 5 monoclonal antibodies against natural Der p 2. The aim of this study was to investigate whether the lack of reactivity with recombinant Der p 2 can be explained by the existence of isoforms. By site-directed mutagenesis three recombinant isoforms of Der p 2 were produced. Reactivity with monoclonal antibodies and human IgE was analysed by means of RAST and RAST-inhibition. All five monoclonals that lacked reactivity with the originally selected isoform, showed reactivity upon replacement of aspartic acid by asparagine at position 114. The other two substitutions (at position 26 and 47) had no effect. Binding of human IgE (n = 10) was not significantly influenced by the isogenetic variation at position 114. Monoclonal antibodies raised against natural Der p 2 can sometimes discriminate between different isoforms, allowing the study of the natural occurrence of isoforms. For application in allergen-measurement assays, non-discriminating monoclonal antibodies should be selected.

  5. Allergens in veterinary medicine.

    PubMed

    Mueller, R S; Janda, J; Jensen-Jarolim, E; Rhyner, C; Marti, E

    2016-01-01

    Allergic diseases in animals are increasingly gaining importance in veterinary practice and as research models. For intradermal testing and allergen immunotherapy, a good knowledge of relevant allergens for the individual species is of great importance. Currently, the knowledge about relevant veterinary allergens is based on sensitization rates identified by intradermal testing or serum testing for allergen-specific IgE; crude extracts are the basis for most evaluations. Only a few studies provide evidence about the molecular structure of (particularly) dust mite, insect and mould allergens in dogs and horses, respectively. In those species, some major allergens differ from those in humans. This position paper summarizes the current knowledge about relevant allergens in dogs, cats and horses.

  6. IgE binding reactivity of peptide fragments of Bla g 4, a major German cockroach allergen.

    PubMed

    Shin, Kwang Hyun; Jeong, Kyoung Yong; Hong, Chein-Soo; Yong, Tai-Soon

    2009-03-01

    Cockroaches have been recognized as a major cause of asthma. Bla g 4 is one of the most important German cockroach allergens. The aim of this study is to investigate IgE reactivity to the recombinant Bla g 4 (rBla g 4) in the sera of allergic patients and identify linear IgE binding epitope. For protein expression, full-length Bla g 4 (EF202172) was divided into 5 overlapping peptide fragments (E1: aa 1-100, E2: aa 34-77, E3: aa 74-117, E4: aa 114-156, and E5: aa 153-182). The full-length and 5 peptide fragments of Bla g 4 was generated by PCR and over-expressed in E. coli BL21 (DE3). The IgE binding reactivities of the full-length and peptide fragments were measured by ELISA using 32 serum samples of cockroach allergy. The sera of 8 patients (25%) reacted with rBla g 4. Four sera (100%) showed IgE-binding reactivity to full-length and peptide fragment 4, and 2 sera (50%) reacted with peptide fragment 2. One (20%) serum reacted with peptide fragment 3. The results of ELISA using overlapping recombinant fragments indicated that the epitope region was located at amino acid sequences 34-73 and 78-113, and major IgE epitope of Bla g 4 was located at amino acid sequences 118-152 of C-terminal. B-cell epitope analysis of German cockroach allergen Bla g 4 could contribute to the strategic development of more specific and potentially efficacious immunotherapy.

  7. Analysis of T cell responses to the major allergens from German cockroach: epitope specificity and relationship to IgE production.

    PubMed

    Oseroff, Carla; Sidney, John; Tripple, Victoria; Grey, Howard; Wood, Robert; Broide, David H; Greenbaum, Jason; Kolla, Ravi; Peters, Bjoern; Pomés, Anna; Sette, Alessandro

    2012-07-15

    Bla g allergens are major targets of IgE responses associated with cockroach allergies. However, little is known about corresponding T cell responses, despite their potential involvement in immunopathology and the clinical efficacy of specific immunotherapy. Bioinformatic predictions of the capacity of Bla g 1, 2, 4, 5, 6, and 7 peptides to bind HLA-DR, -DP, and -DQ molecules, and PBMC responses from 30 allergic donors, identified 25 T cell epitopes. Five immunodominant epitopes accounted for more than half of the response. Bla g 5, the most dominant allergen, accounted for 65% of the response, and Bla g 6 accounted for 20%. Bla g 5 induced both IL-5 and IFN-γ responses, whereas Bla g 6 induced mostly IL-5, and, conversely, Bla g 2 induced only IFN-γ. Thus, responses to allergens within a source are independently regulated, suggesting a critical role for the allergen itself, and not extraneous stimulation from other allergens or copresented immunomodulators. In comparing Ab with T cell responses for several donor/allergen combinations, we detected IgE titers in the absence of detectable T cell responses, suggesting that unlinked T cell-B cell help might support development of IgE responses. Finally, specific immunotherapy resulted in IL-5 down modulation, which was not associated with development of IFN-γ or IL-10 responses to any of the Bla g-derived peptides. In summary, the characteristics of T cell responses to Bla g allergens appear uncorrelated with IgE responses. Monitoring these responses may therefore yield important information relevant to understanding cockroach allergies and their treatment.

  8. Association analysis of food allergens.

    PubMed

    Kanagawa, Yoshiyuki; Matsumoto, Shinya; Koike, Soichi; Imamura, Tomoaki

    2009-06-01

    Food allergy patients are known to present with allergic reactions to multiple allergens, but extrapolating these associations is difficult. Data mining, a procedure that analyzes characteristic combinations among large amounts of information, is often used to analyze and predict consumer purchasing behaviour. We applied this technique to the extrapolation of food allergen associations in allergy patients. We sent 1510 families our 'Questionnaire survey for the prevention of food allergies'. Responses noting 6549 allergens came from 878 families with 1383 patients, including 402 with anaphylaxis. Some results of the survey have already been published and here we presented the results of our association analysis of combinations of food allergens. Egg, milk, wheat, peanuts, and buckwheat are the most common food allergens. The most common simultaneous combinations of these allergens were 'egg-milk', 'egg-wheat', and 'milk-wheat'. The occurrence probability of a combination (i.e. one person suffering from a certain allergen also suffers from another) is called 'confidence'. Confidence was higher for 'chicken-egg', 'abalone-salmon eggs', and 'matsutake mushroom-milk'. As well, the combinations of 'crab-shrimp', 'squid-shrimp', and 'squid-crab' also indicated higher values in a statistical examination of the occurrence probabilities of these allergen combinations (Z-score). From the results of the association analysis, we speculated that some food allergens, such as abalone, orange, salmon, chicken, pork, matsutake mushroom, peach and apple did not independently induce food allergies. We also found that combinations, such as 'crab-shrimp', 'squid-shrimp', 'squid-crab', 'chicken-beef', and 'salmon-mackerel' had strong associations.

  9. House dust mite major allergen Der f 1 enhances proinflammatory cytokine and chemokine gene expression in a cell line of canine epidermal keratinocytes.

    PubMed

    Maeda, Shingo; Maeda, Sadatoshi; Shibata, Sanae; Chimura, Naoki; Fukata, Tsuneo

    2009-10-15

    House dust mite (HDM) allergens are the most common allergens involved in the induction of IgE-mediated hypersensitivity. Recently, epicutaneous sensitization with HDM allergens has been emphasized in the development of atopic dermatitis (AD); however, direct stimulation of canine keratinocytes by mite allergens has not been well investigated. In the present study, we investigated the effects of Der f 1, a major allergen of Dermatophagoides farinae, on cytokine and chemokine gene expression in a canine keratinocyte cell line, CPEK. CPEK constitutively expressed mRNA for TNF-alpha, IL-12p35, IL-18, GM-CSF, TGF-beta, IL-8/CXCL8, TARC/CCL17, CTACK/CCL27 and MEC/CCL28. Of all the cytokines and chemokines investigated in CPEK, transcription levels of GM-CSF, IL-8/CXCL8 and TNF-alpha mRNA were significantly enhanced by stimulation with Der f 1. The present results suggest that Der f 1 can directly augment inflammatory cytokine and chemokine production from keratinocytes, and may initiate allergic inflammation independently of Type-I hypersensitivity.

  10. Purification, Crystallization and Preliminary X-ray Characterization of Prunin-1, a Major Component of the Almond (Prunus dulcis) Allergen Amandin

    SciTech Connect

    Albillos, Silvia M.; Jin, Tengchuan; Howard, Andrew; Zhang, Yuzhu; Kothary, Mahendra H.; Fu, Tong-Jen

    2008-08-04

    The 11S globulins from plant seeds account for a number of major food allergens. Because of the interest in the structural basis underlying the allergenicity of food allergens, we sought to crystallize the main 11S seed storage protein from almond (Prunus dulcis). Prunin-1 (Pru1) was purified from defatted almond flour by water extraction, cryoprecipitation, followed by sequential anion exchange, hydrophobic interaction, and size exclusion chromatography. Single crystals of Pru1 were obtained in a screening with a crystal screen kit, using the hanging-drop vapor diffusion method. Diffraction quality crystals were grown after optimization. The Pru1 crystals diffracted to at least 3.0 {angstrom} and belong to the tetragonal space group P4{sub 1}22, with unit cell parameters of a = b = 150.912 {angstrom}, c = 165.248 {angstrom}. Self-rotation functions and molecular replacement calculations showed that there are three molecules in the asymmetry unit with water content of 51.41%. The three Pru1 protomers are related by a noncrystallographic 3-fold axis and they form a doughnut-shaped trimer. Two prunin trimers form a homohexamer. Elucidation of prunin structure will allow further characterization of the allergenic features of the 11S protein allergens at the molecular level.

  11. Purification, crystallization and preliminary X-ray characterization of prunin-1, a major component of the almond (Prunus dulcis) allergen amandin.

    PubMed

    Albillos, Silvia M; Jin, Tengchuan; Howard, Andrew; Zhang, Yuzhu; Kothary, Mahendra H; Fu, Tong-Jen

    2008-07-09

    The 11S globulins from plant seeds account for a number of major food allergens. Because of the interest in the structural basis underlying the allergenicity of food allergens, we sought to crystallize the main 11S seed storage protein from almond ( Prunus dulcis). Prunin-1 (Pru1) was purified from defatted almond flour by water extraction, cryoprecipitation, followed by sequential anion exchange, hydrophobic interaction, and size exclusion chromatography. Single crystals of Pru1 were obtained in a screening with a crystal screen kit, using the hanging-drop vapor diffusion method. Diffraction quality crystals were grown after optimization. The Pru1 crystals diffracted to at least 3.0 A and belong to the tetragonal space group P4(1)22, with unit cell parameters of a = b = 150.912 A, c = 165.248 A. Self-rotation functions and molecular replacement calculations showed that there are three molecules in the asymmetry unit with water content of 51.41%. The three Pru1 protomers are related by a noncrystallographic 3-fold axis and they form a doughnut-shaped trimer. Two prunin trimers form a homohexamer. Elucidation of prunin structure will allow further characterization of the allergenic features of the 11S protein allergens at the molecular level.

  12. Correlation between airborne Olea europaea pollen concentrations and levels of the major allergen Ole e 1 in Córdoba, Spain, 2012-2014

    NASA Astrophysics Data System (ADS)

    Plaza, M. P.; Alcázar, P.; Galán, C.

    2016-12-01

    Olea europaea L. pollen is the second-largest cause of pollinosis in the southern Iberian Peninsula. Airborne-pollen monitoring networks provide essential data on pollen dynamics over a given study area. Recent research, however, has shown that airborne pollen levels alone do not always provide a clear indicator of actual exposure to aeroallergens. This study sought to evaluate correlations between airborne concentrations of olive pollen and Ole e 1 allergen levels in Córdoba (southern Spain), in order to determine whether atmospheric pollen concentrations alone are sufficient to chart changes in hay fever symptoms. The influence of major weather-related variables on local airborne pollen and allergen levels was also examined. Monitoring was carried out from 2012 to 2014. Pollen sampling was performed using a Hirst-type sampler, following the protocol recommended by the Spanish Aerobiology Network. A multi-vial cyclone sampler was used to collect aeroallergens, and allergenic particles were quantified by ELISA assay. Significant positive correlations were found between daily airborne allergen levels and atmospheric pollen concentrations, although there were occasions when allergen was detected before and after the pollen season and in the absence of airborne pollen. The correlation between the two was irregular, and pollen potency displayed year-on-year variations and did not necessarily match pollen-season-intensity.

  13. Standardization of RP-HPLC methods for the detection of the major peanut allergens Ara h 1, Ara h 2 and Ara h 3

    USDA-ARS?s Scientific Manuscript database

    Crude peanut extract (CPE) was analyzed for three major allergens (Ara h 1, h 2, and h 3) using a C12 and a C18 column at two wavelengths (280 and 220 nm) and under different solvent conditions. HPLC profiles were compared for retention time, resolution, and peak heights. CPE samples were spiked wit...

  14. Quantification of Art v 1 and Act c 1 being major allergens of mugwort pollen and kiwi fruit extracts in mass-units by ion-exchange HPLC-UV method.

    PubMed

    Blanusa, Milan; Perovic, Iva; Popovic, Milica; Polovic, Natalija; Burazer, Lidija; Milovanovic, Mina; Gavrovic-Jankulovic, Marija; Jankov, Ratko; Cirkovic Velickovic, Tanja

    2007-10-01

    A simple ion-exchange HPLC-UV method was developed for determination of major allergens from mugwort pollen and kiwi fruit extracts in mass-units. The separation of Art v 1 and Act c 1 from other components in the extracts was achieved in one step. The extinction coefficients used in the study were theoretically determined and compared to the extinction coefficients determined by gravimetry. We also reported a close correlation of the major allergen contents with the overall allergenic potency of the extracts determined by inhibition ELISA. This method could be a useful tool for standardization of allergenic extracts for clinical use.

  15. Pin p 1 is a major allergen in pine nut and the first food allergen described in the plant group of gymnosperms.

    PubMed

    Cabanillas, Beatriz; Crespo, Jesus F; Maleki, Soheila J; Rodriguez, Julia; Novak, Natalija

    2016-11-01

    This study aimed to report the complete sequence of a 2S albumin purified from pine nut and to analyze its allergenic properties. Individual recognition of this protein by serum IgE from pine nut-allergic patients was assessed. IgE cross-linking capacity was analyzed in a basophil activation test. Inhibition of IgE-binding and stability to heating was also assessed. The complete nucleotide sequence was obtained and a phylogenetic study was carried out. 2S albumin from pine nut (registered as Pin p 1.0101) was recognized by IgE of 75% of sera. The allergen was heat-stable and had a robust capacity to inhibit IgE-binding to whole pine nut extract. The IgE cross-linking capacity of Pin p 1 on basophils was also demonstrated. Despite the low homology of Pin p 1 sequence with other allergenic 2S albumins from angiosperms, Pin p 1 contains the typical skeleton of 8 cysteine residues, important for its α-helixes enriched structure.

  16. Analysis of glabrous canary seeds by ELISA, mass spectrometry, and Western blotting for the absence of cross-reactivity with major plant food allergens.

    PubMed

    Boye, Joyce Irene; Achouri, Allaoua; Raymond, Nancy; Cleroux, Chantal; Weber, Dorcas; Koerner, Terence B; Hucl, Pierre; Patterson, Carol Ann

    2013-06-26

    Glabrous (hairless) canary seed belongs to the Poaceae (Gramineae) family and could serve as an alternative source of gluten-free cereal grain. In this study, allergenic cross-reactivities between hairless, dehulled canary seeds (Phalaris canariensis) and major allergenic proteins from gluten, soy, peanuts, tree nuts, sesame, and mustard were studied using commercial enzyme-linked immune sorbent assay (ELISA) kits specific for these target allergens. Mass spectrometry (MS) and immunoblotting were further used to assess for the presence of gluten-specific protein fragments. MS results revealed the likely presence of proteins homologous with rice, oat, corn, carrot, tomato, radish, beet, and chickpea. However, no presence of celiac-related gluten fragments from wheat, rye, barley, or their derivatives was found. Immunoblotting studies yielded negative results, further confirming the absence of gluten in the canary seed samples tested. No cross-reactivities were detected between canary seeds and almond, hazelnut, mustard, peanut, sesame, soy, walnut, and gluten using ELISA.

  17. The structural requirements of epitopes with IgE binding capacity demonstrated by three major allergens from fish, egg and tree pollen.

    PubMed

    Elsayed, S; Apold, J; Holen, E; Vik, H; Florvaag, E; Dybendal, T

    1991-01-01

    Three major allergens from cod fish, egg white and tree pollen, were characterized by studies on their allergenic and antigenic structures. The major allergen of cod fish, Allergen M "parvalbumins pI 4.75", is composed of 113 amino acid residues with a molecular weight of 12,328 daltons. It comprised three domains, AB, CD and EF, consisting of 3 helices interspaced by one loop. Each of the loops of the CD and EF domains each coordinates one Ca2+. The antigenicity and allergenicity of Allergen M was deduced from studying the modified protein and some particular synthetic peptides. Three sites were encompassing IgE binding epitopes namely peptides 33-44, 65-74 and 88-96. A novel peptide (49-64), of the CD-domain, was demonstrated to be allergenically/antigenically active and cross reactive with birch pollen allergen, which incidentally was used as a negative control. This site encompassed two repetitive sequences (D-E-D-K) and (D-E-L-K), suggested to be mutually critical for the specificity of antibody binding. This hypothesis was reconfirmed by SPPS of several analogous peptides of region 39-64. Furthermore, peptide 88-103 of the EF-domain was similarly synthesized; it functioned as a monovalent hapten, blocking and not eliciting allergic reaction. Moreover, peptide 13-32 of domain AB, the non-calcium binding domain, was thoroughly tested. The results of PK inhibition showed clear activity and the peptide was found to function at the level of a divalent determinant. Ovalbumin (OA) is the most dominant of five major allergens of egg white and universally used as model protein. OA allergenic epitopes were shown to be mainly determined by the primary structure and depend on certain peptide chain length. The N-terminal decapeptide (OA 1-10) was shown to react with reaginic IgE. Direct skin test on egg allergic patients, showed no activity and the site was therefore concluded to encompasses one single Ig binding haptenic epitope. Peptide OA 323-339, was demonstrated to

  18. Stability of major allergen tropomyosin and other food proteins of mud crab (Scylla serrata) by in vitro gastrointestinal digestion.

    USDA-ARS?s Scientific Manuscript database

    Stability in simulated gastric fluid is regarded as an important parameter for the estimation of food allergenicity. In this study, the digestive stability of allergenic protein tropomyosin (TM) and other food proteins from mud crab in simulated gastric fluid (SGF), and simulated intestinal fluid (S...

  19. Sensitization to common allergens among patients with allergies in major Iranian cities: a systematic review and meta-analysis.

    PubMed

    Moghtaderi, Mozhgan; Hosseini Teshnizi, Saeed; Farjadian, Shirin

    2017-01-01

    Various allergens are implicated in the pathogenesis of allergic diseases in different regions. This study attempted to identify the most common allergens among patients with allergies based on the results of skin prick tests in different parts of Iran. Relevant studies conducted from 2000 to 2016 were identified from the MEDLINE database. Six common groups of allergen types, including animal, cockroach, food, fungus, house dust mite, and pollen were considered. Subgroup analysis was performed to determine the prevalence of each type of allergen. The Egger test was used to assess publication bias. We included 44 studies in this meta-analysis. The overall prevalence of positive skin test results for at least one allergen was estimated to be 59% in patients with allergies in various parts of Iran. The number of patients was 11,646 (56% male and 44% female), with a mean age of 17.46±11.12 years. The most common allergen sources were pollen (47.0%), mites (35.2%), and food (15.3%). The prevalence of sensitization to food and cockroach allergens among children was greater than among adults. Pollen is the most common allergen sensitization in cities of Iran with a warm and dry climate; however, sensitization to house dust mites is predominant in northern and southern coastal areas of Iran.

  20. Allergen nomenclature*

    PubMed Central

    1994-01-01

    The revised nomenclature for allergens is presented together with proposed nomenclatures for (a) allergen genes, mRNAs and cDNAs, and (b) recombinant and synthetic peptides of allergenic interest. PMID:7955031

  1. Molecular characterization of human IgG monoclonal antibodies specific for the major birch pollen allergen Bet v 1. Anti-allergen IgG can enhance the anaphylactic reaction.

    PubMed

    Denépoux, S; Eibensteiner, P B; Steinberger, P; Vrtala, S; Visco, V; Weyer, A; Kraft, D; Banchereau, J; Valenta, R; Lebecque, S

    2000-01-07

    We report the molecular characterization of five human monoclonal antibodies, BAB1-5 (BAB1: IgG(1); BAB4: IgG(2); BAB2, 3, 5: IgG(4)), with specificity for the major birch pollen allergen, Bet v 1. BAB1-5 were obtained after immunotherapy and contained a high degree of somatic mutations indicative of an antigen-driven affinity maturation process. While BAB1 inhibited the binding of patients IgE to Bet v 1, BAB2 increased IgE recognition of Bet v 1, and, even as Escherichia coli-expressed Fab, augmented Bet v 1-induced immediate type skin reactions. The demonstration that IgG antibodies can enhance allergen-induced allergic reactions is likely to explain the unpredictability of specific immunotherapy.

  2. Confirmation of immuno-reactivity of the recombinant major birch pollen allergen Bet v 1a by affinity-CIEF.

    PubMed

    Dullnig, Verena; Weiss, Richard; Amon, Sabine; Rizzi, Andreas; Stutz, Hanno

    2009-07-01

    Affinity-CIEF has been applied to characterize a recombinant product of the major birch pollen allergen Betula verrucosa isoform 1a (Bet v 1a) immuno-chemically. For this purpose mAbs of the IgG-type have been produced in-lab from two murine hybridoma lines, specified as clones 2 and 5.1. Both IgG clones were characterized by SDS-PAGE, MALDI-TOF-MS and CIEF. The purified IgG solutions had to be dialysed against 10 mmol/L phosphate (pH 7.4) to prevent IgG precipitation and to ensure appropriate CIEF separation. Both tested monoclonal IgGs (mIgGs) comprised four constituents covering pI ranges of 6.98-7.09 and 6.78-7.03 for clones 2 and 5.1 with major peaks at pI 7.09 and 7.03, respectively. When increasing amounts of Bet v 1a (pI 4.95) were incubated with 2.0 mumol/L mIgG, novel peaks were progressively induced in a pI range slightly more acidic than the focusing region of mIgGs. These peaks grew on the expense of original mIgG peaks. All pI values were calculated using two pI marker compounds with a repeatability of better than 0.03 units. New peaks represent complexes between Bet v 1a and mIgG either of 1:1 or of 2:1 binding stoichiometry. At a molar ratio of 2:1, saturation of both IgG paratopes with allergen (Ag) molecules was achieved as indicated by unbound Bet v 1a. The current CIEF approach addresses the proof of single epitope integrity in the course of immuno-chemical characterization of Bet v 1a. Contrary to traditional immunoassays, affinity CIEF allows for a distinction and relative quantification of mAbs, Ag-antibody complexes and Ag variants coexisting in one sample.

  3. Association of HLA-DR1 with the allergic response to the major mugwort pollen allergen: molecular background

    PubMed Central

    2012-01-01

    Background Mugwort pollen allergens represent the main cause of pollinosis in late summer. The major allergen, Art v 1, contains only one single immunodominant, solely HLA-DR-restricted T cell epitope (Art v 125-36). The frequency of HLA-DRB1*01 is highly increased in mugwort-allergic individuals and HLA-DR1 serves as restriction element for Art v 125-36. However, Art v 125-36 also binds to HLA-DR4 with high affinity and DR1-restricted Art v 125-36 -specific T cell receptors can be activated by HLA-DR4 molecules. To understand the predominance of HLA-DR1 in mugwort allergy in spite of the degeneracy in HLA/peptide-binding and TCR-recognition, we investigated the molecular background of Art v 125-36 /MHC/TCR interactions in the context of HLA-DR1 compared to -DR4. Results The majority of Art v 125-36 -specific T cell lines and clones from HLA-DR1 carrying, mugwort pollen-allergic donors reacted to synthetic and naturally processed Art v 1–peptides when presented by HLA-DR1 or HLA-DR4 expressing antigen presenting cells. However, at limiting peptide concentrations DR1 was more effective in T cell stimulation. In addition, the minimal epitope for 50% of Art v 125-36 -specific T cells was shorter for DR1 than for DR4. In vitro binding assays of Art v 125-36 mutant peptides to isolated DR1- and DR4-molecules indicated similar binding capacities and use of the same register. In silico simulation of Art v 125-36 binding to HLA-DR1 and -DR4 suggested similar binding of the central part of the peptide to either molecule, but a higher flexibility of the N- and C-terminal amino acids and detachment at the C-terminus in HLA-DR1. Conclusions The predominance of HLA-DR1 in the response to Art v 125-36 may be explained by subtle conformation changes of the peptide bound to DR1 compared to DR4. Computer simulation supported our experimental data by demonstrating differences in peptide mobility within the HLA-DR complex that may influence TCR-binding. We suggest that the minor

  4. Structures of Two Major Allergens, Bla g 4 and Per a 4, From Cockroaches and Their IgE Binding Epitopes

    SciTech Connect

    Tan, Y.; Chan, S; Ong, T; Yit, L; Tiong, Y; Chew, F; Sivaraman, J; Mok, Y

    2009-01-01

    Inhalant allergens from cockroaches are an important cause of asthma to millions of individuals worldwide. Here we report for the first time the structures of two major cockroach allergens, Bla g 4 and Per a 4, that adopt a typical lipocalin fold but with distinct structural features as compared with other known lipocalin allergens. Both Bla g 4 and Per a 4 contain two long-range disulfide bonds linking the N and C termini to a beta-barrel. The C-terminal helix of Bla g 4 is bent and greatly extended toward the N terminus. Bla g 4 is found to be a monomer, whereas Per a 4 exists as a dimer in solution with a novel dimeric interface involving residues from loops at the top and bottom of the beta-barrel. Putative ligand binding sites of both allergens are determined by docking of the juvenile hormone III inside the beta-barrel and found to interact with the ligand using non-conserved residues. Bla g 4 and Per a 4 are found to be cross-reactive in sera IgE binding, at least in the Singaporean Chinese population tested. A major IgE binding epitope unique to Per a 4 is found on the loops at the bottom of the beta-barrel that may aid the development of hypoallergens for immunotherapy.

  5. Common food allergens and their IgE-binding epitopes.

    PubMed

    Matsuo, Hiroaki; Yokooji, Tomoharu; Taogoshi, Takanori

    2015-10-01

    Food allergy is an adverse immune response to certain kinds of food. Although any food can cause allergic reactions, chicken egg, cow's milk, wheat, shellfish, fruit, and buckwheat account for 75% of food allergies in Japan. Allergen-specific immunoglobulin E (IgE) antibodies play a pivotal role in the development of food allergy. Recent advances in molecular biological techniques have enabled the efficient analysis of food allergens. As a result, many food allergens have been identified, and their molecular structure and IgE-binding epitopes have also been identified. Studies of allergens have demonstrated that IgE antibodies specific to allergen components and/or the peptide epitopes are good indicators for the identification of patients with food allergy, prediction of clinical severity and development of tolerance. In this review, we summarize our current knowledge regarding the allergens and IgE epitopes in the well-researched allergies to chicken egg, cow's milk, wheat, shrimp, and peanut.

  6. 27 CFR 5.32a - Voluntary disclosure of major food allergens.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... AND TRADE BUREAU, DEPARTMENT OF THE TREASURY ALCOHOL LABELING AND ADVERTISING OF DISTILLED SPIRITS... refined oil derived from a food specified in paragraph (a)(1)(i) of this section and any ingredient derived from such highly refined oil; or (B) A food ingredient that is exempt from major food...

  7. 27 CFR 7.22a - Voluntary disclosure of major food allergens.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... AND TRADE BUREAU, DEPARTMENT OF THE TREASURY LIQUORS LABELING AND ADVERTISING OF MALT BEVERAGES... refined oil derived from a food specified in paragraph (a)(1)(i) of this section and any ingredient derived from such highly refined oil; or (B) A food ingredient that is exempt from major food...

  8. 27 CFR 7.22a - Voluntary disclosure of major food allergens.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... AND TRADE BUREAU, DEPARTMENT OF THE TREASURY ALCOHOL LABELING AND ADVERTISING OF MALT BEVERAGES... refined oil derived from a food specified in paragraph (a)(1)(i) of this section and any ingredient derived from such highly refined oil; or (B) A food ingredient that is exempt from major food...

  9. 27 CFR 7.22a - Voluntary disclosure of major food allergens.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... AND TRADE BUREAU, DEPARTMENT OF THE TREASURY LIQUORS LABELING AND ADVERTISING OF MALT BEVERAGES... refined oil derived from a food specified in paragraph (a)(1)(i) of this section and any ingredient derived from such highly refined oil; or (B) A food ingredient that is exempt from major food...

  10. 27 CFR 5.32a - Voluntary disclosure of major food allergens.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... AND TRADE BUREAU, DEPARTMENT OF THE TREASURY LIQUORS LABELING AND ADVERTISING OF DISTILLED SPIRITS... refined oil derived from a food specified in paragraph (a)(1)(i) of this section and any ingredient derived from such highly refined oil; or (B) A food ingredient that is exempt from major food...

  11. 27 CFR 7.22a - Voluntary disclosure of major food allergens.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... AND TRADE BUREAU, DEPARTMENT OF THE TREASURY LIQUORS LABELING AND ADVERTISING OF MALT BEVERAGES... refined oil derived from a food specified in paragraph (a)(1)(i) of this section and any ingredient derived from such highly refined oil; or (B) A food ingredient that is exempt from major food...

  12. 27 CFR 5.32a - Voluntary disclosure of major food allergens.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... AND TRADE BUREAU, DEPARTMENT OF THE TREASURY LIQUORS LABELING AND ADVERTISING OF DISTILLED SPIRITS... refined oil derived from a food specified in paragraph (a)(1)(i) of this section and any ingredient derived from such highly refined oil; or (B) A food ingredient that is exempt from major food...

  13. 27 CFR 5.32a - Voluntary disclosure of major food allergens.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... AND TRADE BUREAU, DEPARTMENT OF THE TREASURY LIQUORS LABELING AND ADVERTISING OF DISTILLED SPIRITS... refined oil derived from a food specified in paragraph (a)(1)(i) of this section and any ingredient derived from such highly refined oil; or (B) A food ingredient that is exempt from major food...

  14. Atopic donor status does not influence the uptake of the major grass pollen allergen, Phl p 5, by dendritic cells.

    PubMed

    Ashjaei, Kazem; Palmberger, Dieter; Bublin, Merima; Bajna, Erika; Breiteneder, Heimo; Grabherr, Reingard; Ellinger, Isabella; Hoffmann-Sommergruber, Karin

    2015-09-01

    Dendritic cells (DCs) are sentinels of the immune system for antigen recognition and uptake, as well as presentation to naïve T cells for stimulation or priming. Internalization and endocytic degradation of allergens by DCs are important steps required for T cell priming. In the current study we investigated binding and internalization of purified recombinant non-glycosylated grass pollen allergen, Phl p 5, and natural non-specific lipid transfer protein from sunflower, SF-nsLTP to human monocyte derived dendritic cells (MoDCs). Colocalization of Phl p 5 with low affinity (CD23) or high affinity receptor (FcεRI) was investigated by immunofluorescence staining. Likewise, localization of the allergens in early (EE) and late endosomes (LE) was detected by co-staining for early endosome antigen (EEA1) and lysosomal-associated membrane protein 1 (LAMP1). In our experimental setting we could demonstrate that Phl p 5 as well as SF-nsLTP bound to MoDCs from both, grass pollen allergic and non-allergic individuals. Competitive allergen uptake experiments demonstrated non-preferential and simultaneous uptake of Phl p 5 and SF-nsLTP by MoDCs. No overlap of signals from Phl p 5 and CD23 or FcεRI was detectable, excluding IgE-mediated uptake for this allergen. Both allergens, Phl p 5 and SF-nsLTP, were localized in early and late endosomes. The present study applied a set of methods to assess the allergen uptake by MoDCs in an in vitro model. No qualitative and quantitative differences in the allergen uptake of both, Phl p 5 and SF-nsLTP were detected in single and competitive assays.

  15. Immunological differences in the global release of the major cat allergen Fel d 1 are influenced by sex and behaviour.

    PubMed

    Bienboire-Frosini, Cécile; Cozzi, Alessandro; Lafont-Lecuelle, Céline; Vervloet, Daniel; Ronin, Catherine; Pageat, Patrick

    2012-07-01

    The biological function of Fel d 1, the major cat allergen released in the environment, is still unclear despite studies suggesting a putative role in chemical communication. Structural and immunological polymorphisms of Fel d 1 have been described. This study examined how Fel d 1 immunological polymorphism may have a physiological origin by estimating a potential relationship with the sex of cats and cat-human interactions. Samples from bath washes of 21 cats were screened to study antibody binding to Fel d 1 using an ELISA. Personality and Tolerance Handling scores were used to assess the behaviour of the cats. In the washes, Fel d 1 concentrations were significantly lower in females than in males (P<0.05). Slopes from the ELISA dose-dependent curves varied among the cats: males secreted Fel d 1 variants with higher antibody recognition than females (P<0.01). Females that were aggressive and difficult to handle displayed a diminished slope value, and therefore a weaker Fel d 1 immunoreactivity in global washes, compared to females that were sociable (P=0.09) and easy to handle (P=0.07). This study shows a variable immunological polymorphism of Fel d 1 within a cat population, particularly between males and females, and this polymorphism appears to be related to cat-human interactions. Copyright © 2011 Elsevier Ltd. All rights reserved.

  16. Genomic, RNAseq, and Molecular Modeling Evidence Suggests That the Major Allergen Domain in Insects Evolved from a Homodimeric Origin

    PubMed Central

    Randall, Thomas A.; Perera, Lalith; London, Robert E.; Mueller, Geoffrey A.

    2013-01-01

    The major allergen domain (MA) is widely distributed in insects. The crystal structure of a single Bla g 1 MA revealed a novel protein fold in which the fundamental structure was a duplex of two subsequences (monomers), which had diverged over time. This suggested that the evolutionary origin of the MA structure may have been a homodimer of this smaller subsequence. Using publicly available genomic data, the distribution of the basic unit of this class of proteins was determined to better understand its evolutionary history. The duplication and divergence is examined at three distinct levels of resolution: 1) within the orders Diptera and Hymenoptera, 2) within one genus Drosophila, and 3) within one species Aedes aegypti. Within the family Culicidae, we have found two separate occurrences of monomers as independent genes. The organization of the gene family in A. aegypti shows a common evolutionary origin for its monomer and several closely related MAs. Molecular modeling of the A. aegypti monomer with the unique Bla g 1 fold confirms the distant evolutionary relationship and supports the feasibility of homodimer formation from a single monomer. RNAseq data for A. aegypti confirms that the monomer is expressed in the mosquito similar to other A. aegypti MAs after a blood meal. Together, these data support the contention that the detected monomer shares similar functional characteristics to related MAs in other insects. An extensive search for this domain outside of Insecta confirms that the MAs are restricted to insects. PMID:24253356

  17. Genomic, RNAseq, and molecular modeling evidence suggests that the major allergen domain in insects evolved from a homodimeric origin.

    PubMed

    Randall, Thomas A; Perera, Lalith; London, Robert E; Mueller, Geoffrey A

    2013-01-01

    The major allergen domain (MA) is widely distributed in insects. The crystal structure of a single Bla g 1 MA revealed a novel protein fold in which the fundamental structure was a duplex of two subsequences (monomers), which had diverged over time. This suggested that the evolutionary origin of the MA structure may have been a homodimer of this smaller subsequence. Using publicly available genomic data, the distribution of the basic unit of this class of proteins was determined to better understand its evolutionary history. The duplication and divergence is examined at three distinct levels of resolution: 1) within the orders Diptera and Hymenoptera, 2) within one genus Drosophila, and 3) within one species Aedes aegypti. Within the family Culicidae, we have found two separate occurrences of monomers as independent genes. The organization of the gene family in A. aegypti shows a common evolutionary origin for its monomer and several closely related MAs. Molecular modeling of the A. aegypti monomer with the unique Bla g 1 fold confirms the distant evolutionary relationship and supports the feasibility of homodimer formation from a single monomer. RNAseq data for A. aegypti confirms that the monomer is expressed in the mosquito similar to other A. aegypti MAs after a blood meal. Together, these data support the contention that the detected monomer shares similar functional characteristics to related MAs in other insects. An extensive search for this domain outside of Insecta confirms that the MAs are restricted to insects.

  18. Allergen nomenclature.

    PubMed Central

    Marsh, D. G.; Goodfriend, L.; King, T. P.; Lowenstein, H.; Platts-Mills, T. A.

    1986-01-01

    This article presents a nomenclature system for allergens which has been officially recommended by the International Union of Immunological Societies (IUIS). The nomenclature is based on proposals of the IUIS Sub-Committee for Allergen Nomenclature and is applicable to highly purified, well-characterized allergens and to non-purified or partially purified allergenic extracts. PMID:3492310

  19. Symptomatic efficacy of buckwheat products in Non-Celiac Gluten Sensitivity (NCGS).

    PubMed

    Dinu, Monicau; Macchia, Donatella; Pagliai, Giuditta; Gori, Anna Maria; Cesari, Francesca; Marcucci, Rossella; Sofi, Francesco; Casini, Alessandro

    Buckwheat (Fagopyrum esculentum) is a gluten-free grain with acclaimed benefi-cial effects on human health. Our aim was to assess the effect of buckwheat products on intestinal/extra-intestinal symptoms and biochemical parameters in patients with Non-Celiac Gluten Sensitivity (NCGS). A randomized, crossover trial with two intervention phases was conducted on 19 NCGS patients over a 12 week-period. The participants were assigned to consume products made from buckwheat or to maintain their normal gluten-free diet for 6 weeks in a random order. Symptoms due to NCGS were evaluated using two questionnaires. During the intervention period with buckwheat products, patients experienced a signifi-cant decrease in the severity of abdominal pain and bloating (p=0.03). In contrast, the control group showed a significant worsening trend for the majority of NCGS symptoms such as nausea, headache, joint/muscle pain, and attention disorders. The replacement diet with buckwheat products also resulted in a significant increase of serum magnesium (+4.7%) and a significant reduction in the circulating levels of some pro-inflammatory cytokines such as interferon gamma (-33.3%) and monocyte chemotactic protein-1 (-46.5%). The study supports the positive effects of buckwheat for NCGS patients, showing that this alternative cereal can contribute to the re-duction of both negative gastro-intestinal and related symptoms, and nutritional deficiencies, and lead to an im-provement in inflammatory profile.

  20. Differential protein expression by dendritic cells from atopic and non-atopic individuals after stimulation by the major house dust mite allergen Der p 1.

    PubMed

    Horlock, C; Shakib, F; Jones, N S; Sewell, H F; Ghaemmaghami, A M

    2009-01-01

    Dendritic cells (DCs) are sentinels of the immune system and are known to play a key role in allergic responses. However, it is not clear how DCs that have been exposed to an allergen support Th2 type immune responses. It is possible that DCs from atopic individuals are inherently programmed to support allergic disease, or it is the exposure of dendritic cells to allergens that is key to the development of allergic sensitisation. We used 2D gel electrophoresis and MALDI mass spectrometry to compare the proteome of DCs from atopic and non-atopic individuals in both the resting state and after stimulation with the major house dust mite allergen Der p 1. Our data show that unstimulated DCs from atopic and non-atopic individuals are very similar at the whole cell proteome level, showing few differentially expressed proteins. However, upon stimulation with Der p 1, a number of additional proteins are differentially expressed, and of these several were of potential relevance to Th2 cell differentiation and the allergic response, including GTP-binding regulatory protein Gi alpha-2, frabin and cathepsin D. Whilst there are inherent differences between DCs from atopic and non-atopic individuals, it seems that exposure to allergen plays a key role in differential expression of proteins by these key immune cells. Further studies should now focus on establishing the biological relevance of these proteins as biomarkers in house dust mite allergy and their role in allergen induced Th2 cell differentiation. Copyright 2009 S. Karger AG, Basel.

  1. A major allergen from pollen defines a novel family of plant proteins and shows intra- and interspecies [correction of interspecie] cross-reactivity.

    PubMed

    Barral, Patricia; Batanero, Eva; Palomares, Oscar; Quiralte, Joaquín; Villalba, Mayte; Rodríguez, Rosalía

    2004-03-15

    Olive tree (Olea europaea) pollen is a main cause of allergy associated with extensive areas of Europe and North America. Ole e 10, a small (10.8 kDa) and acidic (pI 5.8) protein, has been identified as a major allergen from the olive pollen, isolated, and characterized. Circular dichroism analysis gave 17% alpha helix, 33% beta sheet, and 21% beta turn for its secondary structure. Based on amino acid sequences of tryptic peptides, the protein was cloned and sequenced. The allergen consists of a single polypeptide chain of 102 aa, with a signal peptide of 21 residues. Ole e 10 showed homology with the C-terminal domain of another olive allergen, Ole e 9 (1,3-beta-glucanase, 53% identity), with deduced sequences from Arabidopsis thaliana genes (42-46% identity) and with polypeptide segments (Cys boxes) of proteins involved in yeast development (Epd1/Gas-1p/Phr2 families; 42-43% similarity). Ole e 10 showed 55% prevalence for olive-allergic patients and exhibited an IgE response dependent on its conformation. Remarkable IgE cross-reactivity was detected with Ole e 9, but no correlation was observed between the individual IgE responses to both allergens. Ole e 10 shares IgE B cell epitopes with proteins from Oleaceae, Gramineae, Betulaceae, Chenopodiaceae, Cupressaceae, Ambrosia, and Parietaria pollens, latex, and vegetable foods, such as tomato, kiwi, potato, and peach. These data indicate that Ole e 10 is a new pan-allergenic plant protein that shows notable intra- and interspecie IgE cross-reactivity and is a powerful candidate to be involved in pollen-latex-fruit syndrome.

  2. Human IgE against the major allergen Bet v 1 – defining an epitope with limited cross-reactivity between different PR-10 family proteins

    PubMed Central

    Levin, M; Davies, A M; Liljekvist, M; Carlsson, F; Gould, H J; Sutton, B J; Ohlin, M

    2014-01-01

    Background The interaction between IgE and allergen is a key event at the initiation of an allergic response, and its characteristics have substantial effects on the clinical manifestation. Despite this, the molecular details of the interaction between human IgE and the major birch allergen Bet v 1, one of the most potent tree allergens, still remain poorly investigated. Objective To isolate Bet v 1-specific human monoclonal IgE and characterize their interaction with the allergen. Methods Recombinant human IgE were isolated from a combinatorial antibody fragment library and their interaction with Bet v 1 assessed using various immunological assays. The structure of one such IgE in the single-chain fragment variable format was determined using X-ray crystallography. Results We present four novel Bet v 1-specific IgE, for one of which we solve the structure, all with their genetic origin in the IGHV5 germline gene, and demonstrate that they target two non-overlapping epitopes on the surface of Bet v 1, thereby fulfilling the basic criteria for FcεRI cross-linkage. We further define these epitopes and for one epitope pinpoint single amino acid residues important for the interaction with human IgE. This provides a potential explanation, at the molecular level, for the differences in recognition of isoforms of Bet v 1 and other allergens in the PR-10 protein family displayed by IgE targeting this epitope. Finally, we present the first high-resolution structure of a human allergen-specific IgE fragment in the single-chain fragment variable (scFv) format. Conclusions and Clinical Relevance We here display the usefulness of allergen-specific human monoclonal IgE as a tool in studies of the crucial molecular interaction taking place at the initiation of an allergic response. Such studies may aid us in development of better diagnostic tools and guide us in the development of new therapeutic compounds. PMID:24447087

  3. Cloning and characterization of recombinant tropomyosin of giant freshwater shrimp M. rosenbergii to determine major allergens causing allergic reactions among shrimp-allergic children.

    PubMed

    Kumjim, Sasaros; Jirapongsananuruk, Orathai; Piboonpocanun, Surapon

    2016-09-01

    Seawater and freshwater shrimp are some of the most common causes of food allergy among children in Thailand. Tropomyosin has been reported as a major allergen for shrimp allergic populations around the world. Despite a high number of shrimp-allergic Thai children, however, it is unknown whether shrimp tropomyosin is a major cause of allergic reactions. To clone and characterize tropomyosin of giant freshwater shrimp Macrobrachium rosenbergii (Mr) and determine whether this tropomyosin is a major cross-reactive allergen for Thai children with shrimp allergy. Recombinant shrimp Mr tropomyosin (Mac r1.0101) was expressed in yeast Pichia pastoris. Secondary structure composition of purified recombinant Mac r1.0101 was determined by Circular Dichroism. IgE reactivity was examined by immunoblot, direct binding ELISA and inhibition of IgE ELISA using serum from shrimp-allergic children. The amino acid sequence of Mac r1.0101 showed 2 polymorphic amino acids (F44 and S45) indicating a variant of tropomyosin. Purified recombinant Mac r1.0101 obtained a nature-like α-helix structure which can be bound by serum-specific IgE. The binding affinity of serum-specific IgE to Mac r1.0101 based on the IC50 value was ~1.8 ng/ml. Ten of 13 shrimp-allergic Thai children had serum-specific IgE against Mac r1.0101, but at different levels. Results of the inhibition of specific IgE using Mac r1.0101 showed that 7 of the tested serum samples also had specific IgE against other shrimp allergens in addition to IgE against Mac r1.0101. Tropomyosin therefore appears to be a major cross-reactive allergen for Thai children who are allergic to both seawater and giant freshwater shrimp.

  4. Fagopyrins and Protofagopyrins: Detection, Analysis, and Potential Phototoxicity in Buckwheat.

    PubMed

    Benković, Eva Tavčar; Kreft, Samo

    2015-06-24

    Buckwheat contains many healthy nutrients, and its consumption is therefore increasing. Buckwheat also contains fluorescent phototoxic fagopyrins. A systematic review of fagopyrins and the phototoxicity of buckwheat found that reliable quantitative data on fagopyrin toxicity are not yet available. Generally, buckwheat seeds, flour, and teas are safe in normal amounts. Diets extensively composed of buckwheat sprouts, herbs, and particularly flowers or of fagopyrin-rich buckwheat extracts may cause fagopyrism. A reference standard is needed, as it would enable the accurate evaluation of fagopyrin content in buckwheat products and would allow proper testing of their as yet unknown physical, chemical, and biological characteristics.

  5. IgE and allergen-specific immunotherapy-induced IgG4 recognize similar epitopes of Bet v 1, the major allergen of birch pollen.

    PubMed

    Groh, N; von Loetzen, C S; Subbarayal, B; Möbs, C; Vogel, L; Hoffmann, A; Fötisch, K; Koutsouridou, A; Randow, S; Völker, E; Seutter von Loetzen, A; Rösch, P; Vieths, S; Pfützner, W; Bohle, B; Schiller, D

    2017-05-01

    Allergen-specific immunotherapy (AIT) with birch pollen generates Bet v 1-specific immunoglobulin (Ig)G4 which blocks IgE-mediated hypersensitivity mechanisms. Whether IgG4 specific for Bet v 1a competes with IgE for identical epitopes or whether novel epitope specificities of IgG4 antibodies are developed is under debate. We sought to analyze the epitope specificities of IgE and IgG4 antibodies from sera of patients who received AIT. 15 sera of patients (13/15 received AIT) with Bet v 1a-specific IgE and IgG4 were analyzed. The structural arrangements of recombinant (r)Bet v 1a and rBet v 1a_11x , modified in five potential epitopes, were analyzed by circular dichroism and nuclear magnetic resonance spectroscopy. IgE binding to Bet v 1 was assessed by ELISA and mediator release assays. Competitive binding of monoclonal antibodies specific for Bet v 1a and serum IgE/IgG4 to rBet v 1a and serum antibody binding to a non-allergenic Bet v 1-type model protein presenting an individual epitope for IgE was analyzed in ELISA and western blot. rBet v 1a_11x had a Bet v 1a - similar secondary and tertiary structure. Monomeric dispersion of rBet v 1a_11x was concentration and buffer-dependent. Up to 1500-fold increase in the EC50 for IgE-mediated mediator release induced by rBet v 1a_11x was determined. The reduction of IgE and IgG4 binding to rBet v 1a_11x was comparable in 67% (10/15) of sera. Bet v 1a-specific monoclonal antibodies inhibited binding of serum IgE and IgG4 to 66.1% and 64.9%, respectively. Serum IgE and IgG4 bound specifically to an individual epitope presented by our model protein in 33% (5/15) of sera. Patients receiving AIT develop Bet v 1a-specific IgG4 which competes with IgE for partly identical or largely overlapping epitopes. The similarities of epitopes for IgE and IgG4 might stimulate the development of epitope-specific diagnostics and therapeutics. © 2016 John Wiley & Sons Ltd.

  6. IgE-binding reactivity of peptide fragments of Bla g 1.02, a major German cockroach allergen.

    PubMed

    Yi, Myung-Hee; Jeong, Kyoung Yong; Kim, Chung-Ryul; Yong, Tai-Soon

    2009-01-01

    Cockroaches cause allergic diseases and are closely linked with the development of asthma. Bla g 1 is one of the major allergen proteins produced by German cockroaches. It consists of tandem repeats of approximately 100 amino acids. The aim of the present study was to identify linear IgE-binding epitopes of Bla g 1.02. RT-PCR was used to clone a cDNA sequence encoding Bla g 1.02 (EF202179) which shared 98.6-99.8% identity with a previously reported Bla g 1.02 (AF072220). To investigate IgE binding regions, five separate but overlapping Bla g 1.02 peptide fragments (A: aa 1-111, B: aa 102-215, C: aa 206-299, D: aa 289-403, E: aa 394-491) were amplified and cloned. The full-length and five peptide fragments were overexpressed in Pichia pastoris and E. coli, respectively, and their IgE binding reactivities were measured by ELISA using 37 serum samples isolated from cockroach-sensitized patients. The sera of 24 patients (64.9%) recognized the full-length Bla g 1.02 recombinant protein. Among 19 selected serum samples, 11 sera (57.9%) reacted to peptide fragment A, 5 sera (31.3%) to B, 4 sera (21.1%) to C, 9 sera (47.4%) to D, and 10 sera (52.6%) to peptide fragment E. IgE-binding epitopes are found to be distributed to each tandem repeat of Bla g 1. The combination of peptide fragments A, D, and E may able to detect all Bla g 1-sensitized subjects. We suggest that these peptide fragments may be useful in allergy diagnosis and the design of novel immunotherapeutics.

  7. Plant guide: Parsnipflower buckwheat: Eriogonum heracleoides Nutt

    Treesearch

    Derek Tilley; Dan Ogle; Loren St. John

    2007-01-01

    Parsnipflower buckwheat is a perennial forb to subshrub with a branching woody stem. Leaves are covered with dense white hairs making the herbage appear a light green to blue-grayish color. The flowers are a creamy-yellow color and have six petals which are borne in simple or compound umbels. Plants of parsnipflower buckwheat can be distinguished from other closely...

  8. Assessment of the Olea pollen and its major allergen Ole e 1 concentrations in the bioearosol of two biogeographical areas

    NASA Astrophysics Data System (ADS)

    Moreno-Grau, S.; Aira, M. J.; Elvira-Rendueles, B.; Fernández-González, M.; Fernández-González, D.; García-Sánchez, A.; Martínez-García, M. J.; Moreno, J. M.; Negral, L.; Vara, A.; Rodríguez-Rajo, F. J.

    2016-11-01

    The Olea pollen is currently an important allergy source. In some regions of Southern Spain, olive pollen is the main cause of allergic sensitization exceeding 40% of the sensitized individuals. Due to the scarce presence of olive trees in Northern Spain, limited to some cultivated fields in the South of the Galicia region where they also grow wild, only 8% of the sensitized individuals showed positive results for Olea pollen. The aim of the paper was to assess the behaviour pattern of the Olea pollen and its aeroallergens in the atmosphere, as this information could help us to improve the understanding and prevention of clinical symptoms. Airborne Olea pollen and Ole e 1 allergens were quantified in Cartagena (South-eastern Spain) and Ourense (North-western Spain). A volumetric pollen trap and a Burkard Cyclone sampler were used for pollen and allergen quantification. The Olea flowering took place in April or May in both biometeorological sampling areas. The higher concentrations were registered in the Southern area of Spain, for both pollen and Ole e 1, with values 8 times higher for pollen concentrations and 40 times higher for allergens. An alternate bearing pattern could be observed, characterized by years with high pollen values and low allergen concentrations and vice versa. Moreover, during some flowering seasons the allergen concentrations did not correspond to the atmospheric pollen values. Variations in weather conditions or Long Distance Transport (LDT) processes could explain the discordance. The back trajectory analysis shows that the most important contributions of pollen and allergens in the atmosphere are coincident with air masses passing through potential source areas. The exposure to olive pollen may not be synonym of antigen exposure.

  9. A mimotope gene encoding the major IgE epitope of allergen Phl p 5 for epitope-specific immunization.

    PubMed

    Wallmann, J; Proell, M; Stepanoska, T; Hantusch, B; Pali-Schöll, I; Thalhamer, T; Thalhamer, J; Jensen-Jarolim, E; Hartl, A

    2009-01-29

    A gene vaccine based on a mammalian expression vector containing the sequence of a peptide mimotope of Phl p 5 was constructed. To test whether mimotope gene vaccines can induce allergen-specific antibody responses via molecular mimicry, BALB/c mice were immunized using the mimotope construct with or without a tetanus toxin T-helper epitope. Moreover, intradermal injection was compared to epidermal application via gene gun immunization. Immunization with both mimotope gene constructs elicited allergen-specific antibody responses. As expected, gene gun bombardment induced a Th2-biased immune response, typically associated with IgG1 and IgE antibody production. In contrast, intradermal injection of the vaccine triggered IgG2a antibody expression without any detectable IgE levels, thus biasing the immune response towards Th1. In an RBL assay, mimotope-specific IgG antibodies were able to prevent cross-linking of allergen-specific IgE by Phl p 5. A construct coding for the complete Phl p 5 induced T-cell activation, IFN-gamma and IL-4 production. In contrast, the mimotope-DNA construct being devoid of allergen-specific T-cell epitopes had no capacity to activate allergen-specific T cells. Taken together, our data show that it is feasible to induce blocking IgG antibodies with a mimotope-DNA construct when applied intradermally. Thus the mimotope-DNA strategy has two advantages: (1) the avoidance of IgE induction and (2) the avoidance of triggering allergen-specific T-lymphocytes. We therefore suggest that mimotope gene vaccines are potential candidates for epitope-specific immunotherapy of type I allergy.

  10. Impact of heat processing on the detection of the major shellfish allergen tropomyosin in crustaceans and molluscs using specific monoclonal antibodies.

    PubMed

    Kamath, Sandip D; Abdel Rahman, Anas M; Komoda, Toshikazu; Lopata, Andreas L

    2013-12-15

    The major heat-stable shellfish allergen, tropomyosin, demonstrates immunological cross-reactivity, making specific differentiation of crustaceans and molluscs for food labelling very difficult. The aim of this study was to evaluate the application of allergen-specific monoclonal antibodies in differential detection of shellfish-derived tropomyosin in 11 crustacean and 7 mollusc species, and to study the impact of heating on its detection. Cross-reactive tropomyosin was detected in all crustacean species, with partial detection in molluscs: mussels, scallops and snails but none in oyster, octopus and squid. Furthermore, we have demonstrated that heating of shellfish has a profound effect on tropomyosin detection. This was evident by the enhanced recognition of multiple tropomyosin variants in the analysed shellfish species. Specific monoclonal antibodies, targetting the N-terminal region of tropomyosin, must therefore be developed to differentiate tropomyosins in crustaceans and molluscs. This can help in correct food labelling practices and thus protection of consumers.

  11. Molecular cloning, expression and IgE-immunoreactivity of phospholipase A1, a major allergen from Polybia paulista (Hymenoptera: Vespidae) venom.

    PubMed

    Perez-Riverol, Amilcar; Campos Pereira, Franco Dani; Musacchio Lasa, Alexis; Romani Fernandes, Luis Gustavo; Santos-Pinto, José Roberto Aparecido Dos; Justo-Jacomini, Débora Lais; Oliveira de Azevedo, Gabriel; Bazon, Murilo Luiz; Palma, Mario Sergio; Zollner, Ricardo de Lima; Brochetto-Braga, Márcia Regina

    2016-12-15

    Polybia paulista (Hymenoptera: Vespidae) is a clinically relevant social wasp that frequently causes stinging accidents in southeast Brazil. To date, diagnosis and specific immunotherapy (SIT) of allergy are based on the use of crude venom extracts. Production of recombinant forms of major allergens from P. paulista venom will improve diagnosis and SIT of allergic patients by reducing the incidence of cross-reactivity and non-specific sensitization. Here, we describe the molecular cloning, heterologous expression, purification and IgE-mediated immunodetection of phospholipase A1 (Poly p 1), a major allergen from P. paulista venom. The cDNA of Poly p 1 was extracted from venom glands and then cloned, and further expression of the recombinant allergen (rPoly p 1) was achieved in Escherichia coli BL21 (DE3) cells. Purification of rPoly p 1 was performed using immobilized Ni(2+) metal affinity chromatography. Also, a single-step chromatographic method allowed the purification of native Poly p 1 (nPoly p 1) from the wasp's venom glands. We used western blotting to evaluate IgE-reactivity of the sera from 10 P. paulista venom-allergic patients to rPoly p 1 and nPoly p 1. High levels of insoluble rPoly p 1 were obtained during heterologous expression. After solubilization of inclusion bodies and purification of the recombinant protein, a unique band of ∼34 kDa was detected in SDS-PAGE analysis. Allergen-specific IgE (sIgE) from allergic patients' sera recognized rPoly p 1, nPoly p 1 and crude venom extract to a similar extent. Our results showed that rPoly p 1 could be used for development of component-resolved diagnosis (CRD) and molecular-defined SIT of P. paulista venom allergy.

  12. Major allergen from Amaranthus palmeri pollen is a profilin: Isolation, partial characterisation and IgE recognition.

    PubMed

    Landa-Pineda, C M; Arroyo-Becerra, A; Rosas-Alvarado, A; Terán, L M; Garcia-Cruz, M L; Marchat, L A; Reyes-López, C A

    2016-01-01

    Pollens represent a rich source of proteins that are also potential elicitors of IgE-mediated pollen allergy. Sensitisation to panallergens could play an important role in diagnosis and specific immunotherapy, because these molecules are present in different plant pollens and plant foods and have marked structural similarity in different species. Profilins are one of the most common panallergens to be studied because they are responsible for a large number of sensitisations and are clearly related to cross-reactivity and co-sensitisation. This study aimed to isolate and characterise a new allergen of Amaranthus palmeri pollen and to determine its allergenicity. A. palmeri pollen profilin was purified using poly-l-proline-Sepharose affinity chromatography followed by anion exchanger chromatography. Identification of purified protein was carried out by mass spectrometry. Specific IgE was estimated in sera of patients with positive skin prick test to A. palmeri pollen extract, by enzyme-linked immunosorbent assay (ELISA). Purified protein appeared as a single band at 14 kDa in SDS-PAGE gel. Mass spectrometric analysis of the gel band identified two highly conserved peptides corresponding to allergenic profilins from pollen of other plants. Sera from about 60% of allergic patients have IgE that recognises the purified A. palmeri protein. A 14 kDa protein of A. palmeri pollen was purified and identified as allergenic profilin, which was recognised by sera from pollen allergic patients. Copyright © 2015 SEICAP. Published by Elsevier Espana. All rights reserved.

  13. Emerging pollen allergens.

    PubMed

    Rodríguez, Rosalía; Villalba, Mayte; Batanero, Eva; Palomares, Oscar; Salamanca, Guillermo

    2007-01-01

    Numerous pollen allergens have been reported over the last few years. Most of them belong to well-known families of proteins but some others constitute the first member of new allergenic families. Some of the factors that can contribute to the detection and identification of new pollen allergens are: a) advances in the technology tools for molecular analysis; and b) the deep knowledge of many allergenic sources. The combination of these factors has provided vast information on the olive pollen allergogram and the identification of minor allergens that become major ones for a significant population. The close taxonomical relationship between olive tree and ash -both Oleaceae- has permitted to identify Fra e 1 (the Ole e 1-like allergen) in ash pollen and to detect the presence of protein homologues of Ole e 3 and Ole e 6. In the other hand, extensive areas of south Europe are suffering an increasing desertification. As a consequence of this, new botanical species are spontaneously growing in these areas or being used in greening ground programs: Chenopodium album and Salsola kali are some examples recently recognized as allergenic woods. The identification of the complete panel of allergens from the hypersensitizing sources might help to develop more accurate diagnosis, and efficient and safer therapy tools for Type-I allergic diseases.

  14. [New aero-allergens].

    PubMed

    De Blay, F; Bessot, J C; Pauli, G

    1996-01-01

    As the number of proteins recognized as causing allergic respiratory diseases increases, new aero allergens have appeared in the animal and vegetable realms, both in home and professional environments. Lepidoglyphus destructor and Blomia tropicalis, two mites found in storage areas, are particularly important in agricultural areas and in homes. Over the last ten years, the frequency of reactions to cockroaches has also increased in several countries. The allergenicity of non-biting insects is a frequent cause of allergy in certain countries including Japan. Chironomides cause respiratory diseases in professional and outdoor environments. The important role of Alternaria, a mold, in producing severe asthma has also been demonstrated. The pathophysiology of pollen-induced asthma has been shown to result from pollen allergens carried by particles less than 5 microns in diameter. Cyprus and ash tree pollen also cause an increasing number of pollinoses and flowers can cause rhinitis and asthma. Respiratory allergy to Ficus benjamina inaugurated a new type of allergies caused airborne allergens from non-pollinating plants. Allergy to latex raises a particular problem for health care workers. The immunochemical structures of the major and minor airborne allergens are now better known and the homologous structures of different allergens largely explains certain cross-reactions. In the future, recombinant allergens will probably be used to better understand the role of allergens in inducing and maintaining the allergic reaction and should help in our approach to diagnosis and therapy.

  15. Cross-reactivity between major IgE core epitopes on Cry j 2 allergen of Japanese cedar pollen and relevant sequences on Cha o 2 allergen of Japanese cypress pollen.

    PubMed

    Miyaji, Kazuki; Okamoto, Noriaki; Saito, Akemi; Yasueda, Hiroshi; Takase, Yukari; Shimakura, Hidekatsu; Saito, Saburo; Sakaguchi, Masahiro

    2016-07-01

    Cry j 2 and Cha o 2 are major allergens in Japanese cedar (Cryptomeria japonica; CJ) and Japanese cypress (Chamaecyparis obtusa; CO) pollen, respectively. Here, we assessed the epitopes related to the cross-reactivity between Cry j 2 and Cha o 2 using in vitro analyses. Peptides were synthesized based on Cry j 2 sequential epitopes and relevant Cha o 2 amino acid sequences. Four representative monoclonal antibodies (mAbs) against Cry j 2 were used according to their epitope recognitions. Serum samples were collected from 31 patients with CJ pollinosis. To investigate cross-reactivity between Cry j 2 and Cha o 2, ELISA and inhibition ELISA were performed with mAbs and sera from patients with CJ pollinosis. Two of four mAbs had reactivity to both Cry j 2 and Cha o 2. Of these two mAbs, one mAb (T27) recognized the amino acid sequence (169)KVVNGRTV(176) on Cha o 2. This is related to the core epitope (169)KWVNGREI(176) on Cry j 2, which is an important IgE epitope. In addition, we found that these correlative sequences and purified allergens showed cross-reactivity between Cry j 2 and Cha o 2 in IgE of CJ patients. We demonstrated the importance of (169)KVVNGRTV(176) in Cha o 2 for cross-reactivity with the Cry j 2 epitope (169)KWVNGREI(176), which plays an important role in allergenicity in CJ pollinosis. Our results are useful for the development of safer and more efficient therapeutic strategies for the treatment of CJ and CO pollen allergies. Copyright © 2016 Japanese Society of Allergology. Production and hosting by Elsevier B.V. All rights reserved.

  16. The Major Birch Pollen Allergen Bet v 1 Induces Different Responses in Dendritic Cells of Birch Pollen Allergic and Healthy Individuals

    PubMed Central

    Smole, Ursula; Radauer, Christian; Lengger, Nina; Svoboda, Martin; Rigby, Neil; Bublin, Merima; Gaier, Sonja; Hoffmann-Sommergruber, Karin; Jensen-Jarolim, Erika; Mechtcheriakova, Diana; Breiteneder, Heimo

    2015-01-01

    Dendritic cells play a fundamental role in shaping the immune response to allergens. The events that lead to allergic sensitization or tolerance induction during the interaction of the major birch pollen allergen Bet v 1 and dendritic cells are not very well studied. Here, we analyzed the uptake of Bet v 1 and the cross-reactive celery allergen Api g 1 by immature monocyte-derived dendritic cells (iMoDCs) of allergic and normal donors. In addition, we characterized the allergen-triggered intracellular signaling and transcriptional events. Uptake kinetics, competitive binding, and internalization pathways of labeled allergens by iMoDCs were visualized by live-cell imaging. Surface-bound IgE was detected by immunofluorescence microscopy and flow cytometry. Allergen- and IgE-induced gene expression of early growth response genes and Th1 and Th2 related cytokines and chemokines were analyzed by real-time PCR. Phosporylation of signaling kinases was analyzed by Western blot. Internalization of Bet v 1 by iMoDCs of both donor groups, likely by receptor-mediated caveolar endocytosis, followed similar kinetics. Bet v 1 outcompeted Api g 1 in cell surface binding and uptake. MoDCs of allergic and healthy donors displayed surface-bound IgE and showed a pronounced upregulation of Th2 cytokine- and NFκB-dependent genes upon non-specific Fcε receptor cross-linking. In contrast to these IgE-mediated responses, Bet v 1-stimulation increased transcript levels of the Th2 cytokines IL-4 and IL-13 but not of NFκB-related genes in MoDCs of BP allergic donors. Cells of healthy donors were either unresponsive or showed elevated mRNA levels of Th1-promoting chemokines. Moreover, Bet v 1 was able to induce Erk1/2 and p38 MAPK activation in BP allergics but only a slight p38 activation in normal donors. In conclusion, our data indicate that Bet v 1 favors the activation of a Th2 program only in DCs of BP allergic individuals. PMID:25635684

  17. Other Allergens

    MedlinePlus

    ... Basics Facts and Statistics NIAID Resources Allergens Peanut Tree Nuts Milk Egg Wheat Soy Fish Shellfish Sesame ... Basics Facts and Statistics NIAID Resources Allergens Peanut Tree Nuts Milk Egg Wheat Soy Fish Shellfish Sesame ...

  18. Expression and purification of a major allergen, Pla a 1, from Platanus acerifolia pollen and the preparation of its monoclonal antibody.

    PubMed

    Ni, Wei-Wei; Huang, Wen; Wu, De-Qin; Zhou, Yan-Jun; Ji, Chun-Mei; Cao, Meng-Da; Guo, Miao; Sun, Jin-Lu; Wei, Ji-Fu

    2017-09-01

    Platanus acerifolia pollen is considered an important source of airborne allergens in numerous cities. Pla a 1 is a major allergen from P. acerifolia pollen. The present study aimed to express and purify Pla a 1, and to prepare its monoclonal antibody. In the present study, the Pla a 1 gene was subcloned into a pET‑28a vector and transformed into the ArcticExpress™ (DE3) RP Escherichia coli host strain. The purified Pla a 1 was then used to immunize BALB/c mice. When serum detection was positive, spleen cells were isolated from the mice and fused with SP2/0 myeloma cells at a ratio of 10:1. Hybridoma cells were screened by indirect ELISA and limiting dilution. Positive cells were used to induce the formation of antibody‑containing ascites fluid, and the antibodies were purified using protein A‑agarose. The results of the present study demonstrated that recombinant Pla a 1 was successfully expressed and purified, and exhibited positive immunoglobulin E‑binding to serum from patients allergic to P. acerifolia. A total of 11 hybridomas that steadily secreted anti‑Pla a 1 antibody were obtained and an immunoblotting analysis indicated that all of these monoclonal antibodies specifically recognized the Pla a 1 protein. These results suggested that specific anti‑Pla a 1 antibodies may be obtained, which can be used for the rapid detection of Pla a 1 allergens and in the preparation of vaccines against P. acerifolia pollen.

  19. Characterization of the protective and therapeutic efficiency of a DNA vaccine encoding the major birch pollen allergen Bet v 1a.

    PubMed

    Hartl, A; Hochreiter, R; Stepanoska, T; Ferreira, F; Thalhamer, J

    2004-01-01

    An estimated 100 million individuals suffer from birch pollen allergy. More than 95% of birch pollen-allergic subjects react with the major birch pollen allergen Bet v 1a, and almost 60% of them are sensitized exclusively to this allergen. DNA immunization using the Bet v 1a gene was evaluated with respect to its prophylactic and therapeutic efficacy. A DNA vaccine containing the entire Bet v 1a cDNA under the control of a CMV-promoter was constructed. In order to estimate the protective efficiency, animals received three injections of this vaccine prior to sensitization with recombinant Bet v 1a. Vice versa, in a therapeutic approach, sensitization was followed by treatment with the DNA vaccine. The Bet v 1a DNA vaccine induced strong Bet v 1-specific antibody responses with a Th1-biased response type. Animals which received the DNA vaccine were protected against a following allergic sensitization with Bet v 1a. The protective effect was characterized by suppression of Bet v 1-specific immunoglobulin (Ig)E production, lack of basophil activation and enhanced interferon (IFN)-gamma expression. In a therapeutic situation, treatment of sensitized animals with DNA vaccines decreased IgE production, IgE-mediated basophil release and drastically reduced anaphylactic activity as measured by passive cutaneous anaphylaxis assays. Concerning the cellular immune response, DNA immunization induced a sustaining and dominant shift from a Th2 type response towards a balanced Th1/Th2 type response as indicated by increased IFN-gamma but unchanged IL-5 levels in lymphoproliferation assays. The results demonstrate the allergen-specific protective and therapeutic efficacy of a DNA vaccine encoding the clinically highly relevant allergen Bet v 1a indicating the suitability of this concept for the treatment of allergic diseases.

  20. Peanut oral immunotherapy modifies IgE and IgG4 responses to major peanut allergens

    PubMed Central

    Vickery, Brian P.; Lin, Jing; Kulis, Michael; Fu, Zhiyan; Steele, Pamela H.; Jones, Stacie M.; Scurlock, Amy M.; Gimenez, Gustavo; Bardina, Ludmilla; Sampson, Hugh A.; Burks, A. Wesley

    2012-01-01

    Background Peanut-allergic subjects have highly stable pathologic antibody repertoires to the immunodominant B cell epitopes of the major peanut allergens Ara h 1-3. Objective We used a peptide microarray technique to analyze the effect of treatment with peanut oral immunotherapy (OIT) on such repertoires. Methods Measurements of total peanut-specific IgE (psIgE) and psIgG4 were made with CAP-FEIA. We analyzed sera from 22 OIT subjects and 6 controls and measured serum specific IgE and IgG4 binding to epitopes of Ara h 1-3 using a high-throughput peptide microarray technique. Antibody affinity was measured using a competitive peptide microarray as previously described. Results At baseline, psIgE and psIgG4 diversity were similar between subjects and controls, and there was broad variation in epitope recognition. After a median 41 months of OIT, polyclonal psIgG4 increased from a median 0.3 mcg/mL (IQR 0.1-0.43) at baseline to 10.5 mcg/mL (3.95-45.48) (p<0.0001) and included de novo specificities. PsIgE was reduced from a median baseline of 85.45 kUA/L (23.05-101.0) to 7.75 kUA/L (2.58-30.55) (p<0.0001). Affinity was unaffected. Although the psIgE repertoire contracted in most OIT-treated subjects, several subjects generated new IgE specificities even as the total psIgE decreased. Global epitope-specific shifts from IgE to IgG4 binding occurred, including at an informative epitope of Ara h 2. Conclusion OIT differentially alters Ara h 1-3 binding patterns. These changes are variable between subjects, not observed in controls, and include a progressive polyclonal increase in IgG4, with concurrent reduction in IgE amount and diversity. PMID:23199605

  1. Intranasal Coadministration of Live Lactococci Producing Interleukin-12 and a Major Cow's Milk Allergen Inhibits Allergic Reaction in Mice▿

    PubMed Central

    Cortes-Perez, Naima G.; Ah-Leung, Sandrine; Bermúdez-Humarán, Luis G.; Corthier, Gérard; Wal, Jean-Michel; Langella, Philippe; Adel-Patient, Karine

    2007-01-01

    The Th1/Th2 balance deregulation toward a Th2 immune response plays a central role in allergy. We previously demonstrated that administration of recombinant Lactococcus lactis strains expressing bovine β-lactoglobulin (BLG), a major cow's milk allergen, partially prevents mice from sensitization. In the present study, we aimed to improve this preventive effect by coadministration of L. lactis BLG and a second recombinant L. lactis strain producing biologically active interleukin-12 (IL-12). This L. lactis strain producing IL-12 was previously used to enhance the Th1 immune response in a tumoral murine model (L. G. Bermúdez-Humarán et al., J. Immunol. 175:7297-7302, 2005). A comparison of the administration of either BLG alone or BLG in the presence of IL-12 was conducted. A BLG-specific primary Th1 immune response was observed only after intranasal coadministration of both L. lactis BLG and IL-12-producing L. lactis, as demonstrated by the induction of serum-specific immunoglobulin G2a (IgG2a) concomitant with gamma interferon secretion by splenocytes, confirming the adjuvanticity of IL-12-producing L. lactis. Immunized mice were further sensitized by intraperitoneal administration of purified BLG, and the allergic reaction was elicited by intranasal challenge with purified BLG. Mice pretreated with BLG in either the presence or the absence of IL-12 were rendered completely tolerant to further allergic sensitization and elicitation. Pretreatment with either L. lactis BLG or L. lactis BLG and IL-12-producing L. lactis induces specific anti-BLG IgG2a production in serum and bronchoalveolar lavage (BAL) fluid. Although specific serum IgE was not affected by these pretreatments, the levels of eosinophilia and IL-5 secretion in BAL fluid were significantly reduced after BLG challenge in the groups pretreated with L. lactis BLG and L. lactis BLG-IL-12-producing L. lactis, demonstrating a decreased allergic reaction. Our data demonstrate for the first time (i) the

  2. Intranasal coadministration of live lactococci producing interleukin-12 and a major cow's milk allergen inhibits allergic reaction in mice.

    PubMed

    Cortes-Perez, Naima G; Ah-Leung, Sandrine; Bermúdez-Humarán, Luis G; Corthier, Gérard; Wal, Jean-Michel; Langella, Philippe; Adel-Patient, Karine

    2007-03-01

    The Th1/Th2 balance deregulation toward a Th2 immune response plays a central role in allergy. We previously demonstrated that administration of recombinant Lactococcus lactis strains expressing bovine beta-lactoglobulin (BLG), a major cow's milk allergen, partially prevents mice from sensitization. In the present study, we aimed to improve this preventive effect by coadministration of L. lactis BLG and a second recombinant L. lactis strain producing biologically active interleukin-12 (IL-12). This L. lactis strain producing IL-12 was previously used to enhance the Th1 immune response in a tumoral murine model (L. G. Bermúdez-Humarán et al., J. Immunol. 175:7297-7302, 2005). A comparison of the administration of either BLG alone or BLG in the presence of IL-12 was conducted. A BLG-specific primary Th1 immune response was observed only after intranasal coadministration of both L. lactis BLG and IL-12-producing L. lactis, as demonstrated by the induction of serum-specific immunoglobulin G2a (IgG2a) concomitant with gamma interferon secretion by splenocytes, confirming the adjuvanticity of IL-12-producing L. lactis. Immunized mice were further sensitized by intraperitoneal administration of purified BLG, and the allergic reaction was elicited by intranasal challenge with purified BLG. Mice pretreated with BLG in either the presence or the absence of IL-12 were rendered completely tolerant to further allergic sensitization and elicitation. Pretreatment with either L. lactis BLG or L. lactis BLG and IL-12-producing L. lactis induces specific anti-BLG IgG2a production in serum and bronchoalveolar lavage (BAL) fluid. Although specific serum IgE was not affected by these pretreatments, the levels of eosinophilia and IL-5 secretion in BAL fluid were significantly reduced after BLG challenge in the groups pretreated with L. lactis BLG and L. lactis BLG-IL-12-producing L. lactis, demonstrating a decreased allergic reaction. Our data demonstrate for the first time (i) the

  3. Assessment of the Allergenic Potential of Transgenic Wheat (Triticum aestivum) with Reduced Levels of ω5-Gliadins, the Major Sensitizing Allergen in Wheat-Dependent Exercise-Induced Anaphylaxis.

    PubMed

    Altenbach, Susan B; Tanaka, Charlene K; Pineau, Florence; Lupi, Roberta; Drouet, Martine; Beaudouin, Etienne; Morisset, Martine; Denery-Papini, Sandra

    2015-10-28

    The ω5-gliadins are the major sensitizing allergens in wheat-dependent exercise-induced anaphylaxis (WDEIA). In this study, two-dimensional immunoblot analysis was used to assess the allergenic potential of two transgenic wheat lines in which ω5-gliadin genes were silenced by RNA interference. Sera from 7 of 11 WDEIA patients showed greatly reduced levels of immunoglobulin E (IgE) reactivity to ω5-gliadins in both transgenic lines. However, these sera also showed low levels of reactivity to other gluten proteins. Sera from three patients showed the greatest reactivity to proteins other than ω5-gliadins, either high-molecular-weight glutenin subunits (HMW-GSs), α-gliadins, or non-gluten proteins. The complexity of immunological responses among these patients suggests that flour from the transgenic lines would not be suitable for individuals already diagnosed with WDEIA. However, the introduction of wheat lacking ω5-gliadins could reduce the number of people sensitized to these proteins and thereby decrease the overall incidence of this serious food allergy.

  4. Recombinant allergens

    PubMed Central

    Jutel, Marek; Solarewicz-Madejek, Katarzyna; Smolinska, Sylwia

    2012-01-01

    Allergen specific immunotherapy (SIT) is the only known causative treatment of allergic diseases. Recombinant allergen-based vaccination strategies arose from a strong need to both to improve safety and enhance efficacy of SIT. In addition, new vaccines can be effective in allergies including food allergy or atopic dermatitis, which poorly respond to the current treatment with allergen extracts. A number of successful clinical studies with both wild-type and hypoallergenic derivatives of recombinant allergens vaccines have been reported for the last decade. They showed high efficacy and safety profile as well as very strong modulation of T and B cell responses to specific allergens. PMID:23095874

  5. Assignment of the disulfide bonds of Ole e 1, a major allergen of olive tree pollen involved in fertilization.

    PubMed

    González, E; Monsalve, R I; Puente, X S; Villalba, M; Rodríguez, R

    2000-01-01

    The most prevalent allergen from olive tree pollen, Ole e 1, consists of a single polymorphic polypeptide chain of 145 amino acids which includes six cysteine residues at positions 19, 22, 43, 78, 90 and 131. By using an homogeneous form of the allergen expressed in Pichia pastoris, the array of the disulfide bridges has been elucidated. Specific proteolysis with thermolysin and reverse-phase HPLC separation of the peptides allowed the determination of the disulfide bond between Cys43 and Cys78. Another thermolytic product, which contained three peptides linked by the remaining four cysteines, was digested with Glu-specific staphylococcal V8 protease and the products isolated by reverse-phase HPLC. Amino acid compositions and Edman degradation of the peptide products indicated the presence of the disulfide bonds at Cys19-Cys90 and Cys22-Cys131. These data can help in the analysis of the three-dimensional structure of the protein as well as in studies of its allergenic determinants.

  6. Structural stability of Amandin, a major allergen from almond (Prunus dulcis), and its acidic and basic polypeptides.

    PubMed

    Albillos, Silvia M; Menhart, Nicholas; Fu, Tong-Jen

    2009-06-10

    Information relating to the resistance of food allergens to thermal and/or chemical denaturation is critical if a reduction in protein allergenicity is to be achieved through food-processing means. This study examined the changes in the secondary structure of an almond allergen, amandin, and its acidic and basic polypeptides as a result of thermal and chemical denaturation. Amandin ( approximately 370 kDa) was purified by cryoprecipitation followed by gel filtration chromatography and subjected to thermal (13-96 degrees C) and chemical (urea and dithiothreitol) treatments. Changes in the secondary structure of the protein were followed using circular dichroism spectroscopy. The secondary structure of the hexameric amandin did not undergo remarkable changes at temperatures up to 90 degrees C, although protein aggregation was observed. In the presence of a reducing agent, irreversible denaturation occurred with the following experimental values: T(m) = 72.53 degrees C (transition temperature), DeltaH = 87.40 kcal/mol (unfolding enthalpy), and C(p) = 2.48 kcal/(mol degrees C) (heat capacity). The concentration of urea needed to achieve 50% denaturation was 2.59 M, and the Gibbs free energy of chemical denaturation was calculated to be DeltaG = 3.82 kcal/mol. The basic and acidic polypeptides of amandin had lower thermal stabilities than the multimeric protein.

  7. Solution structure of the C-terminal domain of Ole e 9, a major allergen of olive pollen

    PubMed Central

    Treviño, Miguel Á.; Palomares, Oscar; Castrillo, Inés; Villalba, Mayte; Rodríguez, Rosalía; Rico, Manuel; Santoro, Jorge; Bruix, Marta

    2008-01-01

    Ole e 9 is an olive pollen allergen belonging to group 2 of pathogenesis-related proteins. The protein is composed of two immunological independent domains: an N-terminal domain (NtD) with 1,3-β-glucanase activity, and a C-terminal domain (CtD) that binds 1,3-β-glucans. We have determined the three-dimensional structure of CtD-Ole e 9 (101 amino acids), which consists of two parallel α-helices forming an angle of ∼55°, a small antiparallel β-sheet with two short strands, and a 3–10 helix turn, all connected by long coil segments, resembling a novel type of folding among allergens. Two regions surrounded by aromatic residues (F49, Y60, F96, Y91 and Y31, H68, Y65, F78) have been localized on the protein surface, and a role for sugar binding is suggested. The epitope mapping of CtD-Ole e 9 shows that B-cell epitopes are mainly located on loops, although some of them are contained in secondary structural elements. Interestingly, the IgG and IgE epitopes are contiguous or overlapped, rather than coincident. The three-dimensional structure of CtD-Ole e 9 might help to understand the underlying mechanism of its biochemical function and to determine possible structure–allergenicity relationships. PMID:18096638

  8. IgE-mediated allergy to corn: a 50 kDa protein, belonging to the Reduced Soluble Proteins, is a major allergen.

    PubMed

    Pasini, G; Simonato, B; Curioni, A; Vincenzi, S; Cristaudo, A; Santucci, B; Peruffo, A Dal Belin; Giannattasio, M

    2002-02-01

    Although corn is often cited as an allergenic food, very few studies have been devoted to the identification of corn allergens and corn allergy has been rarely confirmed by double-blind, placebo-controlled food challenge (DBPCFC). Recently, Pastorello et al. (1) identified some salt-soluble IgE-binding proteins of corn flour as potential allergens. One of these, corresponding to corn Lipid Transfer Protein (LTP), appeared to be the major one. The aim of this study was to verify the clinical significance of the skin prick test (SPT) and CAP-FEIA CAP-System IgE fluozoenzyme immunosorbent assay (Pharmacia Diagnostic, Uppsala, Sweden) positivities to corn and to identify the presence of IgE-binding proteins in the corn flour salt-insoluble protein fractions (comprising up to 96% of the total protein) using sera of patients with DBPCFC-documented food allergy to corn. In addition the effect of cooking and proteolytic digestion on the corn allergens was investigated. Sixteen subjects with SPT and CAP-FEIA positivities to corn flour were examined. Only six of them complained of suffering from urticaria and/or other symptoms after ingestion of corn-based foods. The patients were food challenged with cooked corn flour (polenta). IgE-binding proteins were detected by immunoblotting. The digestibility of the IgE-binding proteins was examined during a pepsin attack followed by a pancreatin digestion performed on a cooked corn flour sample. Oral challenge was positive only for six patients with symptoms after ingestion of corn. A 50 kDa protein, belonging to the corn Reduced Soluble Protein (RSP) fraction was recognized by the serum IgE of all the DBPCFC-positive subjects and resulted to be resistant to both heating and peptic/pancreatic digestion. SPT with the purified RSP fraction gave positive results for all of the DBPCFC-positive patients examined. SPT and CAP-FEIA positivities to corn flour had no clinical significance for most of the patients and food allergy to corn has

  9. Retagging Identifies Dendritic Cell-specific Intercellular Adhesion Molecule-3 (ICAM3)-grabbing Non-integrin (DC-SIGN) Protein as a Novel Receptor for a Major Allergen from House Dust Mite*

    PubMed Central

    Emara, Mohamed; Royer, Pierre-Joseph; Mahdavi, Jafar; Shakib, Farouk; Ghaemmaghami, Amir M.

    2012-01-01

    Dendritic cells (DCs) have been shown to play a key role in the initiation and maintenance of immune responses to microbial pathogens as well as to allergens, but the exact mechanisms of their involvement in allergic responses and Th2 cell differentiation have remained elusive. Using retagging, we identified DC-SIGN as a novel receptor involved in the initial recognition and uptake of the major house dust mite and dog allergens Der p 1 and Can f 1, respectively. To confirm this, we used gene silencing to specifically inhibit DC-SIGN expression by DCs followed by allergen uptake studies. Binding and uptake of Der p 1 and Can f 1 allergens was assessed by ELISA and flow cytometry. Intriguingly, our data showed that silencing DC-SIGN on DCs promotes a Th2 phenotype in DC/T cell co-cultures. These findings should lead to better understanding of the molecular basis of allergen-induced Th2 cell polarization and in doing so paves the way for the rational design of novel intervention strategies by targeting allergen receptors on innate immune cells or their carbohydrate counterstructures on allergens. PMID:22205703

  10. Buckwheat allergy: a potential problem in 21st century Britain

    PubMed Central

    Sammut, David; Dennison, Patrick; Venter, Carina; Kurukulaaratchy, Ramesh J

    2011-01-01

    Buckwheat is commonly consumed in many parts of the world and has recently become more available in the UK. Buckwheat allergy is well recognised in parts of mainland Europe and Asia, typically associated with consumption of specific regional foods. No adult cases of buckwheat allergy in the UK have been reported in the literature. The authors present two cases of buckwheat allergy that presented to our UK allergy service recently. A 57-year-old man presented with anaphylaxis after eating home-baked bread prepared using buckwheat flour bought in France. In the second case, a 63-year-old lady presented with bronchospasm and urticaria after consuming health-food muesli. Sensitisation was confirmed in both cases by positive skin prick testing and specific IgE to buckwheat. Given the growing popularity of foods that may contain buckwheat, including ethnic and health-food ranges, buckwheat allergy is likely to become increasingly common in the UK. PMID:22674117

  11. Characterization of the 33-kilodalton major allergen of Penicillium citrinum by using MoAbs and N-terminal amino acid sequencing.

    PubMed

    Shen, H D; Lin, W L; Liaw, S F; Tam, M F; Han, S H

    1997-01-01

    The 33 kD component has been identified as a major allergen of Penicillium citrinum, the most prevalent Penicillium species in the Taipei area of Taiwan. This study analyses the isoforms, antigenic cross-reactivity and the N-terminal amino acid sequence of the 33 kD allergen of P. citrinum. The composition of isoforms and antigenic cross-reactivity was analysed by SDS-PAGE and 2D-immunoblotting using MoAbs generated. The N-terminal sequence was analysed by using an automatic gas/liquid phase sequencer. Two MoAbs (55A and 34H) against the 33 kD allergen were generated in the present study. In addition to the 33 kD component, MoAb 34H also showed immunoblot reactivity to other components in the crude extract of P. citrinum. Analysed by 2D-immunoblotting, at least six different isoforms of the 33 kD component with pI values ranging from 6.75 to greater than 7.0 were shown to be reactive to both MoAbs and IgE antibodies in serum of an asthmatic patient. Different immunoblot patterns were observed when both MoAbs were reacted with four different strains of P. citrinum used in the present study. Among another six different Penicillium and four different Aspergillus species tested, only an immunoblot reactivity of MoAb 55A to the 33 kD component of P. brevicompactum was observed. In 2D-immunoblotting, components of P. brevicompactum with an MW of about 33 kD and pI values similar to those of the 33 kD component of P. citrinum reacted with MoAb 55A and IgE antibodies in serum of the asthmatic patient. The N-terminal amino acid sequence of the 33 kD component of P. citrinum was determined to be ANVVQSNVP which was identical to the first 9 N-terminal amino acids of a heat-labile alkaline serine proteinase from P. citrinum. Results obtained in the present study suggest that the 33 kD major allergen of P. citrinum may be an alkaline serine proteinase.

  12. How far can we simplify in vitro diagnostics for grass pollen allergy?: A study with 17 whole pollen extracts and purified natural and recombinant major allergens.

    PubMed

    van Ree, R; van Leeuwen, W A; Aalberse, R C

    1998-08-01

    Current diagnostics for grass pollen allergy are composed of mixtures of pollen of different grass species. Their complex composition hampers accurate standardization. The aim of the study was to investigate whether mixtures of grass pollen extracts can be replaced by a single pollen species and whether a single pollen species can be replaced by a limited number of purified natural or recombinant major allergens. Sera (n = 800) were selected on the basis of a general suspicion for inhalant allergy and tested in a RAST for IgE reactivity with pollen from 17 different grass species. Cross-reactivity of IgE responses was studied by means of RAST inhibition. Sera with positive test results for grass pollen were tested in a RAST for natural Lol p 1 and Lol p 5 and recombinant Phl p 1 and Phl p 5. Specific IgE antibodies against one or more of the 17 pollen species were detected in 209 of 800 sera (26.1%). The highest responses were observed against Poa pratensis followed by Festuca rubra, Phleum pratense, and Dactylis glomerata. IgE responses were clearly lower (approximately by a factor of 5) against only three species (Phragmites communis, Cynodon dactylon, and Zea mays). With the exception of a few low-responder sera, no sera were found to have negative test results to the high responder species and positive results to any of the other species. Sera with positive test results for grass pollen (n = 154) were tested with purified Lol p 1 and Lol p 5. IgE anti-Lol p 1 and Lol p 5 accounted for an average of 81% +/- 7% of total anti-grass pollen IgE. For 14 sera (all with low anti-grass pollen IgE titers), a RAST with purified allergens resulted in a false-negative diagnosis for grass pollen allergy. With recombinant Phl p 1 and Phl p 5, the mean IgE reactivity was 57% +/- 6% of the anti-grass pollen IgE response (n = 141), with 13 false-negative results. One grass species is sufficient for in vitro diagnosis of grass pollen allergy. With purified natural Lol p 1 and Lol

  13. Localization of transcripts corresponding to the major allergen from olive pollen (Ole e I) by electron microscopic non-radioactive in situ RT-PCR.

    PubMed

    Alché, J D; Castro, A J; Rodríguez-García, M I

    2002-01-01

    In situ reverse transcription-PCR of mRNAs corresponding to the olive major allergen (Ole e I) has been tested at the ultrastructural level in mature olive pollen. The transcripts were present in the cytoplasm of both the vegetative and the generative cells, frequently associated to ribosomes in the endoplasmic reticulum. No labeling was detected in the pollen wall, nor in vacuoles, lipid bodies, plastids or mitochondria. Localization of the major olive allergen at ultrastructural level showed the protein present mainly in the lumen of the endoplasmic reticulum vesicles or pockets scattered in the cytoplasm, and in the outer region of the pollen exine. The results confirm the rough endoplasmic reticulum as the cell system involved in both the synthesis and storage of this protein. This is the first report of in situ RT-PCR on plant material at the ultrastructural level. The method described for mRNA amplification and detection is confirmed as a valuable tool for studying gene expression in plant material.

  14. Rabbit IgG directed to a synthetic C-terminal peptide of the major grass pollen allergen Lol p I inhibits human basophil histamine release induced by natural Lol p I.

    PubMed

    van Ree, R; Aalberse, R C

    1995-03-01

    The potential role of allergen-specific IgG antibodies as 'blocking' antibodies in allergen-induced human basophil histamine release was investigated. This was studied in a model with the major grass pollen allergen Lol p I and polyclonal rabbit antisera directed against this allergen and against a synthetic peptide of its C terminus. When allergen and antibodies were allowed to preincubate, Lol p I induced histamine release was inhibited up to 85% by the antiserum against Lol p I. By omitting preincubation, and thereby more closely mimicking an in vivo situation, up to 55% inhibition was realized. This indicates that allergen-specific IgG can act as 'blocking' antibody without preincubation. Immunization of rabbits with a synthetic C-terminal peptide of Lol p I resulted in antibodies reactive with natural Lol p I. Despite their 100-fold lower avidity for Lol p I (as compared with antinatural Lol p I), these antibodies had the capacity to inhibit Lol p I induced histamine release for > 90% (up to 50% without preincubation). This indicates that it is possible to block histamine release induced by a major allergen with low-avidity IgG antibodies directed against a minor proportion of the allergen (25 amino acids). IgE antibodies from the donors studied were unreactive with this synthetic peptide, indicating that for blocking activity identical epitope specificity of IgE and IgG is not essential. This opens interesting perspectives for application of synthetic peptides in immunotherapy, distinct from their effects on T cell reactivity.

  15. Dominating IgE-binding epitope of Bet v 1, the major allergen of birch pollen, characterized by X-ray crystallography and site-directed mutagenesis.

    PubMed

    Spangfort, Michael D; Mirza, Osman; Ipsen, Henrik; Van Neerven, R J Joost; Gajhede, Michael; Larsen, Jørgen N

    2003-09-15

    Specific allergy vaccination is an efficient treatment for allergic disease; however, the development of safer vaccines would enable a more general use of the treatment. Determination of molecular structures of allergens and allergen-Ab complexes facilitates epitope mapping and enables a rational approach to the engineering of allergen molecules with reduced IgE binding. In this study, we describe the identification and modification of a human IgE-binding epitope based on the crystal structure of Bet v 1 in complex with the BV16 Fab' fragment. The epitope occupies approximately 10% of the molecular surface area of Bet v 1 and is clearly conformational. A synthetic peptide representing a sequential motif in the epitope (11 of 16 residues) did not inhibit the binding of mAb BV16 to Bet v 1, illustrating limitations in the use of peptides for B cell epitope characterization. The single amino acid substitution, Glu(45)-Ser, was introduced in the epitope and completely abolished the binding of mAb BV16 to the Bet v 1 mutant within a concentration range 1000-fold higher than wild type. The mutant also showed up to 50% reduction in the binding of human polyclonal IgE, demonstrating that glutamic acid 45 is a critical amino acid also in a major human IgE-binding epitope. By solving the three-dimensional crystal structure of the Bet v 1 Glu(45)-Ser mutant, it was shown that the change in immunochemical activity is directly related to the Glu(45)-Ser substitution and not to long-range structural alterations or collapse of the Bet v 1 mutant tertiary structure.

  16. Navigating through the Jungle of Allergens: Features and Applications of Allergen Databases.

    PubMed

    Radauer, Christian

    2017-01-01

    The increasing number of available data on allergenic proteins demanded the establishment of structured, freely accessible allergen databases. In this review article, features and applications of 6 of the most widely used allergen databases are discussed. The WHO/IUIS Allergen Nomenclature Database is the official resource of allergen designations. Allergome is the most comprehensive collection of data on allergens and allergen sources. AllergenOnline is aimed at providing a peer-reviewed database of allergen sequences for prediction of allergenicity of proteins, such as those planned to be inserted into genetically modified crops. The Structural Database of Allergenic Proteins (SDAP) provides a database of allergen sequences, structures, and epitopes linked to bioinformatics tools for sequence analysis and comparison. The Immune Epitope Database (IEDB) is the largest repository of T-cell, B-cell, and major histocompatibility complex protein epitopes including epitopes of allergens. AllFam classifies allergens into families of evolutionarily related proteins using definitions from the Pfam protein family database. These databases contain mostly overlapping data, but also show differences in terms of their targeted users, the criteria for including allergens, data shown for each allergen, and the availability of bioinformatics tools. © 2017 S. Karger AG, Basel.

  17. Major grass pollen allergens and components detected in a southern Chinese cohort of patients with allergic rhinitis and/or asthma.

    PubMed

    Luo, Wenting; Huang, Huimin; Zheng, Peiyan; Wei, Nili; Luo, Jiaying; Sun, Baoqing; Zeng, Guangqiao

    2016-10-01

    There is so far a paucity of data about allergen component-resolved diagnosis, and the prevalence of grass pollen allergen components in China, in contrast to those from western countries. Even in this country, allergies to grass pollen allergen components in the vast south are inadequately described. This study aimed to determine the major sensitizing grass pollens in Guangzhou, the largest city in Guangdong province of southern China. Included in this study were 258 patients having allergic rhinitis with or without asthma and 88 healthy controls. ImmunoCap100 was used to examine the serum samples for sIgE to Bermuda, Timothy, and Humulus scandens. Subjects who tested positive were further examined for sIgE to Bermuda antigen Cyn d 1, Timothy antigens Phl p 1/4/5/6/7/11/12, and CCD. The relationship of grass pollen allergy to specific antigen sensitization was assessed. As a result, 22.5% of patients with allergic rhinitis and/or asthma were positive for Bermuda-sIgE, 13.6% for Timothy-sIgE, and 7.0% for Humulus scandens-sIgE. These patients were more likely to be sensitized compared with controls (P<0.001). Of the Bermuda-sIgE positive patients, 53.4% were Cyn d 1 positive and 60.3% were Timothy-sIgE positive. Of the Timothy positive patients, 100% were positive for Phl p 4, 17.1% were positive for Phl p 1 and 8.6% tested positive for Phl p 5/6/7/11/12. Patients with high Bermuda-sIgE levels were more likely to be positive for other grasses. In 41.4% of Bermuda grass positive patients, CCD-sIgE was also positive. Sensitization to Phl p 4 was significantly correlated with CCD (rs=0.928).In summary, we found that these southern Chinese patients with allergic rhinitis and/or asthma tested positive for Bermuda, Timothy, and Humulus scandens IgE. A high Bermuda-sIgE level may predict sensitization to other grasses. Correlations between sensitization to CCD and grass pollen allergens suggested a likelihood of cross-reactivity. Further in vitro inhibition assays are

  18. Mutants of the major ryegrass pollen allergen, Lol p 5, with reduced IgE-binding capacity: candidates for grass pollen-specific immunotherapy.

    PubMed

    Swoboda, Ines; De Weerd, Nicole; Bhalla, Prem L; Niederberger, Verena; Sperr, W R; Valent, Peter; Kahlert, Helga; Fiebig, Helmut; Verdino, Petra; Keller, Walter; Ebner, Christof; Spitzauer, Susanne; Valenta, Rudolf; Singh, Mohan B

    2002-01-01

    More than 400 million individuals are sensitized to grass pollen allergens. Group 5 allergens represent the most potent grass pollen allergens recognized by more than 80 % of grass pollen allergic patients. The aim of our study was to reduce the allergenic activity of group 5 allergens for specific immunotherapy of grass pollen allergy. Based on B- and T-cell epitope mapping studies and on sequence comparison of group 5 allergens from different grasses, point mutations were introduced by site-directed mutagenesis in highly conserved sequence domains of Lol p 5, the group 5 allergen from ryegrass. We obtained Lol p 5 mutants with low IgE-binding capacity and reduced allergenic activity as determined by basophil histamine release and by skin prick testing in allergic patients. Circular dichroism analysis showed that these mutants exhibited an overall structural fold similar to the recombinant Lol p 5 wild-type allergen. In addition, Lol p 5 mutants retained the ability to induce proliferation of group 5 allergen-specific T cell lines and clones. Our results demonstrate that a few point mutations in the Lol p 5 sequence yield mutants with reduced allergenic activity that represent potential vaccine candidates for immunotherapy of grass pollen allergy.

  19. The NH2-terminal amino acid sequence of the immunochemically partial identical major allergens of Alder (Alnus glutinosa) Aln g I, birch (Betula verrucosa) Bet v I, hornbeam (Carpinus betulus) Car b I and oak (Quercus alba) Que a I pollens.

    PubMed

    Ipsen, H; Hansen, O C

    1991-11-01

    The tree pollen major allergens (Aln g I), Bet v I, Car b I and Que a I, were purified to near homogeneity. Rabbit antibodies raised towards the major allergen Bet v I react with the major allergens of alder, hazel, hornbeam and oak, although with distinct variation in affinity. Immunochemically the major allergens seem to resemble one another more within than between the botanically established families of the fagale order. The physico-chemical parameters (molecular weight, pI values, amino acid composition and NH2-terminal amino acid sequence) of the major allergens exhibit a higher degree of consistency than do the immunochemical parameters, indicating that nuances in the allergens' secondary and tertiary structures are likely to govern the immunochemical differences.

  20. Hypoallergenic Variant of the Major Egg White Allergen Gal d 1 Produced by Disruption of Cysteine Bridges.

    PubMed

    Dhanapala, Pathum; Withanage-Dona, Dulashi; Tang, Mimi L K; Doran, Tim; Suphioglu, Cenk

    2017-02-21

    Gal d 1 (ovomucoid) is the dominant allergen in the chicken egg white. Hypoallergenic variants of this allergen can be used in immunotherapy as an egg allergy treatment approach. We hypothesised that disruption of two of the nine cysteine-cysteine bridges by site-directed mutagenesis will allow the production of a hypoallergenic variant of the protein; Methods: Two cysteine residues at C192 and C210 in domain III of the protein were mutated to alanine using site-directed mutagenesis, to disrupt two separate cysteine-cysteine bridges. The mutated and non-mutated proteins were expressed in Escherichia coli (E. coli) by induction with isopropyl β-d-1-thiogalactopyranoside (IPTG). The expressed proteins were analysed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting to confirm expression. Immunoglobulin E (IgE) reactivity of the two proteins was analysed, by immunoblotting, against a pool of egg-allergic patients' sera. A pool of non-allergic patients' sera was also used in a separate blot as a negative control; Results: Mutant Gal d 1 showed diminished IgE reactivity in the immunoblot by showing lighter bands when compared to the non-mutated version, although there was more of the mutant protein immobilised on the membrane when compared to the wild-type protein. The non-allergic negative control showed no bands, indicating an absence of non-specific binding of secondary antibody to the proteins; Conclusion: Disruption of two cysteine bridges in domain III of Gal d 1 reduces IgE reactivity. Following downstream laboratory and clinical testing, this mutant protein can be used in immunotherapy to induce tolerance to Gal d 1 and in egg allergy diagnosis.

  1. Involvement of the mannose receptor in the uptake of Der p 1, a major mite allergen, by human dendritic cells.

    PubMed

    Deslée, Gaëtan; Charbonnier, Anne-Sophie; Hammad, Hamida; Angyalosi, Gerhild; Tillie-Leblond, Isabelle; Mantovani, Alberto; Tonnel, André-Bernard; Pestel, Joël

    2002-11-01

    Immature dendritic cells (DCs) take up antigens in peripheral tissues and, after antigen processing, mature to efficiently stimulate T cells in secondary lymph nodes. In allergic airway diseases DCs have been shown to be involved in the induction and maintenance of a T(H)2-type profile. The present study was undertaken to determine pathways of Der p 1 (a house dust mite allergen) uptake by human DCs and to compare Der p 1 uptake between DCs from patients with house dust mite allergy and DCs from healthy donors. Monocyte-derived DCs (MD-DCs) were obtained from patients with house dust mite allergy (n = 13) and healthy donors (n = 11). Der p 1 was labeled with rhodamine. Der p 1 uptake by MD-DCs was analyzed by means of flow cytometry and confocal microscopy. Rhodamine- labeled Der p 1 was demonstrated to be taken up by MD-DCs in a dose-, time-, and temperature- dependent manner. The involvement of the mannose receptor (MR) in the Der p 1 uptake was demonstrated by using (1) inhibitors of the MR- mediated endocytosis (mannan and blocking anti-MR mAb), which inhibited the Der p 1 uptake from 40 % to 50 %, and (2) confocal microscopy showing the colocalization of rhodamine-labeled Der p 1 with FITC-dextran. Interestingly, compared with DCs from healthy donors, DCs from allergic patients expressed more MR and were more efficient in Der p 1 uptake. These results suggest that the MR could play a key role in the Der p 1 allergen uptake by DCs and in the pathogenesis of allergic diseases in dust mite -sensitive patients.

  2. Hypoallergenic Variant of the Major Egg White Allergen Gal d 1 Produced by Disruption of Cysteine Bridges

    PubMed Central

    Dhanapala, Pathum; Withanage-Dona, Dulashi; Tang, Mimi L. K.; Doran, Tim; Suphioglu, Cenk

    2017-01-01

    Background: Gal d 1 (ovomucoid) is the dominant allergen in the chicken egg white. Hypoallergenic variants of this allergen can be used in immunotherapy as an egg allergy treatment approach. We hypothesised that disruption of two of the nine cysteine-cysteine bridges by site-directed mutagenesis will allow the production of a hypoallergenic variant of the protein; Methods: Two cysteine residues at C192 and C210 in domain III of the protein were mutated to alanine using site-directed mutagenesis, to disrupt two separate cysteine-cysteine bridges. The mutated and non-mutated proteins were expressed in Escherichia coli (E. coli) by induction with isopropyl β-d-1-thiogalactopyranoside (IPTG). The expressed proteins were analysed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting to confirm expression. Immunoglobulin E (IgE) reactivity of the two proteins was analysed, by immunoblotting, against a pool of egg-allergic patients’ sera. A pool of non-allergic patients’ sera was also used in a separate blot as a negative control; Results: Mutant Gal d 1 showed diminished IgE reactivity in the immunoblot by showing lighter bands when compared to the non-mutated version, although there was more of the mutant protein immobilised on the membrane when compared to the wild-type protein. The non-allergic negative control showed no bands, indicating an absence of non-specific binding of secondary antibody to the proteins; Conclusion: Disruption of two cysteine bridges in domain III of Gal d 1 reduces IgE reactivity. Following downstream laboratory and clinical testing, this mutant protein can be used in immunotherapy to induce tolerance to Gal d 1 and in egg allergy diagnosis. PMID:28230769

  3. Cloning, expression, purification, crystallization and preliminary X-ray diffraction studies of a major group 7 allergen, Der f 7, from the dust mite Dermatophagoides farinae.

    PubMed

    Tan, Kang Wei; Kumar, Sundramurthy; Chew, Fook Tim; Mok, Yu Keung

    2011-12-01

    Der f 7 is a major group 7 allergen from the dust mite Dermatophagoides farinae that shows 86% sequence identity to the homologous allergen Der p 7 from D. pteronyssinus. Der f 7 was successfully overexpressed in an Escherichia coli expression system and purified to homogeneity using Ni-NTA affinity and size-exclusion column chromatography. SeMet-labelled Der f 7 was crystallized by the hanging-drop vapour-diffusion method using a reservoir solution consisting of 0.1 M bis-tris pH 7.4 and 28% polyethylene glycol monomethyl ether 2000 at 293 K. X-ray diffraction data were collected to 2.24 Å resolution using synchrotron radiation. The crystals belonged to the orthorhombic system, space group P2(1)2(1)2(1), with unit-cell parameters a = 50.19, b = 58.67, c = 123.81 Å. Based on the estimated Matthews coefficient (2.16 Å(3) Da(-1)), two molecules of Der f 7 could be present in the asymmetric unit of the crystal lattice.

  4. Mutational epitope analysis of Pru av 1 and Api g 1, the major allergens of cherry (Prunus avium) and celery (Apium graveolens): correlating IgE reactivity with three-dimensional structure.

    PubMed

    Neudecker, Philipp; Lehmann, Katrin; Nerkamp, Jörg; Haase, Tanja; Wangorsch, Andrea; Fötisch, Kay; Hoffmann, Silke; Rösch, Paul; Vieths, Stefan; Scheurer, Stephan

    2003-11-15

    Birch pollinosis is often accompanied by adverse reactions to food due to pollen-allergen specific IgE cross-reacting with homologous food allergens. The tertiary structure of Pru av 1, the major cherry (Prunus avium) allergen, for example, is nearly identical with Bet v 1, the major birch (Betula verrucosa) pollen allergen. In order to define cross-reactive IgE epitopes, we generated and analysed mutants of Pru av 1 and Api g 1.0101, the major celery (Apium graveolens) allergen, by immunoblotting, EAST (enzyme allergosorbent test), CD and NMR spectroscopy. The mutation of Glu45 to Trp45 in the P-loop region, a known IgE epitope of Bet v 1, significantly reduced IgE binding to Pru av 1 in a subgroup of cherry-allergic patients. The backbone conformation of Pru av 1 wild-type is conserved in the three-dimensional structure of Pru av 1 Trp45, demonstrating that the side chain of Glu45 is involved in a cross-reactive IgE epitope. Accordingly, for a subgroup of celery-allergic patients, IgE binding to the homologous celery allergen Api g 1.0101 was enhanced by the mutation of Lys44 to Glu. The almost complete loss of IgE reactivity to the Pru av 1 Pro112 mutant is due to disruption of its tertiary structure. Neither the mutation Ala112 nor deletion of the C-terminal residues 155-159 influenced IgE binding to Pru av 1. In conclusion, the structure of the P-loop partially explains the cross-reactivity pattern, and modulation of IgE-binding by site-directed mutagenesis is a promising approach to develop hypo-allergenic variants for patient-tailored specific immunotherapy.

  5. Mutational epitope analysis of Pru av 1 and Api g 1, the major allergens of cherry (Prunus avium) and celery (Apium graveolens): correlating IgE reactivity with three-dimensional structure.

    PubMed Central

    Neudecker, Philipp; Lehmann, Katrin; Nerkamp, Jörg; Haase, Tanja; Wangorsch, Andrea; Fötisch, Kay; Hoffmann, Silke; Rösch, Paul; Vieths, Stefan; Scheurer, Stephan

    2003-01-01

    Birch pollinosis is often accompanied by adverse reactions to food due to pollen-allergen specific IgE cross-reacting with homologous food allergens. The tertiary structure of Pru av 1, the major cherry (Prunus avium) allergen, for example, is nearly identical with Bet v 1, the major birch (Betula verrucosa) pollen allergen. In order to define cross-reactive IgE epitopes, we generated and analysed mutants of Pru av 1 and Api g 1.0101, the major celery (Apium graveolens) allergen, by immunoblotting, EAST (enzyme allergosorbent test), CD and NMR spectroscopy. The mutation of Glu45 to Trp45 in the P-loop region, a known IgE epitope of Bet v 1, significantly reduced IgE binding to Pru av 1 in a subgroup of cherry-allergic patients. The backbone conformation of Pru av 1 wild-type is conserved in the three-dimensional structure of Pru av 1 Trp45, demonstrating that the side chain of Glu45 is involved in a cross-reactive IgE epitope. Accordingly, for a subgroup of celery-allergic patients, IgE binding to the homologous celery allergen Api g 1.0101 was enhanced by the mutation of Lys44 to Glu. The almost complete loss of IgE reactivity to the Pru av 1 Pro112 mutant is due to disruption of its tertiary structure. Neither the mutation Ala112 nor deletion of the C-terminal residues 155-159 influenced IgE binding to Pru av 1. In conclusion, the structure of the P-loop partially explains the cross-reactivity pattern, and modulation of IgE-binding by site-directed mutagenesis is a promising approach to develop hypo-allergenic variants for patient-tailored specific immunotherapy. PMID:12943529

  6. 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.

    PubMed

    Vieths, S; Barber, D; Chapman, M; Costanzo, A; Daas, A; Fiebig, H; Hanschmann, K M; Hrabina, M; Kaul, S; Ledesma, A; Moingeon, P; Reese, G; Schörner, C; van Ree, R; Weber, B; Buchheit, K H

    2012-04-01

    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

  7. Fungal allergens.

    PubMed Central

    Horner, W E; Helbling, A; Salvaggio, J E; Lehrer, S B

    1995-01-01

    Airborne fungal spores occur widely and often in far greater concentrations than pollen grains. Immunoglobulin E-specific antigens (allergens) on airborne fungal spores induce type I hypersensitivity (allergic) respiratory reactions in sensitized atopic subjects, causing rhinitis and/or asthma. The prevalence of respiratory allergy to fungi is imprecisely known but is estimated at 20 to 30% of atopic (allergy-predisposed) individuals or up to 6% of the general population. Diagnosis and immunotherapy of allergy to fungi require well-characterized or standardized extracts that contain the relevant allergen(s) of the appropriate fungus. Production of standardized extracts is difficult since fungal extracts are complex mixtures and a variety of fungi are allergenic. Thus, the currently available extracts are largely nonstandardized, even uncharacterized, crude extracts. Recent significant progress in isolating and characterizing relevant fungal allergens is summarized in the present review. Particularly, some allergens from the genera Alternaria, Aspergillus, and Cladosporium are now thoroughly characterized, and allergens from several other genera, including some basidiomycetes, have also been purified. The availability of these extracts will facilitate definitive studies of fungal allergy prevalence and immunotherapy efficacy as well as enhance both the diagnosis and therapy of fungal allergy. PMID:7621398

  8. Chemical, nutritional and technological characteristics of buckwheat and non-prolamine buckwheat flours in comparison of wheat flour.

    PubMed

    de Francischi, M L; Salgado, J M; Leitão, R F

    1994-12-01

    Chemical, nutritional, and technological characteristics of buckwheat and non-prolamine buckwheat flours in comparison to wheat for celiac patients use have been studied. The results suggested the following conclusions: the 56.5% extraction value for flour obtention is considered good; the buckwheat flour presents methionine and cystine as first limiting amino acids followed by threonine as the second limiting amino acid; the buckwheat flour presents higher content of lysine amino acids than the wheat flour; the buckwheat flour is superior to the wheat flour regarding iron, copper, and magnesium minerals; the buckwheat flour does not present haemagglutinin activity and the tannin content is negligible. Rheological assays indicate that the buckwheat flour does not contain gluten.

  9. Outdoor allergens.

    PubMed Central

    Burge, H A; Rogers, C A

    2000-01-01

    Outdoor allergens are an important part of the exposures that lead to allergic disease. Understanding the role of outdoor allergens requires a knowledge of the nature of outdoor allergen-bearing particles, the distributions of their source, and the nature of the aerosols (particle types, sizes, dynamics of concentrations). Primary sources for outdoor allergens include vascular plants (pollen, fern spores, soy dust), and fungi (spores, hyphae). Nonvascular plants, algae, and arthropods contribute small numbers of allergen-bearing particles. Particles are released from sources into the air by wind, rain, mechanical disturbance, or active discharge mechanisms. Once airborne, they follow the physical laws that apply to all airborne particles. Although some outdoor allergens penetrate indoor spaces, exposure occurs mostly outdoors. Even short-term peak outdoor exposures can be important in eliciting acute symptoms. Monitoring of airborne biological particles is usually by particle impaction and microscopic examination. Centrally located monitoring stations give regional-scale measurements for aeroallergen levels. Evidence for the role of outdoor allergens in allergic rhinitis is strong and is rapidly increasing for a role in asthma. Pollen and fungal spore exposures have both been implicated in acute exacerbations of asthma, and sensitivity to some fungal spores predicts the existence of asthma. Synergism and/or antagonism probably occurs with other outdoor air particles and gases. Control involves avoidance of exposure (staying indoors, preventing entry of outdoor aerosols) as well as immunotherapy, which is effective for pollen but of limited effect for spores. Outdoor allergens have been the subject of only limited studies with respect to the epidemiology of asthma. Much remains to be studied with respect to prevalence patterns, exposure and disease relationships, and control. PMID:10931783

  10. Allergen databases and allergen semantics.

    PubMed

    Gendel, Steven M

    2009-08-01

    The efficacy of any specific bioinformatic analysis of the potential allergenicity of new food proteins depends directly on the nature and content of the databases that are used in the analysis. A number of different allergen-related databases have been developed, each designed to meet a different need. These databases differ in content, organization, and accessibility. These differences create barriers for users and prevent data sharing and integration. The development and application of appropriate semantic web technologies, (for example, a food allergen ontology) could help to overcome these barriers and promote the development of more advanced analytic capabilities.

  11. The major olive pollen allergen (Ole e I) shows both gametophytic and sporophytic expression during anther development, and its synthesis and storage takes place in the RER.

    PubMed

    de Dios Alché, J; Castro, A J; Olmedilla, A; Fernández, M C; Rodríguez, R; Villalba, M; Rodríguez-García, M I

    1999-08-01

    The distribution of Ole e I (the major olive pollen allergen) and its transcripts was investigated in the anther from premeiotic stages until the dehiscent pollen stage. Crude protein extracts were analyzed by immunoblotting and probed with a monoclonal antibody to Ole e I. The protein, with three variants, was found to accumulate from the early microspore stage onwards. In addition to the previously reported localization of the protein, Ole e I has been immunolocalized for the first time within the pollen wall and in the tapetum. Reverse transcription-polymerase chain reaction analysis using specific oligonucleotides and RNA extracted from whole anthers revealed that the Ole e I gene is expressed from the late tetrad stage onwards. No expression was found in control tissues such as petals, roots or leaves. Light microscopy in situ hybridization on developing flower buds and dehiscent pollen confirmed the transcripts to be present in both the microspores and the sporophytic tissue (tapetum). Labeling was found primarily in the tapetum, reaching the highest concentration in the cytoplasm of the developing and mature pollen, once tapetum started to degenerate. In situ hybridization at the transmission electron microscope level showed the transcripts to accumulate on ribosomes of the rough endoplasmic reticulum. These studies, together with others carried out previously by us, indicated that both synthesis and storage of Ole e I take place in the endoplasmic reticulum, coincidentally with the conspicuous changes suffered by this membrane system during pollen development. This process is most likely controlled at the transcriptional level. The localization of the protein in the pollen ectexine bring new insights into the function of the allergen, which are discussed.

  12. Study of the epitope structure of purified Dac G I and Lol p I, the major allergens of Dactylis glomerata and Lolium perenne pollens, using monoclonal antibodies.

    PubMed

    Mourad, W; Mécheri, S; Peltre, G; David, B; Hébert, J

    1988-11-15

    The use of mAb allowed us to further analyze the cross-reactivity between purified Dac g I and Lol p I, the major allergens of Dactylis glomerata (cocksfoot) and Lolium perenne (Rye grass), respectively. It was first shown, using IEF, followed by immunoprinting, that serum IgE antibodies from most grass-sensitive patients recognize both Dac g I and Lol p I. Second, three different anti-Lol p I mAb, 290A-167, 348A-6, and 539A-6, and one anti-Dac g I mAb, P3B2 were all shown to react with Dac g I and Lol p I, indicating that the two molecules share common epitopes. Epitope specificity of the mAb was determined by competitive binding inhibition of a given labeled mAb to solid phase fixed Dac g I or Lol p I by the mAb. The results indicated that the four mAb are directed against four different and non-overlapping epitopes present on both allergens. Using double-binding RIA, our data strongly suggest that the common epitopes are not repetitive on both molecules. In addition to their similar physicochemical characteristics, such as isolectric points and m.w., Dac g I and Lol p I share four identical epitopes. Binding inhibition of human IgE to Lol p I and Dac g I by the mAb was also assessed. The results indicated that each mAb was able to inhibit such reactions to variable degree but no additive inhibition was observed when two mAb of different specificities were used in combination, suggesting that the human IgE binding site is partially shared by each epitope recognized by the four mAb.

  13. Expression of recombinant T-cell epitopes of major Japanese cedar pollen allergens fused with cholera toxin B subunit in Escherichia coli.

    PubMed

    Hoang, Vinh Van; Zou, Yanshuang; Kurata, Kentaro; Enomoto, Keiichi

    2015-05-01

    Peptides containing T-cell epitopes from allergens, which are not reactive to allergen-specific IgE, are appropriate candidates as antigens for specific immunotherapy against allergies. To develop a vaccine that can be used in practical application to prevent and treat Japanese cedar pollen allergy, four major T-cell epitopes from the Cry j 1 antigen and six from the Cry j 2 antigen were selected to design cry j 1 epi and cry j 2 epi, DNA constructs encoding artificial polypeptides of the selected epitopes. To apply cholera toxin B subunit (CTB) as an adjuvant, cry j 1 epi and cry j 2 epi were linked and then fused to the CTB gene in tandem to construct a fusion gene, ctb-linker-cry j 1 epi- cry j 2 epi-flag. The fusion gene was introduced into a pET-28a(+) vector and expressed in Escherichia coli BL21(DE3). The expressed recombinant protein was purified by a His-tag affinity column and confirmed by western blot analysis using anti-CTB and anti-FLAG antibodies. The purified recombinant protein also proved to be antigenic against anti-Cry j 1 and anti-Cry j 2 antibodies. Expression of the recombinant protein induced with 1mM IPTG reached a maximum in 3-5h, and recovery of the affinity-purified recombinant protein was approximately 120mg/L of culture medium. The present study indicates that production of sufficient amounts of recombinant protein with antigenic epitopes may be possible by recombinant techniques using E. coli or other bacterial strains for protein expression.

  14. Immunologic characterization of monoclonal antibodies that modulate human IgE binding to the major birch pollen allergen Bet v 1.

    PubMed

    Lebecque, S; Dolecek, C; Laffer, S; Visco, V; Denépoux, S; Pin, J J; Guret, C; Boltz-Nitulescu, G; Weyer, A; Valenta, R

    1997-03-01

    Bet v 1 and homologous proteins represent major allergens for almost 95% of patients allergic to tree pollen and approximately 70% of those allergic to fruits and vegetables. As yet, no continuous (sequential) IgE epitopes have been determined for Bet v 1, and evidence has accumulated that Bet v 1 IgE epitopes belong to the conformational (discontinuous) type. A panel of 85 mouse monoclonal anti-Bet v 1 antibodies was raised as a tool with which to study the interaction of human IgE antibodies with Bet v 1. The epitopes of selected monoclonal antibodies (mAbs) were characterized by mapping with synthetic overlapping peptides and by cross-competition experiments. Cross-reactivity of Bet v 1-specific mAbs with tree and plant food allergens was investigated by Western blotting. The influence of Bet v 1-specific mAbs on the IgE-Bet v 1 interaction was studied by competition assays with immobilized purified recombinant Bet v 1 and by basophil histamine release experiments. Antibodies that increased the IgE binding to Bet v 1 up to fivefold could be defined, whereas others inhibited IgE binding to Bet v 1 up to 99% and competed with the Bet v 1-induced histamine release from patients' basophils. The activity of the enhancing antibodies is interpreted as a stabilization of Bet v 1 states/IgE epitopes, which are either more accessible for certain IgE antibodies or are recognized with higher affinity. Those mAbs that competed with the Bet v 1-IgE interaction, if humanized or produced as recombinant antibody fragments, might be considered as potential tools for local allergy therapy.

  15. Serum and salivary IgE, IgA, and IgG4 antibodies to Dermatophagoides pteronyssinus and its major allergens, Der p1 and Der p2, in allergic and nonallergic children.

    PubMed

    Miranda, Diego O; Silva, Deise A O; Fernandes, Jorge F C; Queirós, Meimei G J; Chiba, Hamilton F; Ynoue, Leandro H; Resende, Rafael O; Pena, Janethe D O; Sung, Sun-Sang J; Segundo, Gesmar R S; Taketomi, Ernesto A

    2011-01-01

    Allergic rhinitis (AR) is a public health problem with high prevalence worldwide. We evaluated levels of specific IgE, IgA, and IgG4 antibodies to the Dermatophagoides pteronyssinus (Dpt) house dust mite and to its major allergens (Der p1 and Der p2) in serum and saliva samples from allergic and nonallergic children. A total of 86 children were analyzed, from which 72 had AR and 14 were nonallergic healthy children. Serum IgE and serum/salivary IgG4 levels to Dpt, Der p1, and Der p2 were higher in allergic children whereas serum/salivary IgA levels to all allergens were higher in nonallergic children. IgE levels positively correlated with IgG4 and IgA to all allergens in allergic children, while IgA levels negatively correlated with IgG4 to Dpt and Der p1 in nonallergic children. In conclusion, mite-specific IgA antibodies predominate in the serum and saliva of nonallergic children whereas mite-specific IgE and IgG4 are prevalent in allergic children. The presence of specific IgA appears to have a key role for the healthy immune response to mucosal allergens. Also, specific IgA measurements in serum and/or saliva may be useful for monitoring activation of tolerance-inducing mechanisms during allergen specific immunotherapeutic procedures, especially sublingual immunotherapy.

  16. Der f 34, a Novel Major House Dust Mite Allergen Belonging to a Highly Conserved Rid/YjgF/YER057c/UK114 Family of Imine Deaminases.

    PubMed

    ElRamlawy, Kareem Gamal; Fujimura, Takashi; Baba, Koji; Kim, Ji Won; Kawamoto, Chika; Isobe, Toshihide; Abe, Takuya; Hodge-Hanson, Kelsey; Downs, Diana M; Refaat, Inas Hussein; Beshr Al-Azhary, Diaa; Aki, Tsunehiro; Asaoku, Yoshiko; Hayashi, Takaharu; Katsutani, Takashi; Tsuboi, Shinji; Ono, Kazuhisa; Kawamoto, Seiji

    2016-10-07

    The high prevalence of house dust mite (HDM) allergy is a growing health problem worldwide, and the characterization of clinically important HDM allergens is a prerequisite for the development of diagnostic and therapeutic strategies. Here, we report a novel HDM allergen that belongs structurally to the highly conserved Rid/YjgF/YER057c/UK114 family (Rid family) with imine deaminase activity. Isolated HDM cDNA, named der f 34, encodes 128 amino acids homologous to Rid-like proteins. This new protein belongs to the Rid family and has seven conserved residues involved in enamine/imine deaminase activity. Indeed, we demonstrated that purified Der f 34 had imine deaminase activity that preferentially acted on leucine and methionine. Native Der f 34 showed a high IgE binding frequency as revealed by two-dimensional immunoblotting (62.5%) or ELISA (68%), which was comparable with those of a major HDM allergen Der f 2 (77.5 and 79%, respectively). We also found that Der f 34 showed cross-reactivity with another prominent indoor allergen source, Aspergillus fumigatus This is the first report showing that the Rid family imine deaminase represents an additional important pan-allergen that is conserved across organisms.

  17. Antioxidant and antidiabetic properties of tartary buckwheat rice flavonoids after in vitro digestion*

    PubMed Central

    Bao, Tao; Wang, Ye; Li, Yu-ting; Gowd, Vemana; Niu, Xin-he; Yang, Hai-ying; Chen, Li-shui; Chen, Wei; Sun, Chong-de

    2016-01-01

    Oxidative stress and diabetes have a tendency to alter protein, lipid, and DNA moieties. One of the strategic methods used to reduce diabetes-associated oxidative stress is to inhibit the carbohydrate-digesting enzymes, thereby decreasing gastrointestinal glucose production. Plant-derived natural antioxidant molecules are considered a therapeutic tool in the treatment of oxidative stress and diabetes. The objective of this study was to identify tartary buckwheat rice flavonoids and evaluate the effect of in vitro digestion on their antioxidant and antidiabetic properties. High performance liquid chromatography (HPLC) analysis indicated the presence of rutin as a major component and quercitrin as a minor component of both digested and non-digested flavonoids. Both extracts showed a significant antioxidant capacity, but digested flavonoids showed reduced activity compared to non-digested. There were some decreases of the antioxidant activities (2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt (ABTS), 2,2-diphenyl-1-picrylhydrazy (DPPH) radical, and ferric reducing antioxidant power (FRAP)) of digested tartary buckwheat rice flavonoids compared with non-digested. Flavonoids from both groups significantly inhibited reactive oxygen species (ROS) production and α-glucosidase activity. Both digested and non-digested flavonoids markedly increased glucose consumption and glycogen content in HepG2 cells. Tartary buckwheat rice flavonoids showed appreciable antioxidant and antidiabetic properties, even after digestion. Tartary buckwheat rice appears to be a promising functional food with potent antioxidant and antidiabetic properties. PMID:27921399

  18. Tree nut allergens.

    PubMed

    Roux, Kenneth H; Teuber, Suzanne S; Sathe, Shridhar K

    2003-08-01

    Allergic reactions to tree nuts can be serious and life threatening. Considerable research has been conducted in recent years in an attempt to characterize those allergens that are most responsible for allergy sensitization and triggering. Both native and recombinant nut allergens have been identified and characterized and, for some, the IgE-reactive epitopes described. Some allergens, such as lipid transfer proteins, profilins, and members of the Bet v 1-related family, represent minor constituents in tree nuts. These allergens are frequently cross-reactive with other food and pollen homologues, and are considered panallergens. Others, such as legumins, vicilins, and 2S albumins, represent major seed storage protein constituents of the nuts. The allergenic tree nuts discussed in this review include those most commonly responsible for allergic reactions such as hazelnut, walnut, cashew, and almond as well as those less frequently associated with allergies including pecan, chestnut, Brazil nut, pine nut, macadamia nut, pistachio, coconut, Nangai nut, and acorn. Copyright 2003 S. Karger AG, Basel

  19. Allergenicity of processed food.

    USDA-ARS?s Scientific Manuscript database

    Food allergies have become a major public health issue in many countries. In the U.S. it is estimated that approximately 150 individuals die each year from accidental ingestion of an allergic food. As a result, the federal government recently passed the food allergen labeling law which went into ef...

  20. Structural aspects of fungal allergens.

    PubMed

    Crameri, Reto

    2015-03-01

    Despite the increasing number of solved crystal structures of allergens, the key question why some proteins are allergenic and the vast majority is not remains unanswered. The situation is not different for fungal allergens which cover a wide variety of proteins with different chemical properties and biological functions. They cover enzymes, cell wall, secreted, and intracellular proteins which, except cross-reactive allergens, does not show any evidence for structural similarities at least at the three-dimensional level. However, from a diagnostic point of view, pure allergens biotechnologically produced by recombinant technology can provide us, in contrast to fungal extracts which are hardly producible as standardized reagents, with highly pure perfectly standardized diagnostic reagents.

  1. Preference of Bemisia tabaci biotype B on zucchini squash and buckwheat and the effect of Delphastus catalinae on whitefly populations.

    PubMed

    Razze, Janine M; Liburd, Oscar E; McSorley, Robert

    2016-07-01

    Zucchini squash, Cucurbita pepo L., is an important vegetable crop in Florida. Physiological disorders and insect-transmitted diseases are major problems for squash growers in semi-tropical regions around the world. Bemisia tabaci (Gennadius) biotype B is a significant whitefly pest and is largely responsible for transmitting viruses and causing physiological disorders in squash. Several studies have shown that whitefly populations are reduced when crops are interplanted with non-host cover crops or mulches. The aim of the present study was to determine how the presence of buckwheat, Fagopyrum esculentum Moench, and a key predator, Delphastus catalinae (Horn), affect whitefly colonization on squash. Whitefly densities were higher on squash than on buckwheat. The introduction of D. catalinae on squash significantly reduced whitefly populations. Overall, there were higher densities of D. catalinae on squash where the whitefly pest was more concentrated compared with buckwheat. The study provided preliminary evidence that D. catalinae, when used in conjunction with buckwheat as a living mulch, may aid in reducing whiteflies in squash. This greenhouse experiment highlights the need to investigate a multitactic approach of intercropping buckwheat with squash and the incorporation of D. catalinae in the field to manage populations of whiteflies and whitefly-transmitted diseases. © 2015 Society of Chemical Industry. © 2015 Society of Chemical Industry.

  2. Chemical composition and health effects of Tartary buckwheat.

    PubMed

    Zhu, Fan

    2016-07-15

    Tartary buckwheat (Fagopyrum tataricum) contains a range of nutrients including bioactive carbohydrates and proteins, polyphenols, phytosterols, vitamins, carotenoids, and minerals. The unique composition of Tartary buckwheat contributes to their various health benefits such as anti-oxidative, anti-cancer, anti-hypertension, anti-diabetic, cholesterol-lowering, and cognition-improving. Compared with the more widely cultivated and utilised common buckwheat (F. esculentum), Tartary buckwheat tends to contain higher amounts of certain bioactive components such as rutin, therefore, showing higher efficiency in preventing/treating various disorders. This review summarises the current knowledge of the chemical composition of Tartary buckwheat, and their bio-functions as studied by both in vitro and in vivo models. Tartary buckwheat can be further developed as a sustainable crop for functional food production to improve human health. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. Cross-sensitization between poppy seed and buckwheat in a food-allergic patient with poppy seed anaphylaxis.

    PubMed

    Oppel, Tilmann; Thomas, Peter; Wollenberg, Andreas

    2006-01-01

    The opium poppy, Papaver somniferum L., is the source of both poppy seeds and opium. The commercially available seeds are widely used as ingredients for various kinds of food. IgE-mediated sensitization to poppy seeds is rare, but, if present, clinical symptoms are usually severe. Cross-sensitizations between poppy seeds and other food allergens have been described with sesame, hazelnut, rye grain and kiwi fruit. We report the case of a 17-year-old female with an apparently food-allergic reaction after ingestion of a poppy seed cake. Allergological workup revealed a poppy seed anaphylaxis and led to the identification of a novel cross-sensitization with buckwheat.

  4. A Cross-Reactive Human Single-Chain Antibody for Detection of Major Fish Allergens, Parvalbumins, and Identification of a Major IgE-Binding Epitope

    PubMed Central

    Fuchs, Julian E.; Ackerbauer, Daniela; Moraes, Adolfo H.; Almeida, Fabio C. L.; Lengger, Nina; Hafner, Christine; Ebner, Christof; Radauer, Christian; Liedl, Klaus R.; Valente, Ana Paula; Breiteneder, Heimo

    2015-01-01

    Fish allergy is associated with moderate to severe IgE-mediated reactions to the calcium binding parvalbumins present in fish muscle. Allergy to multiple fish species is caused by parvalbumin-specific cross-reactive IgE recognizing conserved epitopes. In this study, we aimed to produce cross-reactive single chain variable fragment (scFv) antibodies for the detection of parvalbumins in fish extracts and the identification of IgE epitopes. Parvalbumin-specific phage clones were isolated from the human ETH-2 phage display library by three rounds of biopanning either against cod parvalbumin or by sequential biopanning against cod (Gad m 1), carp (Cyp c 1) and rainbow trout (Onc m 1) parvalbumins. While biopanning against Gad m 1 resulted in the selection of clones specific exclusively for Gad m 1, the second approach resulted in the selection of clones cross-reacting with all three parvalbumins. Two clones, scFv-gco9 recognizing all three parvalbumins, and scFv-goo8 recognizing only Gad m 1 were expressed in the E. coli non-suppressor strain HB2151 and purified from the periplasm. scFv-gco9 showed highly selective binding to parvalbumins in processed fish products such as breaded cod sticks, fried carp and smoked trout in Western blots. In addition, the scFv-gco9-AP produced as alkaline phosphatase fusion protein, allowed a single-step detection of the parvalbumins. In competitive ELISA, scFv-gco9 was able to inhibit binding of IgE from fish allergic patients’ sera to all three β-parvalbumins by up to 80%, whereas inhibition by scFv-goo8 was up to 20%. 1H/15N HSQC NMR analysis of the rGad m 1:scFv-gco9 complex showed participation of amino acid residues conserved among these three parvalbumins explaining their cross-reactivity on a molecular level. In this study, we have demonstrated an approach for the selection of cross-reactive parvalbumin-specific antibodies that can be used for allergen detection and for mapping of conserved epitopes. PMID:26579717

  5. LC-MS analysis of phenolic compounds and antioxidant activity of buckwheat at different stages of malting.

    PubMed

    Terpinc, Petra; Cigić, Blaž; Polak, Tomaž; Hribar, Janez; Požrl, Tomaž

    2016-11-01

    The impact of malting on the profile of the phenolic compounds and the antioxidant properties of two buckwheat varieties was investigated. The highest relative increases in phenolic compounds were observed for isoorientin, orientin, and isovitexin, which are consequently major inducible phenolic compounds during malting. Only a minor relative increase was observed for the most abundant phenolic compound, rutin. The radical-scavenging activity of buckwheat seeds was evaluated using ABTS and DPPH assays. A considerable increase in total phenolic compounds and higher antioxidant activity were observed after 64h of germination, whereas kilning resulted in decreased total phenolic compounds and antioxidant activity. Higher antioxidant activities for extracts were found for buffered solvents than for pure methanol and water. Changes in the composition of the phenolic compounds and increased antioxidant content were confirmed by several methods, indicating that buckwheat malt can be used as a food rich in antioxidants.

  6. Mycorrhizal status and diversity of fungal endophytes in roots of common buckwheat (Fagopyrum esculentum) and tartary buckwheat (F. tataricum).

    PubMed

    Likar, Matevz; Bukovnik, Urska; Kreft, Ivan; Chrungoo, Nikhil K; Regvar, Marjana

    2008-09-01

    To determine the mycorrhizal status and to identify the fungi colonising the roots of the plants, common buckwheat (Fagopyrum esculentum) and tartary buckwheat (F. tataricum) were inoculated with an indigenous fungal mixture from a buckwheat field. Root colonisation was characterised by the hyphae and distinct microsclerotia of dark septate endophytes, with occasional arbuscules and vesicles of arbuscular mycorrhizal fungi. Sequences of arbuscular mycorrhizal fungi colonising tartary buckwheat clustered close to the Glomus species group A. Sequences with similarity to the Ceratobasidium/Rhizoctonia complex, a putative dark septate endophyte fungus, were amplified from the roots of both common and tartary buckwheat. To the best of our knowledge, this is the first report of arbuscular mycorrhizal colonisation in tartary buckwheat and the first molecular characterisation of these fungi that can colonise both of these economically important plant species.

  7. Food Allergens: Is There a Correlation between Stability to Digestion and Allergenicity?

    PubMed

    Bøgh, Katrine Lindholm; Madsen, Charlotte Bernhard

    2016-07-03

    Food allergy is a major health problem in the Western countries, affecting 3-8% of the population. It has not yet been established what makes a dietary protein a food allergen. Several characteristics have been proposed to be shared by food allergens. One of these is resistance to digestion. This paper reviews data from digestibility studies on purified food allergens and evaluates the predictive value of digestibility tests on the allergenic potential. We point out that food allergens do not necessarily resist digestion. We discuss how the choice of in vitro digestibility assay condition and the method used for detection of residual intact protein as well as fragments hereof may greatly influence the outcome as well as the interpretation of results. The finding that digests from food allergens may retain allergenicity, stresses the importance of using immunological assays for evaluating the allergenic potential of food allergen digestion products. Studies assessing the allergenicity of digestion products, by either IgE-binding, elicitation or sensitizing capacity, shows that digestion may abolish, decrease, have no effect, or even increase the allergenicity of food allergens. Therefore, the predictive value of the pepsin resistance test for assessing the allergenic potential of novel proteins can be questioned.

  8. New insights into ragweed pollen allergens.

    PubMed

    Bordas-Le Floch, Véronique; Groeme, Rachel; Chabre, Henri; Baron-Bodo, Véronique; Nony, Emmanuel; Mascarell, Laurent; Moingeon, Philippe

    2015-11-01

    Pollen allergens from short ragweed (Ambrosia artemisiifolia) cause severe respiratory allergies in North America and Europe. To date, ten short ragweed pollen allergens belonging to eight protein families, including the recently discovered novel major allergen Amb a 11, have been recorded in the International Union of Immunological Societies (IUIS) allergen database. With evidence that other components may further contribute to short ragweed pollen allergenicity, a better understanding of the allergen repertoire is a requisite for the design of proper diagnostic tools and efficient immunotherapies. This review provides an update on both known as well as novel candidate allergens from short ragweed pollen, identified through a comprehensive characterization of the ragweed pollen transcriptome and proteome.

  9. IgG4 inhibits peanut-induced basophil and mast cell activation in peanut-tolerant children sensitized to peanut major allergens

    PubMed Central

    Santos, Alexandra F.; James, Louisa K.; Bahnson, Henry T.; Shamji, Mohammed H.; Couto-Francisco, Natália C.; Islam, Sabita; Houghton, Sally; Clark, Andrew T.; Stephens, Alick; Turcanu, Victor; Durham, Stephen R.; Gould, Hannah J.; Lack, Gideon

    2015-01-01

    explanation for the absence of clinical reactivity in PS patients sensitized to major peanut allergens. PMID:25670011

  10. An immunodiagnostic assay for quantitation of specific IgE to the major pollen allergen component, Pas n 1, of the subtropical Bahia grass.

    PubMed

    Timbrell, Victoria L; Riebelt, Lindsay; Simmonds, Claire; Solley, Graham; Smith, William B; Mclean-Tooke, Andrew; van Nunen, Sheryl; Smith, Peter K; Upham, John W; Langguth, Daman; Davies, Janet M

    2014-01-01

    Pollens of the Panicoideae subfamily of grasses including Bahia (Paspalum notatum) are important allergen sources in subtropical regions of the world. An assay for specific IgE to the major molecular allergenic component, Pas n 1, of Bahia grass pollen (BaGP) would have immunodiagnostic utility for patients with pollen allergy in these regions. Biotinylated Pas n 1 purified from BaGP was coated onto streptavidin ImmunoCAPs. Subjects were assessed by clinical history of allergic rhinitis and skin prick test (SPT) to aeroallergens. Serum total, BaGP-specific and Pas n 1-specific IgE were measured. Pas n 1 IgE concentrations were highly correlated with BaGP SPT (r = 0.795, p < 0.0001) and BaGP IgE (r = 0.915, p < 0.0001). At 0.23 kU/l Pas n 1 IgE, the diagnostic sensitivity (92.4%) and specificity (93.1%) for the detection of BaGP allergy was high (area under receiver operator curve 0.960, p < 0.0001). The median concentrations of Pas n 1 IgE in non-atopic subjects (0.01 kU/l, n = 67) and those with other allergies (0.02 kU/l, n = 59) showed no inter-group difference, whilst grass pollen-allergic patients with allergic rhinitis showed elevated Pas n 1 IgE (6.71 kU/l, n = 182, p < 0.0001). The inter-assay coefficient of variation for the BaGP-allergic serum pool was 6.92%. Pas n 1 IgE appears to account for most of the BaGP-specific IgE. This molecular component immunoassay for Pas n 1 IgE has potential utility to improve the sensitivity and accuracy of diagnosis of BaGP allergy for patients in subtropical regions. © 2015 S. Karger AG, Basel.

  11. Detection of Ara h 1 (a major peanut allergen) in food using an electrochemical gold nanoparticle-coated screen-printed immunosensor.

    PubMed

    Alves, Rita C; Pimentel, Filipa B; Nouws, Henri P A; Marques, Raquel C B; González-García, María Begoña; Oliveira, M Beatriz P P; Delerue-Matos, Cristina

    2015-02-15

    A gold nanoparticle-coated screen-printed carbon electrode was used as the transducer in the development of an electrochemical immunosensor for Ara h 1 (a major peanut allergen) detection in food samples. Gold nanoparticles (average diameter = 32 nm) were electrochemically generated on the surface of screen-printed carbon electrodes. Two monoclonal antibodies were used in a sandwich-type immunoassay and the antibody-antigen interaction was electrochemically detected through stripping analysis of enzymatically (using alkaline phosphatase) deposited silver. The total time of the optimized immunoassay was 3h 50 min. The developed immunosensor allowed the quantification of Ara h 1 between 12.6 and 2000 ng/ml, with a limit of detection of 3.8 ng/ml, and provided precise (RSD <8.7%) and accurate (recovery >96.6%) results. The immunosensor was successfully applied to the analysis of complex food matrices (cookies and chocolate), being able to detect Ara h 1 in samples containing 0.1% of peanut. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. Nomenclature and structural biology of allergens.

    PubMed

    Chapman, Martin D; Pomés, Anna; Breiteneder, Heimo; Ferreira, Fatima

    2007-02-01

    Purified allergens are named using the systematic nomenclature of the Allergen Nomenclature Sub-Committee of the World Health Organization and International Union of Immunological Societies. The system uses abbreviated Linnean genus and species names and an Arabic number to indicate the chronology of allergen purification. Most major allergens from mites, animal dander, pollens, insects, and foods have been cloned, and more than 40 three-dimensional allergen structures are in the Protein Database. Allergens are derived from proteins with a variety of biologic functions, including proteases, ligand-binding proteins, structural proteins, pathogenesis-related proteins, lipid transfer proteins, profilins, and calcium-binding proteins. Biologic function, such as the proteolytic enzyme allergens of dust mites, might directly influence the development of IgE responses and might initiate inflammatory responses in the lung that are associated with asthma. Intrinsic structural or biologic properties might also influence the extent to which allergens persist in indoor and outdoor environments or retain their allergenicity in the digestive tract. Analyses of the protein family database suggest that the universe of allergens comprises more than 120 distinct protein families. Structural biology and proteomics define recombinant allergen targets for diagnostic and therapeutic purposes and identify motifs, patterns, and structures of immunologic significance.

  13. Dermatophagoides farinae allergens diversity identification by proteomics.

    PubMed

    An, Su; Chen, Lingling; Long, Chengbo; Liu, Xiaoyu; Xu, Xuemei; Lu, Xingre; Rong, Mingqiang; Liu, Zhigang; Lai, Ren

    2013-07-01

    The most important indoor allergens for humans are house dust mites (HDM). Fourteen Dermatophagoides farinae allergens (Der f 1-3, 6, 7, 10, 11, 13-18, and 22) are reported although more than 30 allergens have been estimated in D. farinae. Seventeen allergens belonging to 12 different groups were identified by a procedure of proteomics combined with two-dimensional immunoblotting from D. farina extracts. Their sequences were determined by Edman degradation, mass spectrometry analysis, and cDNA cloning. Their allergenicities were assayed by enzyme-linked immunosorbent assay inhibition tests, immunoblots, basophil activation test, and skin prick tests. Eight of them are the first report as D. farinae allergens. The procedure of using a proteomic approach combined with a purely discovery approach using sera of patients with broad IgE reactivity profiles to mite allergens was an effective method to investigate a more complete repertoire of D. farinae allergens. The identification of eight new D. farinae allergens will be helpful for HDM allergy diagnosis and therapy, especially for patients without response for HDM major allergens. In addition, the current work significantly extendedthe repertoire of D. farinae allergens.

  14. Reduction of rutin loss in buckwheat noodles and their physicochemical characterization

    USDA-ARS?s Scientific Manuscript database

    Rutin in buckwheat flour is converted into quercetin by rutin-degrading enzymes when water is added. Thus, buckwheat was subjected to hydrothermal treatments for minimizing the rutin loss in buckwheat-based foods by water addition. When native buckwheat flour was mixed with water, the rutin conten...

  15. Monoclonal antibodies to the major feline allergen Fel d I. II. Single step affinity purification of Fel d I, N-terminal sequence analysis, and development of a sensitive two-site immunoassay to assess Fel d I exposure.

    PubMed

    Chapman, M D; Aalberse, R C; Brown, M J; Platts-Mills, T A

    1988-02-01

    Two mAb were used to develop new techniques for the purification and quantitation of the major feline salivary allergen, Felis domesticus allergen I (Fel d I). The allergen was purified from aqueous house dust extract with a high Fel d I content by affinity chromatography over a monoclonal immunosorbent and elution with 4 mM HCl, pH 2.5. This single step procedure gave 40 to 50% recovery of 90% pure allergen which, following final purification by size exclusion HPLC, showed a single line on immunodiffusion and crossed immunoelectrophoresis against monospecific anti-Fel d I and polyclonal anti-cat dander antibodies. The m.w. of native Fel d I was 39,000 on size exclusion HPLC, and 17,000 under nonreducing conditions on gel electrophoresis. The N-terminal amino acid sequence (33 residues) showed no homology with other known protein sequences. The combination of the SDS-PAGE and N-terminal sequence data suggests that Fel d I is a non-covalently linked homodimer. A two-site RIA was developed using mAb directed against different epitopes on Fel d I. This assay was species-specific, highly sensitive (0.0004 U/ml), and showed an excellent correlation with a polyclonal inhibition RIA (n = 27, r = 0.93, p less than 0.001). Cat allergen extracts used for immediate skin tests showed marked differences in Fel d I content (from 0.1 to 30 U/ml). Consistently high Fel d I levels were found at monthly intervals in six dust samples from four houses with cats (10 to 100 U/g of dust). Comparisons of Fel d I and mite and pollen allergen levels showed that house dust can contain greater than 100 micrograms/g of either of these allergens and is a potent source of foreign environmental antigens. Monoclonal affinity chromatography provides a major breakthrough in the purification of Fel d I, from a source material that would otherwise have been considered impossible (house dust). The mAb assay for Fel d I is both more sensitive and more easily standardized than existing techniques. These

  16. Serum albumins - unusual allergens

    PubMed Central

    Chruszcz, Maksymilian; Mikolajczak, Katarzyna; Mank, Nicholas; Majorek, Karolina A.; Porebski, Przemyslaw J.; Minor, Wladek

    2015-01-01

    Background Albumins are multifunctional proteins present in the blood serum of animals. They can bind and transport a wide variety of ligands which they accommodate due to their conformational flexibility. Serum albumins are highly conserved both in amino acid sequence and three-dimensional structure. Several mammalian and avian serum albumins (SAs) are also allergens. Sensitization to one of the SAs coupled with the high degree of conservation between SAs may result in cross-reactive antibodies in allergic individuals. Sensitivity to SA generally begins with exposure to an aeroallergen, which can then lead to cross-sensitization to serum albumins present in food. Scope of Review This review focuses on the allergenicity of SAs presented in a structural context. Major Conclusions SA allergenicity is unusual taking into account the high sequence identity and similarity between SA from different species and human serum albumin. Cross-reactivity of human antibodies towards different SAs is one of the most important characteristics of these allergens. General Significance Establishing a relationship between sequence and structure of different SAs and their interactions with antibodies is crucial for understanding the mechanisms of cross-sensitization of atopic individuals. Structural information can also lead to better design and production of recombinant SAs to replace natural proteins in allergy testing and desensitization. Therefore, structural analyses are important for diagnostic and treatment purposes. PMID:23811341

  17. Impact of thermal processing on legume allergens.

    PubMed

    Verma, Alok Kumar; Kumar, Sandeep; Das, Mukul; Dwivedi, Premendra D

    2012-12-01

    Food induced allergic manifestations are reported from several parts of the world. Food proteins exert their allergenic potential by absorption through the gastrointestinal tract and can even induce life threatening anaphylaxis reactions. Among all food allergens, legume allergens play an important role in induction of allergy because legumes are a major source of protein for vegetarians. Most of the legumes are cooked either by boiling, roasting or frying before consumption, which can be considered a form of thermal treatment. Thermal processing may also include autoclaving, microwave heating, blanching, pasteurization, canning, or steaming. Thermal processing of legumes may reduce, eliminate or enhance the allergenic potential of a respective legume. In most of the cases, minimization of allergenic potential on thermal treatment has generally been reported. Thus, thermal processing can be considered an important tool by indirectly prevent allergenicity in susceptible individuals, thereby reducing treatment costs and reducing industry/office/school absence in case of working population/school going children. The present review attempts to explore various possibilities of reducing or eliminating allergenicity of leguminous food using different methods of thermal processing. Further, this review summarizes different methods of food processing, major legumes and their predominant allergenic proteins, thermal treatment and its relation with antigenicity, effect of thermal processing on legume allergens; also suggests a path that may be taken for future research to reduce the allergenicity using conventional/nonconventional methods.

  18. Structural basis for the specific recognition of the major antigenic peptide from the Japanese cedar pollen allergen Cry j 1 by HLA-DP5.

    PubMed

    Kusano, Seisuke; Kukimoto-Niino, Mutsuko; Satta, Yoko; Ohsawa, Noboru; Uchikubo-Kamo, Tomomi; Wakiyama, Motoaki; Ikeda, Mariko; Terada, Takaho; Yamamoto, Ken; Nishimura, Yasuharu; Shirouzu, Mikako; Sasazuki, Takehiko; Yokoyama, Shigeyuki

    2014-08-26

    The major allergen, Cry j 1, was isolated from Japanese cedar Cryptomeria japonica (Cry j) pollen and was shown to react with immunoglobulin E antibodies in the sera from pollinosis patients. We previously reported that the frequency of HLA-DP5 was significantly higher in pollinosis patients and the immunodominant peptides from Cry j 1 bound to HLA-DP5 to activate Th2 cells. In the present study, we determined the crystal structure of the HLA-DP5 heterodimer in complex with a Cry j 1-derived nine-residue peptide, at 2.4Å resolution. The peptide-binding groove recognizes the minimal peptide with 10 hydrogen bonds, including those between the negatively charged P1 pocket and the Lys side chain at the first position in the peptide sequence. We confirmed that HLA-DP5 exhibits the same Cry j 1-binding mode in solution, through pull-down experiments using structure-based mutations of Cry j 1. We also identified the characteristic residues of HLA-DP5 that are responsible for the distinct properties of the groove, by comparing the structure of HLA-DP5 and the previously reported structures of HLA-DP2 in complexes with pDRA of the self-antigen. The comparison revealed that the HLA-DP5·pCry j 1 complex forms several hydrogen bond/salt bridge networks between the receptor and the antigen that were not observed in the HLA-DP2·pDRA complex. Evolutionary considerations have led us to conclude that HLA-DP5 and HLA-DP2 represent two major groups of the HLA-DP family, in which the properties of the P1 and P4 pockets have evolved and acquired the present ranges of epitope peptide-binding specificities.

  19. Eriogonum Michx.: wild-buckwheat, buckwheatbrush

    Treesearch

    Susan E. Meyer

    2008-01-01

    The North American genus Eriogonum - wild-buckwheat, also buckwheatbrush - is made up of about 200 species of annual and perennial herbs and shrubs, most of which are found in the West. About half are woody, at least at the base. The habit of the woody species may be either (a) truly shrubby, (b) subshrubby, with annual renewal of upper shoots, or (c) pulvinate (mat-...

  20. [Allergens causing respiratory allergy: the aeroallergens].

    PubMed

    Deschildre, A

    1999-01-01

    Aeroallergens play a major role in the pathogenesis of allergic diseases, particularly asthma and rhinitis. Indoor allergens, including house dust mites, domestic pets, cockroaches, and molds are of particular importance. Pollens are also recognized as a major source of allergens. The role of these different allergens varies with environment conditions, such as climatic factors, and degree of exposure. Knowledge about allergens has progressed, especially with recent molecular biology studies. Structure and function have been identified. These studies have provided explanations about the relationship between allergic sensitization, allergen exposure, and disease activity, about clinical observations such as allergic cross reactions, and improvement in the production of allergenic extracts (necessary to diagnosis and immunotherapy). Environmental control measures are of particular importance in the prevention and management of allergic diseases.

  1. Postharvest biology, quality and shelf-life of buckwheat microgreens

    USDA-ARS?s Scientific Manuscript database

    Buckwheat microgreens are rich in antioxidants and provitamins/vitamins, including flavonoids, carotenoids, and a-tocopherol. However, short shelf life has limited their commercial use. The purpose of this study was to optimize storage conditions to extend the shelf life of buckwheat microgreens. St...

  2. Postharvest biology, quality and shelf-life of buckwheat microgreens

    USDA-ARS?s Scientific Manuscript database

    Buckwheat microgreens are rich in antioxidants and provitamins/vitamins, including flavonoids, carotenoids, and a-tocopherol. However, short shelf life has limited their commercial use. The purpose of this study was to optimize storage conditions to extend the shelf life of buckwheat microgreens. St...

  3. Molecular cloning and expression of active Ole e 3, a major allergen from olive-tree pollen and member of a novel family of Ca2+-binding proteins (polcalcins) involved in allergy.

    PubMed

    Ledesma, A; Villalba, M; Batanero, E; Rodríguez, R

    1998-12-01

    A cDNA encoding Ole e 3, a major allergen from olive-tree pollen, has been cloned and sequenced. A strategy based on two-step PCR amplification towards the 5' end and 3' end, with an internal specific primer, has been used. The isolated cDNA contains an open reading frame coding for a polypeptide of 84 amino acids, which is in agreement with the composition and molecular mass of the natural allergen, exhibiting two 12-residue segments homologous to Ca2+-binding sites of EF-hand type. The cDNA was inserted into the pET-11b expression vector and over-expressed in Escherichia coli. The purified recombinant protein shows identical secondary structure to that of the natural allergen and is able to bind both IgE from sera of patients allergic to olive pollen and polyclonal antibodies raised against olive-pollen Ole e 3. The capacity of binding Ca2+ has been demonstrated for both natural and recombinant allergens. RNA transcripts of Ole e 3 were only detected in pollen tissue. Northern-blot and Western-blot analyses of poly(A)+ RNA and protein extracts, respectively, obtained from a variety of olive-tree-related and nonrelated mature pollens demonstrated the presence of Ole e 3 homologous proteins. This indicates a sequence conservation and widespread distribution for this family of Ca2+-binding proteins that can be responsible for allergenic cross-reactivity. We suggest the tentative generic name of polcalcins for the members of this family of Ca2+-binding proteins from pollen.

  4. Mapping and conformational analysis of IgE-binding epitopic regions on the molecular surface of the major Ara h 3 legumin allergen of peanut (Arachis hypogaea).

    PubMed

    Rougé, Pierre; Culerrier, Raphaël; Sabatier, Virginie; Granier, Claude; Rancé, Fabienne; Barre, Annick

    2009-03-01

    Eight distinct sequential IgE-binding epitopes were identified along the amino acid sequence of Ara h 3 using the Spot technology. They essentially correspond to preferencially electropositive regions exposed on the molecular surface of the protein. A few IgE-binding epitopes are coalescent to create more extended IgE-binding regions exposed on the surface of the allergen. Ara h 3 contains a core region corresponding to the cupin motifs and predicted to be preserved upon the trypsin and chymotrypsin attack in the gastro-intestinal tract. Some of the identified IgE-binding epitopes should remain unaltered in the core region to subsequently interact with the local immune system. They most probably account for the strong allergenic potency of Ara h 3. Most of the identified IgE-binding epitopes of Ara h 3 readily differ from the corresponding regions of other legume and tree-nut legumin allergens except for epitope #1 and #7 which are rather conserved essentially in other allergens. These structurally related epitopes could account for some cross-reactions occurring between Ara h 3 and other legumin allergens.

  5. Characterization of Allergen Exposure in Homes

    DTIC Science & Technology

    1991-01-17

    dust mixture.6 Dust mite allergens have been associated causatively with asthma, atopic dermatitis , and rhini- tis. 7 Studies from several countries...Asthma: A Controlled Trial. The Lancet 1976; ***:333-335. 10. Tuft L. Importance of Inhalant Allergens in Atopic Dermatitis . The Journal of Investigative...Monoclonal Antibodies to the Major Feline Allergen Fel d 1. 1I. Single Step Affinity Purification of Fel d 1, N-Terminal Sequence Analysis, and Development of

  6. Sulphur interferes with selenium accumulation in Tartary buckwheat plants.

    PubMed

    Golob, Aleksandra; Gadžo, Drena; Stibilj, Vekoslava; Djikić, Mirha; Gavrić, Teofil; Kreft, Ivan; Germ, Mateja

    2016-11-01

    Tartary buckwheat (Fagopyrum tataricum Gaertn.) and common buckwheat (Fagopyrum esculentum Moench.) plants grown in the field were treated foliarly with 126 μM solutions of selenate and/or sulphate in order to study the effect of sulphur (S) on selenium (Se) concentration in plants. In both species, the concentration of Se in all plant parts was similar in control and S treated plants. In Tartary buckwheat the concentration of Se was higher in S and Se treated plants than in plants treated with Se alone. S was shown to enhance Se accumulation in Tartary buckwheat. It was also shown that it is possible to produce grain and herb of Tartary and common buckwheat containing appropriate amounts of Se for food without affecting the yield of the plants.

  7. New contact allergens and allergen sources.

    PubMed

    Rudzki, E; Grzywa, Z; Krajewska, D; Kozłowska, A; Czerwińska-Dihm, I

    1978-01-01

    In the report new contact allergens and allergen sources detected in Warsaw in the period 1975-1977 are described. They are divided into 3 groups: industrial allergens, remaining occupational allergens and cosmetics. There are given some data concerning the substances present in industrial oils, hardeners and epoxy resin solvents, drugs sensitizing nurses, several new sources of chromium allergens, essential oils and synthetic flavours. Results obtained with various star anise oil samples are described. Essential oils and synthetic flavours. Results obtained with various star anise oil samples are described. Essential oils and synthetic flavours are discussed as the main allergens in cosmetics.

  8. Mammalian-derived respiratory allergens - implications for diagnosis and therapy of individuals allergic to furry animals.

    PubMed

    Nilsson, Ola B; van Hage, Marianne; Grönlund, Hans

    2014-03-01

    Furry animals cause respiratory allergies in a significant proportion of the population. A majority of all mammalian allergens are spread as airborne particles, and several have been detected in environments where furry animals are not normally kept. The repertoire of allergens from each source belongs to a restricted number of allergen families. Classification of allergen families is particularly important for the characterization of allergenicity and cross-reactivity of allergens. In fact, major mammalian allergens are taken from only three protein families, i.e. the secretoglobin, lipocalin and kallikrein families. In particular, the lipocalin superfamily harbours major allergens in all important mammalian allergen sources, and cross-reactivity between lipocalin allergens may explain cross-species sensitization between mammals. The identification of single allergen components is of importance to improve diagnosis and therapy of allergic patients using component-resolved diagnostics and allergen-specific immunotherapy (ASIT) respectively. Major disadvantages with crude allergen extracts for these applications emphasize the benefits of careful characterization of individual allergens. Furthermore, detailed knowledge of the characteristics of an allergen is crucial to formulate attenuated allergy vaccines, e.g. hypoallergens. The diverse repertoires of individual allergens from different mammalian species influence the diagnostic potential and clinical efficacy of ASIT to furry animals. As such, detailed knowledge of individual allergens is essential for adequate clinical evaluation. This review compiles current knowledge of the allergen families of mammalian species, and discusses how this information may be used for improved diagnosis and therapy of individuals allergic to mammals.

  9. Indoor allergen exposure and asthma outcomes

    PubMed Central

    Sheehan, William J.; Phipatanakul, Wanda

    2016-01-01

    Purpose of review The aim of the present review is to discuss updates on research regarding the relationship between indoor allergen exposure and childhood asthma with a focus on clinical effects, locations of exposure, and novel treatments. Recent findings Recent data continue to demonstrate that early life sensitization to indoor allergens is a predictor of asthma development later in life. Furthermore, avoidance of exposure to these allergens continues to be important especially given that the vast majority of children with asthma are sensitized to at least one indoor allergen. New research suggests that mouse allergen, more so than cockroach allergen, may be the most relevant urban allergen. Recent evidence reminds us that children are exposed to clinically important levels of indoor allergens in locations away from their home, such as schools and daycare centers. Exposure to increased levels of indoor mold in childhood has been associated with asthma development and exacerbation of current asthma; however, emerging evidence suggests that early exposure to higher fungal diversity may actually be protective for asthma development. Novel treatments have been developed that target TH2 pathways thus decreasing asthmatic responses to allergens. These therapies show promise for the treatment of severe allergic asthma refractory to avoidance strategies and standard therapies. Summary Understanding the relationship between indoor allergens and asthma outcomes is a constantly evolving study of timing, location, and amount of exposure. PMID:27653703

  10. Influence of ultrasonic treatment on the allergenic properties of Shrimp ( Penaeus vannamei) Allergen

    NASA Astrophysics Data System (ADS)

    Li, Zhenxing; Lin, Hong; Cao, Limin

    2006-04-01

    The present study was undertaken to determine whether high intensity ultrasound could reduce the allergic properties of shrimp allergens. Reducing the allergenic properties of these allergens will be beneficial to allergic individuals. Samples of shrimp protein extract and shrimp muscle were treated by high-intensity ultrasound with water bathing at 0°C or 50°C for different time periods. The treated and untreated samples were then analyzed by SDS-PAGE, Western blots and competitive inhibition ELISA (Ci-ELISA) to determine the shrimp allergenicity. The results show that high-intensity ultrasound has no effect on allergenicity when the extracts were treated at 0°C. However, a significant decrease was observed in the level of the major shrimp allergen, Pen a 1, when the samples were treated at 50°C. In the determination of allergenicity with Ci-ELISA, a reduction in IgE binding was also observed.

  11. Ribonuclease activity of buckwheat plant (Fagopyrum esculentum) cultivars with different sensitivities to buckwheat burn virus.

    PubMed

    Sindarovska, Y R; Guzyk, O I; Yuzvenko, L V; Demchenko, O A; Didenko, L F; Grynevych, O I; Spivak, M Ya

    2014-01-01

    Ribonucleases (RNases) are present in base-level amounts in intact plants, but this level is able to increase greatly under stress conditions. The possible cause for such an increase is protection against plant RNA-virus attack. Buckwheat burn virus (BBV) is a highly virulent pathogen that belongs to Rhabdoviridae family. In our study, we have analyzed the correlation between RNase activity and resistance of different buckwheat cultivars to BBV infection. Two cultivars, Kara-Dag and Roksolana, with different sensitivities to BBV have been used. Kara-Dag is a cultivar with medium sensitivity to virus and Roksolana is a tolerant cultivar. It has been shown that the base level of RNase activity in Roksolana cultivar was in most cases higher than the corresponding parameter in Kara-Dag cultivar. Both infected and uninfected plants of Roksolana cultivar demonstrated high RNase activity during two weeks. Whereas infected plants of Kara-Dag cultivar demonstrated unstable levels of RNase activity. Significant decline in RNase activity was detected on the 7th day post infection with subsequent gradual increase in RNase activity. Decline of the RNase activity during the first week could promote the virus replication and therefore more successful infection of upper leaves of plants. Unstable levels of RNase activity in infected buckwheat plants may be explained by insufficiency of virus-resistant mechanisms that determines the medium sensitivity of the cultivar to BBV. Thus, plants of buckwheat cultivar having less sensitivity to virus, displayed in general higher RNase activity.

  12. Identification, expression, and characterization of a major salivary allergen (Cul s 1) of the biting midge Culicoides sonorensis relevant for summer eczema in horses

    USDA-ARS?s Scientific Manuscript database

    Salivary proteins of Culicoides biting midges are thought to play a key role in the induction of summer eczema (SE), a seasonal recurrent allergic dermatitis in horses. The present study describes the identification of a candidate allergen in artificially collected saliva of the North American speci...

  13. Differential analyses of major allergen proteins in wild-type rice and rice producing a fragment of anti-rotavirus antibody.

    PubMed

    Yuki, Yoshikazu; Kurokawa, Shiho; Kozuka-Hata, Hiroko; Tokuhara, Daisuke; Mejima, Mio; Kuroda, Masaharu; Oyama, Masaaki; Nishimaki-Mogami, Tomoko; Teshima, Reiko; Kiyono, Hiroshi

    2016-04-01

    To develop oral antibody therapy against rotavirus infection, we previously produced a recombinant fragment of llama heavy-chain antibody to rotavirus (ARP1) in rice seeds (MucoRice-ARP1). We intend to use a purification-free rice powder for clinical application but needed to check whether MucoRice-ARP1 had increased levels of known allergen proteins. For this purpose, we used two-dimensional fluorescence difference gel electrophoresis to compare the allergen protein levels in MucoRice-ARP1 and wild-type rice. We detected no notable differences, except in the levels of α-amylase/trypsin inhibitor-like family proteins. Because by this approach we could not completely separate ARP1 from the proteins of this family, we confirmed the absence of changes in the levels of these allergens by using shotgun mass spectrometry as well as immunoblot. By using immunoelectron microscopy, we also showed that RAG2, a member of the α-amylase/trypsin inhibitor-like protein family, was relocated from protein bodies II to the plasma membrane or cell wall in MucoRice-ARP1 seed. The relocation did not affect the level of RAG2. We demonstrated that most of the known rice allergens were not considerably upregulated by the genetic modification in MucoRice-ARP1. Our data suggest that MucoRice-ARP1 is a potentially safe oral antibody for clinical application.

  14. Identification of Critical Amino Acids in an Immunodominant IgE Epitope of Pen c 13, a Major Allergen from Penicillium citrinum

    PubMed Central

    Chen, Jui-Chieh; Chiu, Li-Li; Lee, Kuang-Lun; Huang, Wei-Ning; Chuang, Jiing-Guang; Liao, Hsin-Kai; Chow, Lu-Ping

    2012-01-01

    Background Pen c 13, identified as a 33-kDa alkaline serine protease, is a major allergen secreted by Penicillium citrinum. Detailed knowledge about the epitopes responsible for IgE binding would help inform the diagnosis/prognosis of fungal allergy and facilitate the rational design of hypoallergenic candidate vaccines. The goal of the present study was to characterize the IgE epitopes of Pen c 13. Methodology/Principal Findings Serum samples were collected from 10 patients with mold allergy and positive Pen c 13 skin test results. IgE-binding epitopes on rPen c 13 were mapped using an enzymatic digestion and chemical cleavage method, followed by dot-blotting and mass spectrometry. A B-cell epitope-predicting server and molecular modeling were used to predict the residues most likely involved in IgE binding. Theoretically predicted IgE-binding regions were further confirmed by site-directed mutagenesis assays. At least twelve different IgE-binding epitopes located throughout Pen c 13 were identified. Of these, peptides S16 (A148–E166) and S22 (A243–K274) were recognized by sera from 90% and 100% of the patients tested, and were further confirmed by inhibition assays. Peptide S22 was selected for further analysis of IgE-binding ability. The results of serum screening showed that the majority of IgE-binding ability resided in the C-terminus. One Pen c 13 mutant, G270A (T261–K274), exhibited clearly enhanced IgE reactivity, whereas another, K274A, exhibited dramatically reduced IgE reactivity. Conclusions/Significance Experimental analyses confirmed in silico-predicted residues involved in an important antigenic region of Pen c 13. The G270A mutant of Pen c 13 has the potential to serve as an additional tool for the diagnosis/prognosis of mold allergy, and the K274A mutant, as a hypoallergenic form of the epitope, may provide a framework for the design and development of a safe and efficient therapeutic strategy for treating human allergic diseases. PMID

  15. Identification of critical amino acids in an immunodominant IgE epitope of Pen c 13, a major allergen from Penicillium citrinum.

    PubMed

    Chen, Jui-Chieh; Chiu, Li-Li; Lee, Kuang-Lun; Huang, Wei-Ning; Chuang, Jiing-Guang; Liao, Hsin-Kai; Chow, Lu-Ping

    2012-01-01

    Pen c 13, identified as a 33-kDa alkaline serine protease, is a major allergen secreted by Penicillium citrinum. Detailed knowledge about the epitopes responsible for IgE binding would help inform the diagnosis/prognosis of fungal allergy and facilitate the rational design of hypoallergenic candidate vaccines. The goal of the present study was to characterize the IgE epitopes of Pen c 13. Serum samples were collected from 10 patients with mold allergy and positive Pen c 13 skin test results. IgE-binding epitopes on rPen c 13 were mapped using an enzymatic digestion and chemical cleavage method, followed by dot-blotting and mass spectrometry. A B-cell epitope-predicting server and molecular modeling were used to predict the residues most likely involved in IgE binding. Theoretically predicted IgE-binding regions were further confirmed by site-directed mutagenesis assays. At least twelve different IgE-binding epitopes located throughout Pen c 13 were identified. Of these, peptides S16 (A(148)-E(166)) and S22 (A(243)-K(274)) were recognized by sera from 90% and 100% of the patients tested, and were further confirmed by inhibition assays. Peptide S22 was selected for further analysis of IgE-binding ability. The results of serum screening showed that the majority of IgE-binding ability resided in the C-terminus. One Pen c 13 mutant, G270A (T(261)-K(274)), exhibited clearly enhanced IgE reactivity, whereas another, K274A, exhibited dramatically reduced IgE reactivity. Experimental analyses confirmed in silico-predicted residues involved in an important antigenic region of Pen c 13. The G270A mutant of Pen c 13 has the potential to serve as an additional tool for the diagnosis/prognosis of mold allergy, and the K274A mutant, as a hypoallergenic form of the epitope, may provide a framework for the design and development of a safe and efficient therapeutic strategy for treating human allergic diseases.

  16. Heterogeneous responses and cross reactivity between the major peanut allergens Ara h 1, 2,3 and 6 in a mouse model for peanut allergy.

    PubMed

    Smit, Joost J; Pennings, Maarten T; Willemsen, Karina; van Roest, Manon; van Hoffen, Els; Pieters, Raymond H

    2015-01-01

    The relative contribution and the relation between individual peanut allergens in peanut allergic responses is still matter of debate. We determined the individual contribution of peanut proteins to B, T cell and allergic effector responses in a mouse model for peanut allergy. Mice were immunized and challenged by oral gavage with peanut protein extract or isolated allergens Ara h 1, 2, 3 and 6 followed by assessment of food allergic manifestations. In addition, T cell responses to the individual proteins were measured by an in vitro dendritic cell-T cell assay. Sensitization with the individual peanut proteins elicited IgE responses with specificity to the allergen used as expected. However, cross reactivity among Ara h 1, 2, 3 and 6 was observed. T cell re-stimulations with peanut extract and individual peanut proteins also showed cross reactivity between Ara h 1, 2, 3 and 6. Despite the cross reactivity at the IgE level, only Ara h 2 and 6 were able to elicit mast cell degranulation after an oral challenge. However, after systemic challenge, Ara h 1, 2 and 6 and to lesser extent Ara h 3 were able to elicit anaphylactic responses. Ara h 1, 2, 3 and 6 sensitize via the intra-gastric route, but differ in their capacity to cause allergic effector responses. Interestingly, extensive cross reactivity at T cell and antibody level is observed among Ara h 1, 2, 3 and 6, which may have important implications for the diagnosis and therapy of peanut allergy. Awareness about the relative contribution of individual peanut allergens and cross reactivity between these allergens is of importance for current research in diagnostics and therapeutics for and the mechanism of peanut allergy.

  17. New structural information on food allergens (abstract)

    USDA-ARS?s Scientific Manuscript database

    A small number of protein families are responsible for food allergies suffered by the majority of allergy patients. What properties of these proteins make them allergens is not clear at present. Reliable methods for allergen prediction and mitigation are lacking. Most the immediate type of food alle...

  18. Characterization of Three Glucosyltransferase Genes in Tartary Buckwheat and Their Expression after Cold Stress.

    PubMed

    Zhou, Jing; Li, Cheng-Lei; Gao, Fei; Luo, Xiao-Peng; Li, Qing-Qing; Zhao, Hai-Xia; Yao, Hui-Peng; Chen, Hui; Wang, An-Hu; Wu, Qi

    2016-09-21

    Anthocyanins confer the red color in the hypocotyl of tartary buckwheat sprouts. Uridine diphosphate (UDP)-glucose:flavonoid 3-O-glycosyltransferase (UFGT) stabilizes anthocyanin by attaching the glucosyl moiety from UDP-glucose to the C3 hydroxyl of anthocyanin. In this study, we characterized three UFGT-like genes, designated FtUFGT1, 2, and 3 from tartary buckwheat. The results revealed that FtUFGT1, FtUFGT2, and FtUFGT3 can convert cyanidin to cyanidin 3-O-glucoside, with specific activities of 20.01 × 10(-3), 8.93 × 10(-3), and 20.24 × 10(-3) IU/mg, respectively. The active-site residues of the C-terminal domains and the N-terminal domains are important for the donor and acceptor recognition of these proteins. The expression of the three FtUFGTs paralleled the tissue-specific anthocyanin accumulation. After cold treatment, the increased content of anthocyanin was accompanied by the up-regulated expression of the three FtUFGTs. Among these three UGFT gene members, FtUFGT3 showed the highest expression level and the highest specific activity, suggesting that FtUFGT3 might be the major gene involved in anthocyanin biosynthesis. These results suggested that the FtUFGT genes, FtUFGT3 in particular, might be important candidates for anthocyanin formation in tartary buckwheat sprouts.

  19. Structural identification of anthocyanins and analysis of concentrations during growth and flowering in buckwheat (Fagopyrum esculentum Moench) petals.

    PubMed

    Suzuki, Tatsuro; Kim, Sun-Ju; Mohamed, Zaidul Islam Sarker; Mukasa, Yuji; Takigawa, Shigenobu; Matsuura-Endo, Chie; Yamauchi, Hiroaki; Hashimoto, Naoto; Noda, Takahiro; Saito, Tatsuya

    2007-11-14

    The anthocyanin profiles and variety/breeding-line differences of anthocyanin concentrations in petals of common buckwheat flowers have been studied. Four anthocyanins, cyanidin 3-O-glucoside, cyanidin 3-O-rutinoside, cyanidin 3-O-rhamnoside, and cyanidin 3-O-galactosyl-rhamnoside were isolated from the petals of common buckwheat (Fagopyrum esculentum Moench), separated using high performance liquid chromatography and identified using reversed-phase liquid chromatography-electrospray ionization-tandem mass spectrometry techniques. In every variety/breeding line tested, cyanidin 3-O-rutinoside was detected as the major anthocyanin and the next is cyanidin 3-O-glucoside whereas cyanidin 3-O-rhamnoside and cyanidin 3-O-galactosyl-rhamnoside were trace or not detectable in white and pink flowered buckwheat. Of all the varieties/breeding lines tested, Gan-Chao, a Chinese variety, contained the highest amount of anthocyanins. The largest part of cyanidin moiety was presented as a proanthocyanidin form (PAs-Cy). Anthocyanins and PAs-Cy in petals were increased along with increase of flower development stages. Therefore, fully developed petals of red flowered buckwheat, especially Gan-Chao, are promising as a new anthocyanin-rich material for food processing.

  20. In vitro gastrointestinal digestion of the major peach allergen Pru p 3, a lipid transfer protein: molecular characterization of the products and assessment of their IgE binding abilities.

    PubMed

    Cavatorta, Valeria; Sforza, Stefano; Aquino, Giancarlo; Galaverna, Gianni; Dossena, Arnaldo; Pastorello, Elide Anna; Marchelli, Rosangela

    2010-10-01

    A simulated gastrointestinal digestion has been carried out on purified peach lipid transfer protein, one of the main allergens among the population of the Mediterranean area and the major allergen of peach allergic patients. The percentage of intact protein, after extensive digestion, measured by comparison with a non-digestible peptide analogue used as internal standard, was found to be about one-third of the original protein content. The peptides formed in digested fraction were characterized by means of LC/MS. The products of the digestion essentially derived from trypsin action, whereas the protein appeared to be resistant to pepsin and chymotrypsin. The identified peptides could be classified as low molecular weight and high molecular weight peptides. The latter consisted of the full protein, with the disulfide bridges still intact, deprived of the smaller peptides. The different digestion products, including the high and low molecular weight peptides, were purified by LC and assessed, together with the intact protein, by dot-blot analysis with sera of allergic patients, allowing to estimate their potential allergenicity. The intact protein and the high molecular weight peptides were found to be recognized by patients' sera, whereas the small peptides were found to be not reactive.

  1. Extraction and analysis of coffee bean allergens.

    PubMed

    Lehrer, S B; Karr, R M; Salvaggio, J E

    1978-05-01

    Workers in the coffee industry can develop occupational allergic disease upon exposure to dust associated with coffee manufacturing. Since controversy exists as to the source or chemical nature of these allergens, the mouse model of reaginic antibody production was used to assess the potential sources of allergens in samples obtained from a local coffee manufacturing plant. Mice were immunized with extracts of coffee dust and beans and the resulting reaginic antibody response determined by the passive cutaneous anaphylaxis reaction. Cross-reacting allergens were detected in samples of coffee dust, cleaner can debris and green coffee beans, but not in chaff or roasted coffee beans. None of the allergens detected in coffee samples cross-reacted with extract of castor beans, although these extracts contained the potent castor bean allergen. Green coffee bean allergens partially purified by gel filtration were heterogeneous with respect to molecular size, although quite similar in their reactivity with reaginic antiserum. These results suggest that the green coffee bean is the major source of allergen in coffee manufacturing plants. This allergen is heterogeneous with respect to size and heat lability, and is immunochemically different from the castor bean allergen.

  2. Isolation and characterization of genes encoding leucoanthocyanidin reductase (FeLAR) and anthocyanidin reductase (FeANR) in buckwheat (Fagopyrum esculentum).

    PubMed

    Matsui, Katsuhiro; Hisano, Tomomi; Yasui, Yasuo; Mori, Masashi; Walker, Amanda R; Morishita, Toshikazu; Katsu, Kenjiro

    2016-10-20

    Proanthocyanidins (PAs) are a major group of flavonoids synthesized via the phenylpropanoid biosynthesis pathway, however the pathway has not been fully characterized in buckwheat. Anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR) are involved in the last steps of PA biosynthesis. To isolate the genes for these enzymes from buckwheat we performed PCR using degenerate primers and obtained cDNAs of ANR and LAR, which we designated FeANR and FeLAR1. A search for homologs in a buckwheat genome database with both sequences returned two more LAR sequences, designated FeLAR2 and FeLAR3. Linkage analysis with an F2 segregating population indicated that the three LAR loci were not genetically linked. We detected high levels of PAs in roots and cotyledons of buckwheat seedlings and in buds and flowers of mature plants. FeANR and FeLAR1-3 were expressed in most organs but had different expression patterns. Our findings would be useful for breeding and further analysis of PA synthesis and its regulation in buckwheat. Copyright © 2016 Elsevier GmbH. All rights reserved.

  3. Role of the polypeptide backbone and post-translational modifications in cross-reactivity of Art v 1, the major mugwort pollen allergen.

    PubMed

    Gruber, Petra; Gadermaier, Gabriele; Bauer, Roman; Weiss, Richard; Wagner, Stefan; Leonard, Renaud; Breiteneder, Heimo; Ebner, Christof; Ferreira, Fatima; Egger, Matthias

    2009-01-01

    Artemisia vulgaris (mugwort) is one of the main causes of late summer pollinosis in Europe, with >95% of patients sensitized to the glycoallergen Art v 1. Despite the importance of this allergen, little is known about its cross-reactive behavior. Here we investigated the occurrence of conserved Art v 1 antigenic determinants in sources known to display clinically relevant cross-reactivity with mugwort pollen. For this purpose, monoclonal antibodies specific for a cysteine-stabilized epitope of the Art v 1 defensin domain and for carbohydrates attached to the proline domain were produced by hybridoma and phage display technologies. Using polyclonal Art v 1-specific rabbit sera and antibodies against both the Art v 1 carbohydrate and polypeptide moieties, we could identify cross-reactive structures in pollen from botanically related Asteraceae weeds (Artemisia absinthium, Helianthus annuus and Ambrosia sp.). Homologous allergens were also recognized by IgE from mugwort-sensitized patients and the reactivity could be decreased by serum pre-incubation with natural and recombinant Art v 1. As no cross-reactive structures could be found in foods associated with mugwort pollinosis, we conclude that Art v 1 is poorly involved in mugwort cross-reactivity to food allergens.

  4. Ole e 1, the major allergen from olive (Olea europaea L.) pollen, increases its expression and is released to the culture medium during in vitro germination.

    PubMed

    de Dios Alché, Juan; M'rani-Alaoui, Mohamed; Castro, Antonio Jesús; Rodríguez-García, María Isabel

    2004-09-01

    Ole e 1 is a well-characterized allergenic protein from olive pollen. This paper examines its presence and that of its transcripts during in vitro pollen germination and pollen tube growth. A significant increase of the protein was detected after the emergence of the pollen tube, whereas part of the protein was released into the culture medium throughout pollen germination. A slight increase in the number of Ole e 1 transcripts was also detected prior to the described rise in the protein level. Within the pollen tube, the allergen was localized in the subapical region, mainly in the lumen of endoplasmic reticulum cisternae. Ole e 1 was also localized extracellularly in the vicinity of the pollen tube cell wall. These findings are discussed regarding the biological role attributed to the protein during pollen hydration and pollen tube growth and in terms of their importance for the understanding of the allergenic response in humans. On the basis of recent findings for the LAT52 protein in tomato, we propose that the homologous Ole e 1 protein might participate in a similar signal transduction pathway in olive, to control pregermination and pollen tube emergence and guidance.

  5. High Aluminum Resistance in Buckwheat1

    PubMed Central

    Zheng, Shao Jian; Ma, Jian Feng; Matsumoto, Hideaki

    1998-01-01

    High Al resistance in buckwheat (Fagopyrum esculentum Moench. cv Jianxi) has been suggested to be associated with both internal and external detoxification mechanisms. In this study the characteristics of the external detoxification mechanism, Al-induced secretion of oxalic acid, were investigated. Eleven days of P depletion failed to induce secretion of oxalic acid. Exposure to 50 μm LaCl3 also did not induce the secretion of oxalic acid, suggesting that this secretion is a specific response to Al stress. Secretion of oxalic acid was maintained for 8 h by a 3-h pulse treatment with 150 μm Al. A nondestructive method was developed to determine the site of the secretion along the root. Oxalic acid was found to be secreted in the region 0 to 10 mm from the root tip. Experiments using excised roots also showed that secretion was located on the root tip. Four kinds of anion-channel inhibitors showed different effects on Al-induced secretion of oxalic acid: 10 μm anthracene-9-carboxylic acid and 4,4′-diisothiocyanatostilbene-2,2′-disulfonate had no effect, niflumic acid stimulated the secretion 4-fold, and phenylglyoxal inhibited the secretion by 50%. Root elongation in buckwheat was not inhibited by 25 μm Al or 10 μm phenylglyoxal alone but was inhibited by 40% in the presence of Al and phenylglyoxal, confirming that secretion of oxalic acid is associated with Al resistance. PMID:9662517

  6. Rutin, quercetin, and free amino acid analysis in buckwheat (Fagopyrum) seeds from different locations.

    PubMed

    Bai, C Z; Feng, M L; Hao, X L; Zhong, Q M; Tong, L G; Wang, Z H

    2015-12-29

    In this study, five common buckwheats and nine tartary buckwheats grown at different locations were analyzed for the contents of rutin, quercetin, and amino acids by high-performance liquid chromatography and spectrophotometry. The rutin content was higher than quercetin in buckwheat seeds. Rutin content was in the range from 0.05 (0.05 g per 100 g dry seeds) to 1.35% of buckwheat seeds. Quercetin content varied from 0.01 to 0.17% and in some common buckwheats it was even difficult to detect. Comparatively, tartary buckwheat seeds contained more rutin and quercetin than common buckwheat seeds. Meanwhile, the bran has higher rutin content than the farina in tartary buckwheat seeds, with a respective content of 0.45 to 1.19% and 0.14 to 0.67%. It was found that amino acid contents were around 1.79 to 12.65% (farina) and 5.74 to 7.89% (bran) in common buckwheats, and 1.73 to 5.63% (farina) and 2.64 to 16.78% (bran) in tartary buckwheat seeds. The highest total rutin content was found to be 1.35% in tartary buckwheat seeds from Sichuan, China. The highest total amounts of amino acid were detected to be 20.13% in tartary buckwheat seeds from Changzhi, Shanxi Province (China). Our results suggested that food products made of whole-buckwheat flour are healthier than those made of fine white flour.

  7. 66. Buckwheat Launder, date unknown Historic Photograph, Photographer Unknown; Collection ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    66. Buckwheat Launder, date unknown Historic Photograph, Photographer Unknown; Collection of William Everett, Jr. (Wilkes-Barre, PA), photocopy by Joseph E.B. Elliot - Huber Coal Breaker, 101 South Main Street, Ashley, Luzerne County, PA

  8. Allergenic extracts to diagnose and treat sensitivity to insect venoms and inhaled allergens.

    PubMed

    Khurana, Taruna; Bridgewater, Jennifer L; Rabin, Ronald L

    2017-05-01

    To review allergenic extracts used to diagnose or treat insect allergies, including how the extracts are manufactured and their measurements of potency or concentration. Peer-reviewed articles derived from searching PubMed (National Center for Biotechnology Information) about insect allergies and extract preparation. Encyclopedia of Life (http://www.eol.org/) and http://allergome.org/ were also referenced for background information on insects and associated allergens. Search terms used for the PubMed searches included insect allergens and allergies, Apidae, Vespidae, fire ants, cockroach allergies, insect allergen extract preparation, and standardization. Humans may be sensitized to insect allergens by inhalation or through stings. Cockroaches and moths are predominantly responsible for inhalation insect allergy and are a major indoor allergen in urban settings. Bees, fire ants, and wasps are responsible for sting allergy. In the United States, there are multiple insect allergen products commercially available that are regulated by the US Food and Drug Administration. Of those extracts, honeybee venom and insect venom proteins are standardized with measurements of potency. The remaining insect allergen extracts are nonstandardized products that do not have potency measurements. Sensitization to inhalational and stinging insect allergens is reported worldwide. Crude insect allergen extracts are used for diagnosis and specific immunotherapy. A variety of source materials are used by different manufacturers to prepare these extracts, which may result in qualitative differences that are not reflected in measurements of potency or protein concentration. Copyright © 2017 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  9. Identification and characterization of granule bound starch synthase I (GBSSI) gene of tartary buckwheat (Fagopyrum tataricum Gaertn.).

    PubMed

    Wang, Xun; Feng, Bo; Xu, Zhibin; Sestili, Francesco; Zhao, Guojun; Xiang, Chao; Lafiandra, Domenico; Wang, Tao

    2014-01-25

    Tartary buckwheat (Fagopyrum tataricum Gaertn.) is increasingly considered as an important functional food material because of its rich nutraceutical compounds. Reserve starch is the major component of tartary buckwheat seed. However, the gene sequences and the molecular mechanism of tartary buckwheat starch synthesis are unknown so far. In this study, the complete genomic sequence and full-size cDNA coding tartary buckwheat granule-bound starch synthase I (FtGBSSI), which is responsible for amylose synthesis, were isolated and analyzed. The genomic sequence of the FtGBSSI contained 3947 nucleotides and was composed of 14 exons and 13 introns. The cDNA coding sequence of FtGBSSI shared 63.3%-75.1% identities with those of dicots and 56.6%-57.5% identities with monocots (Poaceae). In deduced amino acid sequence of FtGBSSI, eight motifs conserved among plant starch synthases were identified. A cleavage at the site IVC↓G of FtGBSSI protein produces the chloroplast transit sequence of 78 amino acids and the mature protein of 527 amino acids. The FtGBSSI mature protein showed an identity of 73.4%-77.8% with dicot plants, and 67.6%-70.4% with monocot plants (Poaceae). The mature protein was composed of 20 α-helixes and 16 β-strands, and folds into two main domains, N- and C-terminal domains. The critical residues which are involved in ADP and sugar binding were predicted. These results will be useful to modulate starch composition of buckwheat kernels with the aim to produce novel improved varieties in future breeding programs. © 2013.

  10. Molecular basis of pollen-related food allergy: identification of a second cross-reactive IgE epitope on Pru av 1, the major cherry (Prunus avium) allergen.

    PubMed

    Wiche, Regina; Gubesch, Michaela; König, Herbert; Fötisch, Kay; Hoffmann, Andreas; Wangorsch, Andrea; Scheurer, Stephan; Vieths, Stefan

    2005-01-01

    Birch (Betula verrucosa) pollen-associated food allergy is a well-characterized syndrome, which is due to the cross-reactivity of IgE antibodies to homologous allergens in various foods. One crossreacting area on the major birch pollen allergen Bet v 1 and its homologue in cherry (Prunus avium) Pru av 1 has already been identified. This is the so-called 'P-loop' region, which encompasses amino acid residues around position 45 and is found on the two virtually identical tertiary protein structures. We tried to determine an additional IgE cross-reacting patch on Pru av 1 and Bet v 1. The putative IgE-binding region on Pru av 1 was localized with a mAb (monoclonal antibody) that was generated against Bet v 1, and cross-reacts with several Bet v 1 homologues in food and inhibits the binding of patients' IgE to Pru av 1. mAb reactivity pattern was analysed and amino acid positions 28 and 108 of Pru av 1 were selected and mutated by site-directed mutagenesis. The Pru av 1 mutants were produced as recombinant proteins and characterized for their folding, mAb- and IgE-binding capacity and allergenic potency with a cellular assay using the humanized rat basophilic leukaemia cell line RBL-25/30. Amino acid position 28 is involved in a second major IgE-binding region on Pru av 1 and probably on Bet v 1. The identification of this second major IgE-binding region is an essential prerequisite to understand the phenomenon of cross-reactivity and its clinical consequences, and to produce hypoallergenic proteins for an improved immunotherapy of type I allergy.

  11. Molecular basis of pollen-related food allergy: identification of a second cross-reactive IgE epitope on Pru av 1, the major cherry (Prunus avium) allergen

    PubMed Central

    2004-01-01

    Birch (Betula verrucosa) pollen-associated food allergy is a well-characterized syndrome, which is due to the cross-reactivity of IgE antibodies to homologous allergens in various foods. One crossreacting area on the major birch pollen allergen Bet v 1 and its homologue in cherry (Prunus avium) Pru av 1 has already been identified. This is the so-called ‘P-loop’ region, which encompasses amino acid residues around position 45 and is found on the two virtually identical tertiary protein structures. We tried to determine an additional IgE cross-reacting patch on Pru av 1 and Bet v 1. The putative IgE-binding region on Pru av 1 was localized with a mAb (monoclonal antibody) that was generated against Bet v 1, and cross-reacts with several Bet v 1 homologues in food and inhibits the binding of patients' IgE to Pru av 1. mAb reactivity pattern was analysed and amino acid positions 28 and 108 of Pru av 1 were selected and mutated by site-directed mutagenesis. The Pru av 1 mutants were produced as recombinant proteins and characterized for their folding, mAb- and IgE-binding capacity and allergenic potency with a cellular assay using the humanized rat basophilic leukaemia cell line RBL-25/30. Amino acid position 28 is involved in a second major IgE-binding region on Pru av 1 and probably on Bet v 1. The identification of this second major IgE-binding region is an essential prerequisite to understand the phenomenon of cross-reactivity and its clinical consequences, and to produce hypoallergenic proteins for an improved immunotherapy of type I allergy. PMID:15330760

  12. Allergens in celery and zucchini.

    PubMed

    Vieths, Stefan; Lüttkopf, D; Reindl, J; Anliker, M D; Wüthrich, B; Ballmer-Weber, B K

    2002-01-01

    The aim of this study was to confirm allergy to celery tuber and to zucchini, for the first time, by DBPCFC, and to identify the allergens recognized by IgE from DBPCFC-positive patients. Therefore, raw vegetables were hidden in a broccoli drink, and a DBPCFC-procedure was developed that consisted of a spit and swallow protocol, making sure that the procedure was safe for the patients and that reactions strictly localized to the oral cavity as well as systemic reactions could be reproduced by DBPCFC. The allergens in celery and zucchini extract were identified by immunoblot inhibition using allergen extracts, recombinant allergens and purified N-glycans as inhibitors. Celery allergy was confirmed in 69% (22/32) of subjects with a positive case history. Four subjects with a history of allergic reactions to zucchini had a positive DBPCFC to this vegetable. During DBPCFC, systemic reactions were provoked in 50% (11/22) of the patients to celery, and in 3/4 of the zucchini-allergic patients. The Bet v 1-related major celery allergen was detected by IgE of 59% (13/22) of the patients. Cross-reactive carbohydrate epitopes (CCD) bound IgE of 55% (12/22) of the celery-allergic patients and in 2/4 of the subjects with zucchini allergy. Profilin was a food allergen in celery in 23% (5/22) and in zucchini in 2/4 of the cases. A zucchini-specific allergen was detected by IgE from one patient. We conclude that ubiquitous cross-reactive structures are important in allergy to both, celery and zucchini, and that a specific association to birch pollen allergy exists in allergy to celery (mediated by Api g 1), but not in zucchini allergy.

  13. Latex-allergic patients sensitized to the major allergen hevein and hevein-like domains of class I chitinases show no increased frequency of latex-associated plant food allergy

    PubMed Central

    Radauer, Christian; Adhami, Farzaneh; Fürtler, Irene; Wagner, Stefan; Allwardt, Dorothee; Scala, Enrico; Ebner, Christof; Hafner, Christine; Hemmer, Wolfgang; Mari, Adriano; Breiteneder, Heimo

    2011-01-01

    Allergies to certain fruits such as banana, avocado, chestnut and kiwi are described in 30–70% of latex-allergic patients. This association is attributed to the cross-reactivity between the major latex allergen hevein and hevein-like domains (HLDs) from fruit class I chitinases. We aimed to assess the extent of cross-reactivity between hevein and HLDs using sera from latex-allergic patients with and without plant food allergy. Hevein and HLDs of latex, banana, and avocado chitinases were expressed in Escherichia coli as fusion proteins with the maltose-binding protein and purified by affinity chromatography. IgE binding to these proteins was studied in sera from 59 latex-allergic patients and 20 banana-allergic patients without latex allergy by ELISA and ELISA inhibition. Additionally, 16,408 allergic patients’ sera were tested for IgE binding to hevein, latex chitinase, and wheat germ agglutinin using an allergen microarray. Hevein-specific IgE was detected in 34/59 (58%) latex-allergic patients’ sera. HLDs of latex, banana, and avocado chitinases were recognized by 21 (36%), 20 (34%), and 9 (15%) sera, respectively. In contrast, only one of 20 banana-allergic patients without latex allergy was sensitized to chitinase HLDs. In most tested latex-allergic patients’ sera, IgE binding to hevein was only partially reduced by preincubation with HLDs. Among hevein-sensitized, latex-allergic patients, the percentage of plant food allergy (15/34 = 44%) was equal to latex-allergic patients without hevein sensitization (11/25 = 44%). In the general allergic population, 230 of 16,408 sera (1.4%) reacted to hevein and/or a hevein-like allergen. Of these, 128 sera showed an isolated sensitization to hevein, whereas only 17 bound to latex chitinase or wheat germ agglutinin without hevein sensitization. In conclusion, the IgE response to HLDs is elicited by hevein as sensitizing allergen in most cases. Despite considerable cross-reactivity between these allergens, no

  14. Shellfish allergens: tropomyosin and beyond.

    PubMed

    Faber, M A; Pascal, M; El Kharbouchi, O; Sabato, V; Hagendorens, M M; Decuyper, I I; Bridts, C H; Ebo, D G

    2016-12-27

    IgE-mediated shellfish allergy constitutes an important cause of food-related adverse reactions. Shellfish are classified into mollusks and crustaceans, the latter belonging to the class of arthropoda. Among crustaceans, shrimps are the most predominant cause of allergic reactions and thus more extensively studied. Several major and minor allergens have been identified and cloned. Among them, invertebrate tropomyosin, arginine kinase, myosin light chain, sarcoplasmic calcium-binding protein, and hemocyanin are the most relevant. This review summarizes our current knowledge about these allergens.

  15. Household Arthropod Allergens in Korea

    PubMed Central

    Jeong, Kyoung Yong

    2009-01-01

    Arthropods are important in human health, which can transmit pathogens to humans, parasitize, or produce important allergens. Allergy prevalence becomes higher in Korea recently as well as other developed countries in contrast to a decrease of infectious diseases. Allergic diseases caused by household arthropods have increased dramatically during the last few decades since human beings spend more their time for indoor activities in modernized life style. Household arthropods are one of the most common causes of allergic diseases. Biological characterization of household arthropods and researches on their allergens will provide better understanding of the pathogenesis of allergic diseases and suggest new therapeutic ways. Therefore, studies on arthropods of allergenic importance can be considered one of the major research areas in medical arthropodology and parasitology. Here, the biology of several household arthropods, including house dust mites and cockroaches, the 2 most well known arthropods living indoor together with humans worldwide, and characteristics of their allergens, especially the research activities on these allergens performed in Korea, are summarized. PMID:19885330

  16. Survey of Allergenic Substances in Foods in Chiba Prefecture (Fiscal Years 2004-2014).

    PubMed

    Harada, Rie; Hongo, Takeshi; Hashimoto, Hiroyuki

    2016-01-01

    We surveyed the presence of allergenic substances such as egg, milk, wheat, buckwheat, peanut, shrimp and crab in foods produced or distributed in Chiba Prefecture during the fiscal years 2004-2014. Six hundred and ninety-five samples that did not display the target allergenic substances were screened by using ELISA. Three percent of the samples (21/695 samples) were found to be positive. Wheat had the highest positivity rate of 9.7% (10/103 samples). Among the samples tested, advisory labeling was displayed in the case of 109 samples. Among these samples, 77.1%(84/109 samples) contained less than 1.0 μg/g of the specified allergen. Out of the 21 samples that tested positive in the screening-test, 16 did not display the advisory labeling.

  17. Effect of oleic acid on the allergenic properties of peanut and cashew allergens

    USDA-ARS?s Scientific Manuscript database

    Oleic acid is the major fatty acid in peanuts and cashews. There is limited information about its effect on peanut and cashew allergens during heating. The objective was to determine if heat treatment with oleic acid changes the allergenic properties of these nut proteins. Peanut and cashew protein...

  18. Origin and Functional Prediction of Pollen Allergens in Plants.

    PubMed

    Chen, Miaolin; Xu, Jie; Devis, Deborah; Shi, Jianxin; Ren, Kang; Searle, Iain; Zhang, Dabing

    2016-09-01

    Pollen allergies have long been a major pandemic health problem for human. However, the evolutionary events and biological function of pollen allergens in plants remain largely unknown. Here, we report the genome-wide prediction of pollen allergens and their biological function in the dicotyledonous model plant Arabidopsis (Arabidopsis thaliana) and the monocotyledonous model plant rice (Oryza sativa). In total, 145 and 107 pollen allergens were predicted from rice and Arabidopsis, respectively. These pollen allergens are putatively involved in stress responses and metabolic processes such as cell wall metabolism during pollen development. Interestingly, these putative pollen allergen genes were derived from large gene families and became diversified during evolution. Sequence analysis across 25 plant species from green alga to angiosperms suggest that about 40% of putative pollen allergenic proteins existed in both lower and higher plants, while other allergens emerged during evolution. Although a high proportion of gene duplication has been observed among allergen-coding genes, our data show that these genes might have undergone purifying selection during evolution. We also observed that epitopes of an allergen might have a biological function, as revealed by comprehensive analysis of two known allergens, expansin and profilin. This implies a crucial role of conserved amino acid residues in both in planta biological function and allergenicity. Finally, a model explaining how pollen allergens were generated and maintained in plants is proposed. Prediction and systematic analysis of pollen allergens in model plants suggest that pollen allergens were evolved by gene duplication and then functional specification. This study provides insight into the phylogenetic and evolutionary scenario of pollen allergens that will be helpful to future characterization and epitope screening of pollen allergens. © 2016 American Society of Plant Biologists. All rights reserved.

  19. Quantitation of major allergens in dust samples from urban populations collected in different seasons in two climatic areas of the Basque region (Spain).

    PubMed

    Echechipía, S; Ventas, P; Audícana, M; Urrutia, I; Gastaminza, G; Polo, F; Fernández de Corres, L

    1995-06-01

    We present the results of allergen content evaluation in 80 dust samples from 31 homes of atopic patients from two climatic areas (humid and subhumid), collected in two seasons of the year (autumn and winter). Monoclonal antibody-based immunoassays were used to quantify Der p 1, Der f 1, Der 2, Lep d 1, and Fel d 1. The results were compared according to climate, season, and the type of sensitization (Pyroglyphidae mites, storage mites, or grass pollens). We underline the predominance of Dermatophagoides pteronyssinus (89% of samples) over D. farinae (16% of samples) in our environment. Der p 1 rates were higher in the humid area (Mann-Whitney P < 0.001), especially in the autumn (Wilcoxon P < 0.05). Lep d 1 was detected in 23% of samples and Lep d 1 levels were higher in the homes of patients sensitized to storage mites (Mann-Whitney P < 0.05), whereas this allergen was not detected in the homes of pollen-allergic patients. Fel d 1 was detected in nine of the 31 homes (16% of samples) although there was a cat in only one home.

  20. Antigen presentation of the immunodominant T-cell epitope of the major mugwort pollen allergen, Art v 1, is associated with the expression of HLA-DRB1 *01.

    PubMed

    Jahn-Schmid, Beatrice; Fischer, Gottfried F; Bohle, Barbara; Faé, Ingrid; Gadermaier, Gabriele; Dedic, Azra; Ferreira, Fatima; Ebner, Christof

    2005-02-01

    Mugwort pollen allergens are the main cause of pollinosis in late summer in Europe. Ninety-five percent of patients allergic to mugwort are sensitized to the major allergen Art v 1. In contrast to other common pollen allergens that contain multiple T-cell epitopes, Art v 1 contains only 1 immunodominant T-cell epitope (Art v 1 25-36 ). To characterize the minimal epitope of Art v 1 25-36 and to investigate a possible association of Art v 1 reactivity with HLA class II phenotypes. Art v 1-specific T-cell lines and clones were established from 51 patients with clinically defined mugwort pollen allergy and IgE specific for Art v 1. To define minimal epitopes and binding sites within Art v 1 25-36 , truncated and single-substitution analog peptides were used for T-cell stimulation. To study HLA restriction, monoclonal anti-HLA antibodies and antigen-presenting cells with defined HLA-DRB and -DQB1 alleles were used. HLA typing of patients with allergy was performed by hybridization with sequence-specific oligonucleotides, PCR, and nucleotide sequencing. In 96% of the patients, a cellular response to Art v 1 25-36 was obtained, and a core region of 5 to 10 amino acids containing 3 to 5 amino acids essential for T-cell reactivity was defined. The frequency of HLA-DRB1 * 01 in patients recognizing Art v 1 25-36 was significantly increased as compared with healthy controls (69% vs 21%; odds ratio, 8.45; P < 10 -6 ), and HLA-DRB1 * 01 was identified as the main restriction element for the presentation of the immunodominant epitope. Allergy to Art v 1 is characterized by a uniform T-cell response. The disease is apparently associated with the HLA-DR1 phenotype. Therefore, mugwort pollinosis is an ideal candidate for a peptide-based immunotherapy.

  1. Animal Allergens and Their Presence in the Environment

    PubMed Central

    Zahradnik, Eva; Raulf, Monika

    2014-01-01

    Exposure to animal allergens is a major risk factor for sensitization and allergic diseases. Besides mites and cockroaches, the most important animal allergens are derived from mammals. Cat and dog allergies affect the general population; whereas, allergies to rodents or cattle is an occupational problem. Exposure to animal allergens is not limited to direct contact to animals. Based on their aerodynamic properties, mammalian allergens easily become airborne, attach to clothing and hair, and can be spread from one environment to another. For example, the major cat allergen Fel d 1 was frequently found in homes without pets and in public buildings, including schools, day-care centers, and hospitals. Allergen concentrations in a particular environment showed high variability depending on numerous factors. Assessment of allergen exposure levels is a stepwise process that involves dust collection, allergen quantification, and data analysis. Whereas a number of different dust sampling strategies are used, ELISA assays have prevailed in the last years as the standard technique for quantification of allergen concentrations. This review focuses on allergens arising from domestic, farm, and laboratory animals and describes the ubiquity of mammalian allergens in the human environment. It includes an overview of exposure assessment studies carried out in different indoor settings (homes, schools, workplaces) using numerous sampling and analytical methods and summarizes significant factors influencing exposure levels. However, methodological differences among studies have contributed to the variability of the findings and make comparisons between studies difficult. Therefore, a general standardization of methods is needed and recommended. PMID:24624129

  2. Identification, expression and characterization of a major salivary allergen (Cul s 1) of the biting midge Culicoides sonorensis relevant for summer eczema in horses

    PubMed Central

    Langner, Kathrin F. A.; Jarvis, Donald L.; Nimtz, Manfred; Heselhaus, Julia E.; McHolland, Linda E.; Leibold, Wolfgang; Drolet, Barbara S.

    2009-01-01

    Salivary proteins of Culicoides biting midges are thought to play a key role in summer eczema (SE), a seasonal recurrent allergic dermatitis in horses. The present study describes the identification, expression and clinical relevance of a candidate allergen of the North American midge Culicoides sonorensis. Immunoblot analysis of midge saliva revealed a 66 kDa protein (Cul s 1) that was bound by IgE from several SE-affected (SE+) horses. Further characterization by fragmentation, mass spectrometry and bioinformatics identified Cul s 1 as maltase, an enzyme involved in sugar meal digestion. A cDNA encoding Cul s 1 was isolated and expressed as a polyhistidine-tagged fusion protein in a baculovirus/insect cell expression system. The clinical relevance of the affinity-purified recombinant Cul s 1 (rCul s 1) was investigated by immunoblotting, histamine release testing (HRT) and intradermal testing (IDT) in eight SE+ and eight control horses. Seven SE+ horses had rCul s 1-specific IgE, whereas only one control animal had IgE directed against this allergen. Furthermore, the HRT showed rCul s 1 induced basophil degranulation in samples from seven of eight SE+ horses but in none of the control animals. rCul s 1 also induced immediate (7/8), late-phase (8/8) and delayed (1/8) skin reactivity in IDT on all SE+ horses that had a positive test with the whole body extract (WBE) of C. sonorensis. None of the control horses showed immediate or delayed skin reactivity with rCul s 1, and only one control horse had a positive late-phase response, while several non-specific late-phase reactions were observed with the insect WBE. Thus, we believe rCul s 1 is the first specific salivary allergen of C. sonorensis to be described that promises to advance both in vitro and in vivo diagnosis and may contribute to the development of immunotherapy for SE in horses. PMID:18708061

  3. Cholesterol-Lowering Activity of Tartary Buckwheat Protein.

    PubMed

    Zhang, Chengnan; Zhang, Rui; Li, Yuk Man; Liang, Ning; Zhao, Yimin; Zhu, Hanyue; He, Zouyan; Liu, Jianhui; Hao, Wangjun; Jiao, Rui; Ma, Ka Ying; Chen, Zhen-Yu

    2017-03-08

    Previous research has shown that Tartary buckwheat flour is capable of reducing plasma cholesterol. The present study was to examine the effect of rutin and Tartary buckwheat protein on plasma total cholesterol (TC) in hypercholesterolemia hamsters. In the first animal experiment, 40 male hamsters were divided into four groups fed either the control diet or one of the three experimental diets containing 8.2 mmol rutin, 8.2 mmol quercetin, or 2.5 g kg(-1) cholestyramine, respectively. Results showed that only cholestyramine but not rutin and its aglycone quercetin decreased plasma TC, which suggested that rutin was not the active ingredient responsible for plasma TC-lowering activity of Tartary buckwheat flour. In the second animal experiment, 45 male hamsters were divided into five groups fed either the control diet or one of the four experimental diets containing 24% Tartary buckwheat protein, 24% rice protein, 24% wheat protein, or 5 g kg(-1) cholestyramine, respectively. Tartary buckwheat protein reduced plasma TC more effectively than cholestyramine (45% versus 37%), while rice and wheat proteins only reduced plasma TC by 10-13%. Tartary buckwheat protein caused 108% increase in the fecal excretion of total neutral sterols and 263% increase in the fecal excretion of total acidic sterols. real-time polymerase chain reaction and Western blotting analyses showed that Tartary buckwheat protein affected the gene expression of intestinal Niemann-Pick C1-like protein 1 (NPC1L1), acyl CoA:cholesterol acyltransferase 2 (ACAT2), and ATP binding cassette transporters 5 and 8 (ABCG5/8) in a down trend, whereas it increased the gene expression of hepatic cholesterol-7α -hydroxylase (CYP7A1). It was concluded that Tartary buckwheat protein was at least one of the active ingredients in Tartary buckwheat flour to lower plasma TC, mainly mediated by enhancing the excretion of bile acids via up-regulation of hepatic CYP7A1 and also by inhibiting the absorption of dietary

  4. Expression, production and excretion of Bla g 1, a major human allergen, in relation to food intake in the German cockroach, Blattella germanica.

    PubMed

    Gore, J Chad; Schal, C

    2005-06-01

    The German cockroach, Blattella germanica (Linnaeus) (Dictyoptera: Blattellidae), produces several potent human allergens, one of which, Blattella germanica allergen 1 (Bla g 1), is produced in the midgut and excreted in faeces. We tested with descriptive and experimental approaches the hypothesis that Bla g 1 production is related to food intake in adult males and females of the German cockroach. Bla g 1 mRNA expression in the female midgut (assayed by real time quantitative polymerase chain reaction), her Bla g 1 content (assayed by enzyme-linked immunosorbent assay), and the female's faeces production and its Bla g 1 content tracked a cyclic pattern in relation to the gonadotrophic cycle. All four measures rose as food intake increased, declined before oviposition in relation to diminishing food intake, and remained low while the female carried an egg case for 20 days. After her first clutch of embryos hatched, the female resumed feeding, and faeces and Bla g 1 production increased concomitantly. Both Bla g 1 mRNA expression and Bla g 1 protein levels remained low in experimentally starved females. However, when starved females were allowed to feed, Bla g 1 production elevated and the gonadotrophic cycle resumed. Bla g 1 mRNA expression also increased six-fold in response to feeding compared to starved females. By contrast, there were no apparent cycles in the pattern of Bla g 1 production in males, reflecting their low and non-cyclic food intake. Our results therefore demonstrate that Bla g 1 production in B. germanica is modulated in relation to food intake.

  5. Taxonomy of Allergenic Fungi.

    PubMed

    Levetin, Estelle; Horner, W Elliott; Scott, James A

    2016-01-01

    The Kingdom Fungi contains diverse eukaryotic organisms including yeasts, molds, mushrooms, bracket fungi, plant rusts, smuts, and puffballs. Fungi have a complex metabolism that differs from animals and plants. They secrete enzymes into their surroundings and absorb the breakdown products of enzyme action. Some of these enzymes are well-known allergens. The phylogenetic relationships among fungi were unclear until recently because classification was based on the sexual state morphology. Fungi lacking an obvious sexual stage were assigned to the artificial, now-obsolete category, "Deuteromycetes" or "Fungi Imperfecti." During the last 20 years, DNA sequencing has resolved 8 fungal phyla, 3 of which contain most genera associated with important aeroallergens: Zygomycota, Ascomycota, and Basidiomycota. Advances in fungal classification have required name changes for some familiar taxa. Because of regulatory constraints, many fungal allergen extracts retain obsolete names. A major benefit from this reorganization is that specific immunoglobulin E (IgE) levels in individuals sensitized to fungi appear to closely match fungal phylogenetic relationships. This close relationship between molecular fungal systematics and IgE sensitization provides an opportunity to systematically look at cross-reactivity and permits representatives from each taxon to serve as a proxy for IgE to the group. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  6. [Extraction, isolation and immunological identification of allergens from Pseudosciaena crocea].

    PubMed

    Yang, Rui; Wu, Haiqiang; Liu, Zhigang

    2009-01-01

    To extract, identify and purify the major allergens of Pseudosciaena crocea in order to evaluate the immunological activities. Pseudosciaena crocea proteins were extracted by Coca's buffer. Allergens of the Pseudosciaena crocea were identified by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western-Blotting. Major allergens were isolated by ion exchange chromatagraphy (IEC) and their immunological activities were evaluated by Western-Blotting with the serum of patients as 1st antibody. The molecular weight of the Pseudosciaena crocea protein were between 8-116 kD. The molecular weight of the major allergens of Pseudosciaena crocea were 54, 29, 27, 14 kD. The concentration of allergens of Pseudosciaena crocea after isolated by IEC were highly improved and the immunological activities were kept. The allergens of Pseudosciaena crocea were detected and identified by immunological activity.

  7. Complete Chloroplast Genome Sequence of Tartary Buckwheat (Fagopyrum tataricum) and Comparative Analysis with Common Buckwheat (F. esculentum)

    PubMed Central

    Cho, Kwang-Soo; Yun, Bong-Kyoung; Yoon, Young-Ho; Hong, Su-Young; Mekapogu, Manjulatha; Kim, Kyung-Hee; Yang, Tae-Jin

    2015-01-01

    We report the chloroplast (cp) genome sequence of tartary buckwheat (Fagopyrum tataricum) obtained by next-generation sequencing technology and compared this with the previously reported common buckwheat (F. esculentum ssp. ancestrale) cp genome. The cp genome of F. tataricum has a total sequence length of 159,272 bp, which is 327 bp shorter than the common buckwheat cp genome. The cp gene content, order, and orientation are similar to those of common buckwheat, but with some structural variation at tandem and palindromic repeat frequencies and junction areas. A total of seven InDels (around 100 bp) were found within the intergenic sequences and the ycf1 gene. Copy number variation of the 21-bp tandem repeat varied in F. tataricum (four repeats) and F. esculentum (one repeat), and the InDel of the ycf1 gene was 63 bp long. Nucleotide and amino acid have highly conserved coding sequence with about 98% homology and four genes—rpoC2, ycf3, accD, and clpP—have high synonymous (Ks) value. PCR based InDel markers were applied to diverse genetic resources of F. tataricum and F. esculentum, and the amplicon size was identical to that expected in silico. Therefore, these InDel markers are informative biomarkers to practically distinguish raw or processed buckwheat products derived from F. tataricum and F. esculentum. PMID:25966355

  8. Complete Chloroplast Genome Sequence of Tartary Buckwheat (Fagopyrum tataricum) and Comparative Analysis with Common Buckwheat (F. esculentum).

    PubMed

    Cho, Kwang-Soo; Yun, Bong-Kyoung; Yoon, Young-Ho; Hong, Su-Young; Mekapogu, Manjulatha; Kim, Kyung-Hee; Yang, Tae-Jin

    2015-01-01

    We report the chloroplast (cp) genome sequence of tartary buckwheat (Fagopyrum tataricum) obtained by next-generation sequencing technology and compared this with the previously reported common buckwheat (F. esculentum ssp. ancestrale) cp genome. The cp genome of F. tataricum has a total sequence length of 159,272 bp, which is 327 bp shorter than the common buckwheat cp genome. The cp gene content, order, and orientation are similar to those of common buckwheat, but with some structural variation at tandem and palindromic repeat frequencies and junction areas. A total of seven InDels (around 100 bp) were found within the intergenic sequences and the ycf1 gene. Copy number variation of the 21-bp tandem repeat varied in F. tataricum (four repeats) and F. esculentum (one repeat), and the InDel of the ycf1 gene was 63 bp long. Nucleotide and amino acid have highly conserved coding sequence with about 98% homology and four genes--rpoC2, ycf3, accD, and clpP--have high synonymous (Ks) value. PCR based InDel markers were applied to diverse genetic resources of F. tataricum and F. esculentum, and the amplicon size was identical to that expected in silico. Therefore, these InDel markers are informative biomarkers to practically distinguish raw or processed buckwheat products derived from F. tataricum and F. esculentum.

  9. Spectrum of allergens for Japanese cedar pollinosis and impact of component-resolved diagnosis on allergen-specific immunotherapy.

    PubMed

    Fujimura, Takashi; Kawamoto, Seiji

    2015-10-01

    The high prevalence of Japanese cedar pollinosis in Japan is associated with a negative impact on the quality of life of patients, as well as significant loss of productivity among the workforce in early spring, thus representing a serious social problem. Furthermore, the prevalence is increasing, and has risen by more than 10% in this decade. Cry j 1 and Cry j 2 were identified as the major allergens in Japanese cedar pollen (JCP), and in 2004, the existence of other major and minor allergens were revealed by a combination of two-dimensional electrophoresis and immunoblotting analysis. Allergenome analysis identified a chitinase, a lipid transfer protein, a serine protease, and an aspartic protease as novel IgE-reactive allergens in patients with JCP allergy. Thaumatin-like protein (Cry j 3) was shown to be homologous to Jun a 3, a major allergen from mountain cedar pollen. Isoflavone reductase-like protein was also characterized in a study of a JCP cDNA library. The characterization of component allergens is required to clarify the sensitizer or cross-reactive elicitor allergens for component-resolved diagnosis (CRD). Increasing evidence from numerous clinical trials indicates that CRD can be used to design effective allergen-specific immunotherapy. In this review, we summarize the eight characterized JCP allergens and discuss the impact of CRD and characterization of novel allergens on allergen-specific immunotherapy.

  10. Pb hyperaccumulation and tolerance in common buckwheat (Fagopyrum esculentum Moench).

    PubMed

    Tamura, Hideo; Honda, Munechika; Sato, Takeshi; Kamachi, Hiroyuki

    2005-10-01

    Common buckwheat grown in Pb-contaminated soil was found to accumulate a large amount of Pb in its leaves (8,000 mg/kg DW), stem (2,000 mg/kg DW), and roots (3,300 mg/kg DW), without significant damage. This indicates that buckwheat is a newly recognized Pb hyperaccumulator, which is defined as a plant containing over 1,000 mg/kg of Pb in its shoots on a dry-weight basis. Moreover, it was shown that application of the biodegradable chelator methylglycinediacetic acid trisodium salt at concentrations of up to 20 mmol/kg resulted in a more than five times higher concentration of Pb in the shoot without notable growth inhibitation at up to 10 mmol/kg. These results indicate that buckwheat is a potential phytoremediator of Pb-contaminated soils.

  11. High-Density IgE Recognition of the Major Grass Pollen Allergen Phl p 1 Revealed with Single-Chain IgE Antibody Fragments Obtained by Combinatorial Cloning

    PubMed Central

    Madritsch, Christoph; Gadermaier, Elisabeth; Roder, Uwe W.; Lupinek, Christian; Valenta, Rudolf; Flicker, Sabine

    2015-01-01

    The timothy grass pollen allergen Phl p 1 belongs to the group 1 of highly cross-reactive grass pollen allergens with a molecular mass of ~25–30 kDa. Group 1 allergens are recognized by >95% of grass pollen allergic patients. We investigated the IgE recognition of Phl p 1 using allergen-specific IgE-derived single-chain variable Ab fragments (IgE-ScFvs) isolated from a combinatorial library constructed from PBMCs of a grass pollen–allergic patient. IgE-ScFvs reacted with recombinant Phl p 1 and natural group 1 grass pollen allergens. Using synthetic Phl p 1–derived peptides, the binding sites of two ScFvs were mapped to the N terminus of the allergen. In surface plasmon resonance experiments they showed comparable high-affinity binding to Phl p 1 as a complete human IgE-derived Ab recognizing the allergens’ C terminus. In a set of surface plasmon resonance experiments simultaneous allergen recognition of all three binders was demonstrated. Even in the presence of the three binders, allergic patients’ polyclonal IgE reacted with Phl p 1, indicating high-density IgE recognition of the Phl p 1 allergen. Our results show that multiple IgE Abs can bind with high density to Phl p 1, which may explain the high allergenic activity and sensitizing capacity of this allergen. PMID:25637023

  12. Protocol for simultaneous isolation of three important banana allergens.

    PubMed

    Nikolic, Jasna; Mrkic, Ivan; Grozdanovic, Milica; Popovic, Milica; Petersen, Arnd; Jappe, Uta; Gavrovic-Jankulovic, Marija

    2014-07-01

    Banana fruit (Musa acuminata) has become an important food allergen source in recent years. So far, 5 IgE reactive banana proteins have been identified, and the major allergens are: Mus a 2 (a class I chitinase, 31kDa), Mus a 4 (thaumatin-like protein, 21kDa), and Mus a 5 (β-1,3-glucanase, 33kDa). Due to variations in allergen expression levels, diagnostic reagents for food allergy can be improved by using individual allergen components instead of banana allergen extracts. The purpose of this study was to optimize the purification protocol of the three major allergens present in banana fruit: Mus a 2, Mus a 4 and Mus a 5. By employing a three-step purification protocol (a combination of anion-exchange, cation-exchange and reversed-phase chromatography) three important banana allergens were obtained in sufficient yield and high purity. Characterization of the purified proteins was performed by both biochemical (2-D PAGE, mass fingerprint and N-terminal sequencing) and immunochemical (immunoblot) methods. IgE reactivity to the purified allergens was tested by employing sera of five allergic patients. The purified allergens displayed higher sensitivity in IgE detection than the routinely used extracts. The three purified allergens are good candidates for reagents in component-based diagnosis of banana allergy.

  13. Distribution of peanut allergen in the environment.

    PubMed

    Perry, Tamara T; Conover-Walker, Mary Kay; Pomés, Anna; Chapman, Martin D; Wood, Robert A

    2004-05-01

    Patients with peanut allergy can have serious reactions to very small quantities of peanut allergen and often go to extreme measures to avoid potential contact with this allergen. The purpose of this study was to detect peanut allergen under various environmental conditions and examine the effectiveness of cleaning agents for allergen removal. A monoclonal-based ELISA for Arachis hypogaea allergen 1 (Ara h 1; range of detection, 30-2000 ng/mL) was used to assess peanut contamination on cafeteria tables and other surfaces in schools, the presence of residual peanut protein after using various cleaning products on hands and tabletops, and airborne peanut allergen during the consumption of several forms of peanut. After hand washing with liquid soap, bar soap, or commercial wipes, Ara h 1 was undetectable. Plain water and antibacterial hand sanitizer left detectable Ara h 1 on 3 of 12 and 6 of 12 hands, respectively. Common household cleaning agents removed peanut allergen from tabletops, except dishwashing liquid, which left Ara h 1 on 4 of 12 tables. Of the 6 area preschools and schools evaluated, Ara h 1 was found on 1 of 13 water fountains, 0 of 22 desks, and 0 of 36 cafeteria tables. Airborne Ara h 1 was undetectable in simulated real-life situations when participants consumed peanut butter, shelled peanuts, and unshelled peanuts. The major peanut allergen, Ara h 1, is relatively easily cleaned from hands and tabletops with common cleaning agents and does not appear to be widely distributed in preschools and schools. We were not able to detect airborne allergen in many simulated environments.

  14. Analysis of olive allergens.

    PubMed

    Esteve, C; Montealegre, C; Marina, M L; García, M C

    2012-04-15

    Olive pollen is one of the most important causes of seasonal respiratory allergy in Mediterranean countries, where this tree is intensely cultivated. Besides this, some cases of contact dermatitis and food allergy to the olive fruit and olive oil have been also described. Several scientific studies dealing with olive allergens has been reported, being the information available about them constantly increasing. Up to date, twelve allergens have been identified in olive pollen while just one allergen has been identified in olive fruit. This review article describes considerations about allergen extraction and production, also describing the different methodologies employed in the physicochemical and immunological characterization of olive allergens. Finally, a revision of the most relevant studies in the analysis of both olive pollen and olive fruit allergens is carried out.

  15. Molecular approaches to allergen standardization.

    PubMed

    Chapman, Martin D; Briza, Peter

    2012-10-01

    Molecular approaches to allergen standardization include the development of purified natural or recombinant allergen standards whose structural and allergenic properties have been validated, in tandem with certified immunoassays for allergen measurement. Purified allergens can be used individually or incorporated into multiple allergen standards. Multicenter international collaborative studies are required to validate candidate allergen standards and immunoassays, as a prelude to being approved by regulatory agencies. Mass spectrometry is a sophisticated and powerful proteomics tool that is being developed for allergen analysis. Recent results using pollen allergens show that mass spectrometry can identify and measure specific allergens in a complex mixture and can provide precise information of the variability of natural allergen extracts. In future, the combined use of immunoassays and mass spectrometry will provide complete standardization of allergenic products. Molecular standardization will form the basis of new allergy diagnostics and therapeutics, as well as assessment of environmental exposure, and will improve the quality of treatment options for allergic patients.

  16. Discovery and genetic analysis of non-bitter Tartary buckwheat (Fagopyrum tataricum Gaertn.) with trace-rutinosidase activity

    PubMed Central

    Suzuki, Tatsuro; Morishita, Toshikazu; Mukasa, Yuji; Takigawa, Shigenobu; Yokota, Satoshi; Ishiguro, Koji; Noda, Takahiro

    2014-01-01

    In a screening of about 500 lines of Tartary buckwheat, we identified lines that contained no detectable rutinosidase isozymes using an in-gel detection assay. We confirmed that seeds of these individuals had only a trace level of in-vitro rutinosidase activity. To investigate the heritability of the trace-rutinosidase characteristic, we analyzed the progeny of crosses between rutinosidase trace-lines, ‘f3g-162’, and the ‘Hokkai T8’. The F2 progeny clearly divided into two groups: those with rutinosidase activity under 1.5 nkat/g seed (trace-rutinosidase) and those with activity over 400 nkat/g seed (normal rutinosidase). The segregation pattern of this trait in F2 progeny exhibited 1 : 3 ratio (trace-rutinosidase : normal rutinosidase), suggesting that the trace-rutinosidase trait is conferred by a single recessive gene; rutinosidase-trace A (rutA). In addition, sensory panelists evaluated the bitterness of flour from trace-rutinosidase individuals and did not detect bitterness, whereas flour from normal rutinosidase individuals was found to have strong bitterness. Although at least three bitter compounds have been reported in Tartary buckwheat seeds, our present findings indicate that rutin hydrolysis is the major contributing factor to bitterness. In addition, the trace-rutinosidase line identified here, ‘f3g-162’, is a promising material for generating a non-bitter Tartary buckwheat variety. PMID:25914588

  17. Discovery and genetic analysis of non-bitter Tartary buckwheat (Fagopyrum tataricum Gaertn.) with trace-rutinosidase activity.

    PubMed

    Suzuki, Tatsuro; Morishita, Toshikazu; Mukasa, Yuji; Takigawa, Shigenobu; Yokota, Satoshi; Ishiguro, Koji; Noda, Takahiro

    2014-12-01

    In a screening of about 500 lines of Tartary buckwheat, we identified lines that contained no detectable rutinosidase isozymes using an in-gel detection assay. We confirmed that seeds of these individuals had only a trace level of in-vitro rutinosidase activity. To investigate the heritability of the trace-rutinosidase characteristic, we analyzed the progeny of crosses between rutinosidase trace-lines, 'f3g-162', and the 'Hokkai T8'. The F2 progeny clearly divided into two groups: those with rutinosidase activity under 1.5 nkat/g seed (trace-rutinosidase) and those with activity over 400 nkat/g seed (normal rutinosidase). The segregation pattern of this trait in F2 progeny exhibited 1 : 3 ratio (trace-rutinosidase : normal rutinosidase), suggesting that the trace-rutinosidase trait is conferred by a single recessive gene; rutinosidase-trace A (rutA). In addition, sensory panelists evaluated the bitterness of flour from trace-rutinosidase individuals and did not detect bitterness, whereas flour from normal rutinosidase individuals was found to have strong bitterness. Although at least three bitter compounds have been reported in Tartary buckwheat seeds, our present findings indicate that rutin hydrolysis is the major contributing factor to bitterness. In addition, the trace-rutinosidase line identified here, 'f3g-162', is a promising material for generating a non-bitter Tartary buckwheat variety.

  18. Hypolipidemic activity of common (Fagopyrum esculentum Moench) and tartary (Fagopyrum tataricum Gaertn.) buckwheat.

    PubMed

    Tomotake, Hiroyuki; Kayashita, Jun; Kato, Norihisa

    2015-08-15

    Buckwheat grain has well-balanced nutritional value, whereas its digestibility is relatively low. This review summarizes recent advances in studies on the hypolipidemic activity of buckwheat. The most remarkable function is a powerful hypocholesterolemic activity of buckwheat protein in rats, which is far stronger than that of soy protein. The cholesterol-lowering effect is mediated by mechanisms involving higher excretion of fecal sterols and lower digestibility of buckwheat protein. The insoluble fraction of buckwheat protein associates with cholesterol and reduces micelle cholesterol uptake in caco-2 cells. Furthermore, consumption of buckwheat protein suppresses cholesterol-induced gallstones and body fat in rodents. Buckwheat sprouts also have hypolipidemic activity in rats or type 2 diabetic mice. Tartary buckwheat bran extract reduced the serum level of total cholesterol and triglyceride in hyperlipidemic rats. The consumption of buckwheat seed reduced low-density lipoprotein cholesterol in the pastureland Mongolian population. Taken together, buckwheat may be beneficial for prevention of hyperlipidemia. © 2014 Society of Chemical Industry.

  19. Food processing and allergenicity.

    PubMed

    Verhoeckx, Kitty C M; Vissers, Yvonne M; Baumert, Joseph L; Faludi, Roland; Feys, Marcel; Flanagan, Simon; Herouet-Guicheney, Corinne; Holzhauser, Thomas; Shimojo, Ryo; van der Bolt, Nieke; Wichers, Harry; Kimber, Ian

    2015-06-01

    Food processing can have many beneficial effects. However, processing may also alter the allergenic properties of food proteins. A wide variety of processing methods is available and their use depends largely on the food to be processed. In this review the impact of processing (heat and non-heat treatment) on the allergenic potential of proteins, and on the antigenic (IgG-binding) and allergenic (IgE-binding) properties of proteins has been considered. A variety of allergenic foods (peanuts, tree nuts, cows' milk, hens' eggs, soy, wheat and mustard) have been reviewed. The overall conclusion drawn is that processing does not completely abolish the allergenic potential of allergens. Currently, only fermentation and hydrolysis may have potential to reduce allergenicity to such an extent that symptoms will not be elicited, while other methods might be promising but need more data. Literature on the effect of processing on allergenic potential and the ability to induce sensitisation is scarce. This is an important issue since processing may impact on the ability of proteins to cause the acquisition of allergic sensitisation, and the subject should be a focus of future research. Also, there remains a need to develop robust and integrated methods for the risk assessment of food allergenicity. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  20. Removing peanut allergens by tannic acid.

    PubMed

    Chung, Si-Yin; Reed, Shawndrika

    2012-10-01

    Tannic acid (TA) forms insoluble complexes with proteins. The aims here were to remove major peanut allergens as insoluble TA complexes and determine if they would dissociate and release the allergens at pH 2 and 8 (gut pH). Release of the allergens in the gut could lead to absorption and consequently an allergic reaction. TA (0.25, 0.5, 1, and 2 mg/ml) was added to a peanut butter extract (5 mg/ml; pH 7.2), stirred, and centrifuged. The precipitates were then suspended in buffer at pH 2, centrifuged, re-suspended at pH 8, and centrifuged. Supernatants from each step were analysed by SDS-PAGE, ELISA, and Western blots. The effect of NaCl (1M) on complexes was also determined. Results showed that complexes formed at a TA concentration >0.5 mg/ml did not release major peanut allergens at pH 2 and 8, regardless of 1M NaCl being present or not. IgE binding of the extracts was reduced substantially, especially at a TA concentration of 1-2 mg/ml. Animal or clinical studies are still needed before TA can find an application in the development of low-allergen peanut products/beverages or the removal of peanut allergens due to accidental ingestion. Published by Elsevier Ltd.

  1. Allergen immunotherapy and allergic rhinitis: false beliefs

    PubMed Central

    2013-01-01

    Background Over the last 100 years, several persistent misconceptions or ‘false beliefs’ have built up around allergen immunotherapy and its use in allergic rhinitis. This is perhaps because enthusiastic physicians administered complex allergen extracts to a diverse population of patients suffering from heterogeneous atopic conditions. Here, we review evidence that counters seven of these ‘false beliefs.’ Discussion 1. The symptoms of allergic rhinitis can be more heterogeneous, more severe and more troublesome in everyday life than many physicians believe. Large-scale epidemiological surveys show that the majority of allergic rhinitis patients have at least one symptom severe enough to interfere with sleep quality, productivity and/or well-being. 2. Allergen immunotherapy is not necessarily suitable for all allergic rhinitis patients (notably those with mild symptoms). Recent evidence from double-blind, placebo-controlled, randomized clinical trials suggests that the more severe the disease, the greater the treatment effect. 3. Allergen immunotherapy is often accused of lack of efficacy (relative to pharmacotherapy, for example). However, there are now many meta-analyses, systematic reviews and high-quality clinical trials that find overwhelmingly in favor of the efficacy of allergen immunotherapy (including sublingual formulations) in allergic rhinitis induced by pollen and, increasingly, other allergens. 4. Natural-exposure and challenge-chamber trials have shown that symptom relief may become apparent within months or even weeks of the initiation of allergen immunotherapy. 5. In pollen-induced allergic rhinitis, several years of subcutaneous or sublingual allergen immunotherapy are associated with sustained clinical efficacy after subsequent treatment cessation – confirming the disease-modifying nature of this therapy. 6. Most patients seeking treatment for allergic rhinitis are polysensitized, and allergen immunotherapy has proven efficacy in large

  2. Wind-pollination and the roles of pollen allergenic proteins.

    PubMed

    Songnuan, Wisuwat

    2013-12-01

    Over the past few decades, there has been an explosion of understanding of the molecular nature of major allergens contained within pollens from the most important allergenic plant species. Most major allergens belong to only a few protein families. Protein characteristics, cross-reactivity, structures, and IgE binding epitopes have been determined for several allergens. These efforts have led to significant improvements in specific immunotherapy, yet there has been little discussion about the physiological functions of these proteins. Even with large amounts of available information about allergenic proteins from pollens, the incidence of pollen allergy continuously increases worldwide. The reason for this increase is unclear and is most likely due to a combination of factors. One important culprit might be a change in the pollen itself. Knowledge about pollen biology and how pollen is changing as a result of more extreme environmental conditions might improve our understanding of the disease. This review focuses on the characteristics of plants producing allergenic pollens that are relevant to pollen allergy, including the phylogenetic relationships, pollen dispersal distances, amounts of pollen produced, amounts of protein in each type of pollen, and how allergenic proteins are released from pollens. In addition, the physiological roles of major allergenic protein families will be discussed to help us understand why some of these proteins become allergens and why GMO plants with hypoallergenic pollens may not be successful.

  3. Tree pollen allergens-an update from a molecular perspective.

    PubMed

    Asam, C; Hofer, H; Wolf, M; Aglas, L; Wallner, M

    2015-10-01

    It is estimated that pollen allergies affect approximately 40% of allergic individuals. In general, tree pollen allergies are mainly elicited by allergenic trees belonging to the orders Fagales, Lamiales, Proteales, and Pinales. Over 25 years ago, the gene encoding the major birch pollen allergen Bet v 1 was the first such gene to be cloned and its product characterized. Since that time, 53 tree pollen allergens have been identified and acknowledged by the WHO/IUIS allergen nomenclature subcommittee. Molecule-based profiling of allergic sensitization has helped to elucidate the immunological connections of allergen cross-reactivity, whereas advances in biochemistry have revealed structural and functional aspects of allergenic proteins. In this review, we provide a comprehensive overview of the present knowledge of the molecular aspects of tree pollen allergens. We analyze the geographic distribution of allergenic trees, discuss factors pivotal for allergic sensitization, and describe the role of tree pollen panallergens. Novel allergenic tree species as well as tree pollen allergens are continually being identified, making research in this field highly competitive and instrumental for clinical applications.

  4. Worldwide food allergy labeling and detection of allergens in processed foods.

    PubMed

    Taylor, Steve L; Baumert, Joseph L

    2015-01-01

    The labeling of allergenic foods is an important public health measure to assist food-allergic consumers in avoiding foods that can cause allergic reactions. The regulatory framework for such labeling depends upon the selection of priority allergenic foods, which vary among countries. Most countries include milk, eggs, fish, crustacean shellfish, peanuts, tree nuts, soybeans, and cereal sources of gluten on the priority allergenic foods list, as recommended by the Codex Alimentarius Commission. However, a variety of other foods appear on the priority lists of some countries but not on others. Sesame seeds, molluscan shellfish, buckwheat, and mustard are identified in two or more countries. In most countries, all ingredients derived from these priority allergen sources must also be declared on labels by source. However, exemptions exist for some ingredients in some countries but not in others. Detection methods are critical for the enforcement of allergen labeling regulations and for the investigation of allergic reactions in the community by public health officials. The development of detection methods has advanced considerably over the past several decades and will be briefly reviewed in this chapter. Because of the emphasis on labeling and the development of detection methods, the ingredient statement on packaged food labels now contains more information than ever before to assist food-allergic consumers.

  5. Allergen immunotherapy for birch pollen-allergic patients: recent advances.

    PubMed

    Moingeon, Philippe; Floch, Véronique Bordas-Le; Airouche, Sabi; Baron-Bodo, Véronique; Nony, Emmanuel; Mascarell, Laurent

    2016-05-01

    As of today, allergen immunotherapy is performed with aqueous natural allergen extracts. Recombinant allergen vaccines are not yet commercially available, although they could provide patients with well-defined and highly consistent drug substances. As Bet v 1 is the major allergen involved in birch pollen allergy, with more than 95% of patients sensitized to this allergen, pharmaceutical-grade recombinant Bet v 1-based vaccines were produced and clinically tested. Herein, we compare the clinical results and modes of action of treatments based on either a birch pollen extract or recombinant Bet v 1 expressed as hypoallergenic or natural-like molecules. We also discuss the future of allergen immunotherapy with improved drugs intended for birch pollen-allergic patients suffering from rhinoconjunctivitis.

  6. Tartary buckwheat sprout powder lowers plasma cholesterol level in rats.

    PubMed

    Kuwabara, Tomoko; Han, Kyu-Ho; Hashimoto, Naoto; Yamauchi, Hiroaki; Shimada, Ken-Ichiro; Sekikawa, Mitsuo; Fukushima, Michihiro

    2007-12-01

    We examined the effects of different types of buckwheat sprouts on the plasma cholesterol concentration, fecal steroid excretion and hepatic mRNA expression related to cholesterol metabolism in rats. Rats were fed a cholesterol-free diet with 5 g of Kitawasesoba common buckwheat sprout powder (KS)/100 g, 5 g of Hokkai T no. 8 tartary buckwheat sprout powder (HS-8)/100 g or 5 g of Hokkai T no. 9 tartary buckwheat sprout powder (HS-9)/100 g of diet for 4 wk. Control rats were fed a diet with alpha-cornstarch instead of sprout powder for 4 wk. There were no significant differences in food intake, body weight, liver weight or cecal contents among the groups. Plasma total cholesterol concentrations in the HS-8 and HS-9 groups were significantly lower than in the control group, whereas there was no significant difference between the KS and control groups. Fecal bile acid excretion and cecal short-chain fatty acid concentrations in the KS, HS-8 and HS-9 groups were significantly greater than in the control group. Furthermore, fecal matter excretion in the KS, HS-8 and HS-9 groups tended to be increased compared to the control group, with that in the HS-8 group being significantly higher than in the control group. Hepatic cholesterol 7alpha-hydroxylase mRNA expression in the KS, HS-8 and HS-9 groups and hepatic HMG-CoA reductase mRNA expression in the HS-9 group were significantly higher than in the control group. The results suggest that tartary buckwheat sprout powder has a serum cholesterol-lowering function by enhancing fecal bile acid excretion through increased fecal matter excretion or the upregulation of hepatic cholesterol 7alpha-hydroxylase mRNA expression in rats.

  7. Criteria to determine food allergen priority.

    PubMed

    Yeung, J M; Applebaum, R S; Hildwine, R

    2000-07-01

    The emergent health issue of food allergens presents an important challenge to the food industry. More than 170 foods have been reported in the scientific literature as causing allergic reactions. Clearly, it would be impossible to deal with the presence of trace amounts of all these in the context of food labeling. If the decision to classify major allergens is based solely on the knowledge and experience of allergists and food scientists in the field, without scientifically defined criteria, it is likely to lead to a proliferation of lists. Such practices may lead to an unnecessary elimination of foods containing important nutrients. This paper defines food allergy, food intolerance, and food anaphylaxis and identifies criteria for classifying food allergens associated with frequent allergic reactions. A practical list of food allergens that may result in potentially life-threatening allergic reactions is provided. A mechanism-based (i.e., immunoglobulin E mediated), acute life-threatening anaphylaxis that is standardized and measurable and reflects the severity of health risk is proposed as the principal inclusion criterion for food allergen labeling. Where available, prevalence in the population and threshold levels of allergens should be used as an additional guide to identify possible future labeling needs.

  8. Clinical and laboratory findings of childhood buckwheat allergy in a single tertiary hospital

    PubMed Central

    Park, Kyujung; Jeong, Kyunguk

    2016-01-01

    Purpose Buckwheat allergy is one of the most severe types of food allergy in some countries, especially among children. However, few studies have investigated this condition. The aim of this study was to report the clinical and laboratory findings in Korean children with buckwheat allergy. Methods Thirty-seven subjects, aged 1 to 14 years, were enrolled by retrospective medical record review from January 2000 through May 2015 at the Department of Pediatrics in Ajou University Hospital. The demographic profile, previous exposure to buckwheat pillows, clinical symptoms, and laboratory findings of each subject were recorded. Results Twenty-six of the 37 children had immediate-type allergic symptoms to buckwheat, while 11 subjects were tolerant to buckwheat. Seventeen out of 26 buckwheat allergic children (65.4%) had anaphylaxis. The median buckwheat specific IgE level in the buckwheat allergic group (7.71 kUA/L) was significantly higher (P<0.001) than in the buckwheat tolerant group (0.08 kUA/L) with an optimal cutoff value of 1.27 kUA/L (sensitivity 84.6%, specificity 100%). When adjusted for age, the difference between the 2 groups showed no statistical significance (P=0.063). In subjects who had anaphylaxis, buckwheat-specific IgE levels ranged from 0.37 to 100 kUA/L. Conclusion Almost two-thirds of buckwheat-allergic children had anaphylaxis, and a wide-range of buckwheat specific IgE levels were observed in these children. Anaphylaxis occurred in a subject with a remarkably low IgE level (0.37 kUA/L). PMID:27826326

  9. Crosslinking of peanut allergen Ara h 2 by polyphenol oxidase: digestibility and potential allergenicity assessment.

    PubMed

    Wu, Zhihua; Lian, Jun; Han, Yuanlong; Zhou, Ningling; Li, Xin; Yang, Anshu; Tong, Ping; Chen, Hongbing

    2016-08-01

    Peanut is one of the eight major food allergens. Its allergen, Ara h 2, can be recognized by over 90% of serum IgE samples from peanut-allergic patients. Therefore, reducing the allergenicity of Ara h 2 is especially important. In the present study, polyphenol oxidase (PPO), a protein cross-linking reaction catalyst that acts on tyrosine residue, was used to modify Ara h 2. After crosslinking, the microstructure, digestibility, IgG binding capability and IgE binding capability of Ara h 2 were analyzed. Cross-linking decreased the potential allergenicity of Ara h 2 by masking the allergen epitope, while the antigenicity of Ara h 2 changed slightly. After crosslinking, the apparent diameter of Ara h 2 was altered from 300 to 1700 nm or 220 nm, indicating that polymerization could either be inter- or intramolecular. Regarding digestibility, crosslinked Ara h 2 was relatively more easily digested by gastric fluid compared with the untreated Ara h 2, but much more difficult in the intestinal fluid. The crosslinking reaction catalyzed by PPO, as a non-thermal process, may be beneficial for avoiding food allergy. The reaction could mask allergen epitopes, decreasing the allergenicity of Ara h 2. © 2015 Society of Chemical Industry. © 2015 Society of Chemical Industry.

  10. Allergenicity and allergens of amphipods found in nori (dried laver).

    PubMed

    Motoyama, Kanna; Hamada, Yuki; Nagashima, Yuji; Shiomi, Kazuo

    2007-09-01

    Gammaridean and caprellid amphipods, crustaceans of the order Amphipoda, inhabit laver culture platforms and, hence, are occasionally found in nori (dried laver) sheets. Amphipods mixed in nori may cause allergic reactions in sensitized patients, as is the case with other crustaceans, such as shrimp and crab, members of the order Decapoda. In this study, dried samples of amphipods (unidentified) found in nori and fresh samples of gammaridean amphipod (Gammarus sp., not accurately identified) and caprellid amphipod (Caprella equilibra) were examined for allergenicity and allergens using two species of decapods (black tiger prawn and spiny lobster) as references. When analyzed by ELISA, sera from crustacean-allergic patients reacted to extracts from amphipod samples, although less potently than to the extracts from decapods. In IgE-immunoblotting, a 37-kDa protein was found to be the major allergen in amphipods. Based on the molecular mass and the cross-reactivity with decapod tropomyosin evidenced by inhibition ELISA and inhibition immunoblotting, the 37-kDa protein was identified as amphipod tropomyosin.

  11. Characterization of allergens of Penicillium and Aspergillus species.

    PubMed

    Shen, H D; Han, S H

    1998-09-01

    Penicillium and Aspergillus species are common indoor airborne fungi and have been identified to be important causative agents of extrinsic bronchial asthma. However, little was known about allergens of these ubiquitous fungal species. Results from a survey conducted by us showed that P. citrinum was the most prevalent Penicillium species in the Taipei area. Characterization of allergens by SDS-PAGE-immunoblotting using sera from asthmatic patients showed that there was an IgE cross-reactivity among the 33 KDa group major allergens of P. citrinum, P. notatum and P. brevicompactum. Results obtained from N-terminal amino acid sequence analysis suggest that the 33 KDa major allergens of P. citrinum and P. brevicompactum may be the alkaline serine proteinase of individual Penicillium species. In addition, our results suggest that the 34 KDa major allergen of A. oryzae is also an alkaline serine proteinase. IgE cross-reactivity between the major serine proteinase allergens of A. oryzae and P. citrinum has also been detected. Furthermore, results from cDNA cloning suggest that the 68 KDa allergen of P. notatum is a beta-N-acetyl-glucosaminidase. Lastly, a heat shock protein in the hsp70 family has also been identified as an allergen of P. citrinum. Results obtained in these studies will provide important basis for clinical diagnosis and treatment of mould allergy.

  12. Characterization of maize chitinase-A, a tough allergenic molecule

    USDA-ARS?s Scientific Manuscript database

    Food allergy is recognized as a major health concern with a steady increasing trend in Western countries. Food allergens are proteins belonging to a small group of about 30 families, with restricted biochemical functions. This leads to the assumption that allergens must meet specific, but not yet co...

  13. Reducing peanut allergens by high pressure combined with polyphenol oxidase

    USDA-ARS?s Scientific Manuscript database

    Polyphenol oxidase (PPO) has been shown to reduce major peanut allergens (Ara h 1 and Ara h 2). Because high pressure (HP) can increase enzyme activity, we postulated that further reduction of peanut allergens can be achieved through HP combined with PPO. Peanut extracts were treated with each of th...

  14. Buckwheat Honey Attenuates Carbon Tetrachloride-Induced Liver and DNA Damage in Mice

    PubMed Central

    Cheng, Ni; Wu, Liming; Zheng, Jianbin; Cao, Wei

    2015-01-01

    Buckwheat honey, which is widely consumed in China, has a characteristic dark color. The objective of this study was to investigate the protective effects of buckwheat honey on liver and DNA damage induced by carbon tetrachloride in mice. The results revealed that buckwheat honey had high total phenolic content, and rutin, hesperetin, and p-coumaric acid were the main phenolic compounds present. Buckwheat honey possesses super DPPH radical scavenging activity and strong ferric reducing antioxidant power. Administration of buckwheat honey for 10 weeks significantly inhibited serum lipoprotein oxidation and increased serum oxygen radical absorbance capacity. Moreover, buckwheat honey significantly inhibited aspartate aminotransferase and alanine aminotransferase activities, which are enhanced by carbon tetrachloride. Hepatic malondialdehyde decreased and hepatic antioxidant enzymes (superoxide dismutase and glutathione peroxidase) increased in the presence of buckwheat honey. In a comet assay, lymphocyte DNA damage induced by carbon tetrachloride was significantly inhibited by buckwheat honey. Therefore, buckwheat honey has a hepatoprotective effect and inhibits DNA damage, activities that are primarily attributable to its high antioxidant capacity. PMID:26508989

  15. Allergens in School Settings: Results of Environmental Assessments in 3 City School Systems

    ERIC Educational Resources Information Center

    Abramson, Stuart L.; Turner-Henson, Anne; Anderson, Lise; Hemstreet, Mary P.; Bartholomew, L. Kay; Joseph, Christine L. M.; Tang, Shenghui; Tyrrell, Shellie; Clark, Noreen M.; Ownby, Dennis

    2006-01-01

    Environmental allergens are major triggers for pediatric asthma. While children's greatest exposure to indoor allergens is in the home, other public places where children spend a large amount of time, such as school and day care centers, may also be sources of significant allergen encounters. The purpose of this article is to describe schoolroom…

  16. Identification and characterization of a new pecan [Cara illinoinensis (Wangenh.) K. Koch] allergen, Car i 2

    USDA-ARS?s Scientific Manuscript database

    The 7S and 11S seed storage globulins belong to the cupin protein superfamily and are major food allergens in many foods that are constitutors to the “big eight” groups of food allergen sources. Here, pecan vicilin was found to be a new food allergen. The vicilin protein consists a low-complexity re...

  17. Allergens in School Settings: Results of Environmental Assessments in 3 City School Systems

    ERIC Educational Resources Information Center

    Abramson, Stuart L.; Turner-Henson, Anne; Anderson, Lise; Hemstreet, Mary P.; Bartholomew, L. Kay; Joseph, Christine L. M.; Tang, Shenghui; Tyrrell, Shellie; Clark, Noreen M.; Ownby, Dennis

    2006-01-01

    Environmental allergens are major triggers for pediatric asthma. While children's greatest exposure to indoor allergens is in the home, other public places where children spend a large amount of time, such as school and day care centers, may also be sources of significant allergen encounters. The purpose of this article is to describe schoolroom…

  18. Allergen-induced asthma

    PubMed Central

    Cockcroft, Donald W

    2014-01-01

    It was only in the late 19th century that specific allergens, pollen, animal antigens and, later, house dust mite, were identified to cause upper and lower airway disease. Early allergen challenge studies, crudely monitored before measurement of forced expiratory volume in 1 s became widespread in the 1950s, focused on the immediate effects but noted in passing prolonged and/or recurrent asthma symptoms. The late asthmatic response, recurrent bronchoconstriction after spontaneous resolution of the early responses occurring 3 h to 8 h or more postchallenge, has been identified and well characterized over the past 50 years. The associated allergen-induced airway hyper-responsiveness (1977) and allergen-induced airway inflammation (1985) indicate that these late sequelae are important in the mechanism of allergen-induced asthma. Allergens are now recognized to be the most important cause of asthma. A standardized allergen inhalation challenge model has been developed and is proving to be a valuable research tool in the investigation of asthma pathophysiology and of potential new pharmacological agents for the treatment of asthma. PMID:24791256

  19. Cockroach allergens: function, structure and allergenicity.

    PubMed

    Pomés, A; Wünschmann, S; Hindley, J; Vailes, L D; Chapman, M D

    2007-01-01

    Cockroach allergy is a widespread health problem in the world, associated with the development of asthma. The German and American cockroach species are important producers of a wide variety of allergens. Knowledge of their structure and function contributes to understand their role in allergy and to design tools for diagnosis and immunotherapy.

  20. Identification and quantification of aroma compounds of tartary buckwheat (Fagopyrum tataricum Gaertn.) and some of its milling fractions.

    PubMed

    Janeš, Damjan; Prosen, Helena; Kreft, Samo

    2012-07-01

    Tartary buckwheat (Fagopyrum tataricum) seeds have a strong aroma that characteristically differs from the aroma of common buckwheat (Fagopyrum esculentum). Its phytochemical background has only been recently investigated. The aim of this study was to identify and quantify individual compounds responsible for tartary buckwheat aroma. Volatiles from different samples (whole seed, flour, and husks) were extracted with simultaneous extraction and distillation by Likens-Nickerson apparatus and analyzed by GC-MS. A total of 48 compounds were quantified and their odor activity values (OAV) were calculated. OAV of 26 compounds was higher than 10; therefore, they significantly contribute to the overall tartary buckwheat aroma. The compounds with OAV > 500 were: (E,E)-2,4-decadienal, (E)-2-nonenal, 2-phenylethanol, (E,E)-2,4-nonadienal, hexanal, decanal, and nonanal. The most important difference from the aroma of common buckwheat is the absence of salicylaldehyde and presence of naphthalene. Salicylaldehyde could be proposed as a marker to detect contamination/adulteration of tartary buckwheat with common buckwheat. Buckwheat is becoming one of important alternative crops. Its products which are rich in proteins, fiber, vitamins, and antioxidants have been associated with healthy nutrition. Although tartary buckwheat is similar to more familiar common buckwheat, their characteristic aromas differ notably. This study expands recent research on aroma of tartary buckwheat tea to seed, flour, and husks, and suggests how products from different species of buckwheat can be distinguished by analysis of aroma compounds. © 2012 Institute of Food Technologists®

  1. [Outdoor and indoor allergens and the respiratory tract].

    PubMed

    Kanceljak-Macán, B; Macan, J; Plavec, D

    2000-09-01

    This paper gives an overview of common outdoor and indoor allergens which cause sensitisation of the respiratory tract and considers relevant biological and ecological hallmarks and symptoms of allergies. Grass, tree, and weed pollens as well as moulds (Cladosporium and Alternaria species) are a major source of allergens in the outdoor environment whereas mites (Pyroglyphidae, Acaridae, and Glycyphagidae), animals (pets, rodents, and insects), and moulds (Aspergillus, Penicillium, and Mucor species) are a major source in the indoor environment. The paper pays attention to the seasonal, geographical, and climatic influence on the concentration of allergen in the environment. The authors discuss differences between exposure to outdoor and indoor allergens, as well as the impact of pollutants on sensitisation of the respiratory tract. The paper proceeds with a short description of the primary prevention measures such as avoidance of the allergens and the secondary measures which are intended to prevent the occurrence or deterioration of respiratory symptoms in sensitised persons.

  2. Early allergen exposure and atopic eczema.

    PubMed

    Harris, J M; Williams, H C; White, C; Moffat, S; Mills, P; Newman Taylor, A J; Cullinan, P

    2007-04-01

    . The lack of any clear exposure-disease relationship between allergens in early life and subsequent eczema argues against allergen exposure being a major factor causing eczema. If the lower levels of eczema at higher levels of house dust mite are confirmed, then interventions aimed at reducing house dust mite in early infancy could paradoxically increase the risk of subsequent eczema.

  3. [Nasal allergenic provocation test].

    PubMed

    Becerril Angeles, M H; Pérez López, A; Azuara Pliego, E

    2000-01-01

    This is a method to evaluate both specific sensitivity to allergens in the nasal mucosa, IgE-mediated hypersensitivity, and antiinflammatory and antiallergic drugs efficacy, whose objectives are for research in diagnosis and treatment. The method is based in allergen extracts delivery in the nasal mucosa and the post-challenge measurement of rhinitis symptoms, vasoactive mediators release quantification and nasal obstruction degree evaluated by rhinomanometry. Nasal allergen challenge is a procedure of diagnostic and therapeutic evaluation usefulness, that must be performed in selected patients, in adequate facilities, by experts physicians, with standardised allergen dosages, in an specific nasal area, with objective measurements (rhinomanometry, mediators and secretions of the allergic response) and symptoms scoring that allow get reliable results in patients with allergic rhinitis under study.

  4. Allergens in the Lab.

    ERIC Educational Resources Information Center

    Fisher, Thomas M.

    1987-01-01

    Points out the health and legal implications related to laboratory substances that could cause allergic reactions. Presents a list of potential cosmetic allergens and irritants. Includes precautionary measures dealing with allergy situations. (ML)

  5. Allergens in the Lab.

    ERIC Educational Resources Information Center

    Fisher, Thomas M.

    1987-01-01

    Points out the health and legal implications related to laboratory substances that could cause allergic reactions. Presents a list of potential cosmetic allergens and irritants. Includes precautionary measures dealing with allergy situations. (ML)

  6. Allergens and thunderstorm asthma.

    PubMed

    Nasser, Shuaib M; Pulimood, Thomas B

    2009-09-01

    Thunderstorm-related asthma is increasingly recognized in many parts of the world. This review focuses on important advances in the understanding of the mechanism of the role of allergens, in particular fungal spores such as Alternaria, in asthma epidemics associated with thunderstorms. From our observations, we have proposed that the prerequisites for this phenomenon are as follows: 1) a sensitized, atopic, asthmatic individual; 2) prior airway hyperresponsiveness before a sudden, large allergen exposure; 3) a large-scale thunderstorm with cold outflow occurring at a time and location during an allergen season in which large numbers of asthmatics are outdoors; and 4) sudden release of large amounts of respirable allergenic fragments, particularly fungal spores such as Alternaria.

  7. Anti-fatigue properties of tartary buckwheat extracts in mice.

    PubMed

    Jin, Hong-Mei; Wei, Ping

    2011-01-01

    Anti-fatigue properties of tartary buckwheat extracts (TBE) was investigated in male Kunming mice. The animals were divided into four groups. The first group, designated as the control group (control), was administered with distilled water by gavage every day for 28 days. The other three groups, designated as TBE treatment groups, were administered with TBE of 60, 120 and 240 mg/kg body weight, respectively, by gavage every day for 28 days. Exhaustive swimming time, blood lactic acid (BLA), blood urea nitrogen (BUN), tissue glycogen, glutathione peroxidase (GPx) and superoxide dismutase (SOD) of mice after swimming were determined. The results showed that tartary buckwheat extracts had anti-fatigue properties, which extended the exhaustive swimming time of mice, effectively inhibiting the increase of BLA, decreasing the level of BUN, increasing the tissue glycogen content and the activities of SOD and GPx of mice. However, further study is needed to elucidate the exact mechanism of the effect of TBE on fatigue.

  8. The 2S albumin allergens of Arachis hypogaea, Ara h 2 and Ara h 6, are the major elicitors of anaphylaxis and can effectively desensitize peanut-allergic mice

    PubMed Central

    Kulis, Mike; Chen, Xueni; Lew, Jeanney; Wang, Qian; Patel, Ojas P.; Zhuang, Yonghua; Murray, Kerry S.; Duncan, Mark W.; Porterfield, Harry S.; Burks, A. Wesley; Dreskin, Stephen C.

    2012-01-01

    Background Ara h 2 and Ara h 6, co-purified together in a 13-25 kD fraction (Ara h 2/6; 20 kD fraction) on gel filtration chromatography, account for the majority of effector activity in a crude peanut extract (CPE) when assayed with RBL SX-38 cells sensitized with IgE from human peanut allergic sera. Objectives To determine if Ara h 2/6 are the primary peanut allergens responsible for allergic reactions in vivo and to determine if Ara h 2/6 would be sufficient to prevent allergic reactions to a complete CPE. Methods An oral sensitization mouse model of peanut allergy was used to assess the activity of Ara h 2/6 (20 kD) and CPE without the 20 kD fraction (CPE w/o 20 kD) for allergic provocation challenge and immunotherapy. The activity of these preparations was also tested in an assay of histamine release from human basophils in whole blood. Results Compared to mice challenged with control CPE, mice challenged with CPE w/o 20 kD experienced reduced symptoms (p<0.05) and a smaller decrease in body temperature (p<0.01). Results with the basophil histamine release assay corroborated these findings (p<0.01). The mouse model was also used to administer Ara h 2/6 (20 kD) in an immunotherapy protocol, in which peanut-allergic mice treated with the 20 kD fraction experienced significantly reduced symptoms, changes in body temperature, and mast cell protease (MMCP-1) release compared to placebo (p<0.01 for all parameters). Importantly, immunotherapy with the 20 kD fraction was just as effective as treatment with CPE, whereas CPE w/o 20 kD was significantly less effective for higher dose peanut challenges. Conclusions and Clinical Relevance Ara h 2/6 are the most potent peanut allergens in vivo and can be used to desensitize peanut-allergic mice. These results have potential implications for clinical research in the areas of diagnosis and immunotherapy for peanut allergy. PMID:22288514

  9. The 2S albumin allergens of Arachis hypogaea, Ara h 2 and Ara h 6, are the major elicitors of anaphylaxis and can effectively desensitize peanut-allergic mice.

    PubMed

    Kulis, M; Chen, X; Lew, J; Wang, Q; Patel, O P; Zhuang, Y; Murray, K S; Duncan, M W; Porterfield, H S; W Burks, A; Dreskin, S C

    2012-02-01

    Ara h 2 and Ara h 6, co-purified together in a 13-25 kD fraction (Ara h 2/6; 20 kD fraction) on gel filtration chromatography, account for the majority of effector activity in a crude peanut extract (CPE) when assayed with RBL SX-38 cells sensitized with IgE from human peanut allergic sera. To determine if Ara h 2/6 are the primary peanut allergens responsible for allergic reactions in vivo and to determine if Ara h 2/6 would be sufficient to prevent allergic reactions to a complete CPE. An oral sensitization mouse model of peanut allergy was used to assess the activity of Ara h 2/6 (20 kD) and CPE without the 20 kD fraction (CPE w/o 20 kD) for allergic provocation challenge and immunotherapy. The activity of these preparations was also tested in an assay of histamine release from human basophils in whole blood. Compared with mice challenged with control CPE, mice challenged with CPE w/o 20 kD experienced reduced symptoms (P < 0.05) and a smaller decrease in body temperature (P < 0.01). Results with the basophil histamine release assay corroborated these findings (P < 0.01). The mouse model was also used to administer Ara h 2/6 (20 kD) in an immunotherapy protocol, in which peanut-allergic mice treated with the 20 kD fraction experienced significantly reduced symptoms, changes in body temperature, and mast cell protease (MMCP-1) release compared with placebo (P < 0.01 for all parameters). Importantly, immunotherapy with the 20 kD fraction was just as effective as treatment with CPE, whereas CPE w/o 20 kD was significantly less effective for higher dose peanut challenges. Ara h 2/6 are the most potent peanut allergens in vivo and can be used to desensitize peanut-allergic mice. These results have potential implications for clinical research in the areas of diagnosis and immunotherapy for peanut allergy. © 2011 Blackwell Publishing Ltd.

  10. Monitoring for airborne allergens

    SciTech Connect

    Burge, H.A. )

    1992-07-01

    Monitoring for allergens can provide some information on the kinds and levels of exposure experienced by local patient populations, providing volumetric methods are used for sample collection and analysis is accurate and consistent. Such data can also be used to develop standards for the specific environment and to begin to develop predictive models. Comparing outdoor allergen aerosols between different monitoring sites requires identical collection and analysis methods and some kind of rational standard, whether arbitrary, or based on recognized health effects.32 references.

  11. The spectrum of olive pollen allergens.

    PubMed

    Rodríguez, R; Villalba, M; Monsalve, R I; Batanero, E

    2001-07-01

    Olive pollen is one of the most important causes of seasonal respiratory allergy in Mediterranean countries, where this tree is intensely cultivated. Among the high number of protein allergens detected in this pollen, 8 - Ole e 1 to Ole e 8 - have been isolated and characterized. Ole e 1 is the most frequent sensitizing agent, affecting more than 70% of the patients suffering of olive pollinosis, although others, such as Ole e 4 and Ole e 7, have also been shown to be major allergens. In this context, the prevalence of many olive pollen allergens seems to be dependent on the geographical area where the sensitized patients live. Some of the olive allergens have been revealed as members of known protein families: profilin (Ole e 2), Ca(2+)-binding proteins (Ole e 3 and Ole e 8), superoxide dismutase (Ole e 5) and lipid transfer protein (Ole e 7). No biological function has been demonstrated for Ole e 1, whereas Ole e 4 and Ole e 6 are new proteins without homology to known sequences from databases. cDNAs encoding for Ole e 1, Ole e 3 and Ole e 8 have been overproduced in heterologous systems. The recombinant products were correctly folded and exhibited the functional activities of the natural allergens. In addition to the Oleaceae family, other species, such as Gramineae or Betulaceae, contain pollen allergens structurally or immunologically related to those of the olive tree. This fact allows to detect and evaluate antigenic cross-reactivities involving olive allergens. The aim of this research is the development of new diagnostic tools for olive pollinosis and new approaches to improve the classical immunotherapy.

  12. Allergens in corticosteroid vehicles.

    PubMed

    Coloe, Jacquelyn; Zirwas, Matthew J

    2008-01-01

    Whereas allergy to vehicle ingredients (ie, excipients and preservatives) in topical steroid vehicles is well recognized, there are no data regarding which vehicle ingredients are in common use or on which vehicles and active molecules are associated with which ingredients. To produce descriptive data on the use of allergenic vehicle ingredients in prescription topical corticosteroids. The package insert for every steroid in widespread use in the United States was obtained from the manufacturer and used to generate an ingredient list for the product. There are seven vehicle ingredients that are commonly used in topical corticosteroid vehicles that are well-known allergens: propylene glycol, sorbitan sesquioleate, formaldehyde-releasing preservatives, parabens, methylchloroisothiazolinone/methylisothiazolinone, lanolin, and fragrance. Of 166 topical corticosteroids, 128 (including all creams) had at least one of these vehicle ingredients. More generic products were free of allergens than were branded products. Solutions and ointments were the least allergenic vehicles. The most commonly present potential allergens were propylene glycol and sorbitan sesquioleate. Most prescription topical corticosteroids have the potential to cause allergic contact dermatitis owing to vehicle ingredients. Dermatologists should be aware of this possibility and should consider prescribing agents that do not contain potentially allergenic vehicle ingredients.

  13. Passive immunization with allergen-specific IgG antibodies for treatment and prevention of allergy

    PubMed Central

    Flicker, Sabine; Linhart, Birgit; Wild, Carmen; Wiedermann, Ursula; Valenta, Rudolf

    2013-01-01

    IgE antibody-mediated allergies affect more than 25% of the population worldwide. To investigate therapeutic and preventive effects of passive immunization with allergen-specific IgG antibodies on allergy in mouse models we used clinically relevant pollen allergens. In a treatment model, mice were sensitized to the major birch pollen allergen Bet v 1 and to the major grass pollen allergens, Phl p 1 and Phl p 5 and then received passive immunization with rabbit IgG antibodies specific for the sensitizing or an unrelated allergen. In a prevention model, mice obtained passive immunization with allergen-specific rabbit IgG before sensitization. Kinetics of the levels of administered IgG antibodies, effects of administered allergen-specific IgG on allergen-specific IgE reactivity, the development of IgE and IgG responses and on immediate allergic reactions were studied by ELISA, rat basophil leukaemia degranulation assays and skin testing, respectively. Treated mice showed an approximately 80% reduction of allergen-specific IgE binding and basophil degranulation which was associated with the levels of administered allergen-specific IgG antibodies. Preventive administration of allergen-specific IgG antibodies suppressed the development of allergen-specific IgE and IgG1 antibody responses as well as allergen-induced basophil degranulation and skin reactivity. Our results show that passive immunization with allergen-specific IgG antibodies is effective for treatment and prevention of allergy to clinically important pollen allergens in a mouse model and thus may pave the road for the clinical application of allergen-specific antibodies in humans. PMID:23182706

  14. Characterization of cookies made from wheat flour blended with buckwheat flour and effect on antioxidant properties.

    PubMed

    Jan, Ulfat; Gani, Adil; Ahmad, Mudasir; Shah, Umar; Baba, Waqas N; Masoodi, F A; Maqsood, Sajid; Gani, Asir; Wani, Idress Ahmed; Wani, S M

    2015-10-01

    Buckwheat flour was incorporated into wheat flour at different levels (0, 20, 40, 60, 80, and 100 %) and the physicochemical, functional and antioxidant properties of the blended flour were studied. This study also investigated the effect of buckwheat on the retention of antioxidant properties of cookies during baking. The results showed significant variation in physicochemical and functional properties of the blended flour. The addition of buckwheat flour into wheat flour also increased the antioxidant properties of blended flour proportionally, but metal chelating properties decreased. The incorporation of buckwheat in wheat flour helped in better retention of antioxidant potential of cookies during baking process as buckwheat cookies (100 % buckwheat) showed greater percentage increase in antioxidant properties than control (100 % wheat). Quality characteristics of cookies such as hardness and spread ratio decreased, while as non-enzymatic browning (NEB) increased significantly with increase in the proportion of buckwheat flour in wheat flour. The Overall acceptability of cookies by sensory analysis was highest at 40 % level of blending. This study concluded that addition of buckwheat in wheat flour, may not only improve the physico-chemical and functional properties of the blended flour but may also enhance the nutraceutical potential of the product prepared from it.

  15. [New aero-allergens. Interaction between allergens and the environment].

    PubMed

    Charpin, D; Vervloet, D

    1997-11-01

    There is a qualitative as well as a quantitative change in allergen exposure. From a qualitative viewpoint, the relevance of some allergens (domestic allergens i.e. cockroaches, outdoor allergens i.e. plane tree, chestnut and ash tree pollens) has been established. The role of some other allergens has been, strictly speaking, discovered: latex from Hevea and ficus, trichophyton mold, some occupational allergens and very recently transgenic allergens. From a quantitative viewpoint, the concentration and/or distribution of some allergens has increased. Plantation of numerous trees or fortuitous introduction of weeds has led to an increased specific sensitization. In like manner, introduction of new foods or food processing procedures has created new food allergens and allergy. Besides, the distribution of some well-known aero-allergens is better known since discovery of ELISA--technology allowing measurements of minute amounts of these allergens. A burning issue today is to know whether irritant factors could modify allergens. Few data, sometimes contradictory, are available in the field of interaction between air pollutants and allergens.

  16. The Contribution of Buckwheat Genetic Resources to Health and Dietary Diversity

    PubMed Central

    Sytar, Oksana; Brestic, Marian; Zivcak, Marek; Tran, Lam-Son Phan

    2016-01-01

    Despite several reports on the beneficial effects of buckwheat in prevention of human diseases, little attention has been devoted to the variability of biochemical and physiological traits in different buckwheat genetic resources. This review describes the biochemical evaluation of buckwheat genetic resources and the identification of elite genotypes for plant breeding and exploitation. The various types of bioactive compounds present in different varieties provide basic background information needed for the efficient production of buckwheat foods with added value. In this review, we will provide an integrated view of the biochemistry of bioactive compounds of buckwheat plants of different origin, especially of fagopyrin, proteins and amino acids, as well as of other phenolic compounds including rutin and chlorogenic acid. In addition to the genetic background, the effect of different growth conditions is discussed. The health effects of fagopyrin, phenolic acids, specific proteins and rutin are also presented. PMID:27252586

  17. [Different uses of Fagopyrum esculentum Moench (buckwheat) in Japan and China: what ancient medical documents reveal].

    PubMed

    Tatsumi, Nami; Marui, Eiji

    2012-03-01

    The purpose of this thesis is to demonstrate that buckwheat has been recognized, both in Japan and China, as a crop that is useful in many ways: as an agricultural crop, and for the healing powers and properties that, according to traditional Chinese medicine, it has. A comparative study of ancient documents pertaining to medicine in these countries has made it clear that this is the case. Buckwheat, however, has been used quite differently in each country. As is shown in some ancient Chinese documents pertaining to medicine, China has treated buckwheat primarily as a medicine for clinical use rather than as an edible crop. Nowadays, buckwheat is eaten only in some regions of China. Although it came to Japan from China as a medicine, in Japan buckwheat gradually became a popular food crop. It has become an important component of traditional Japanese cuisine thanks in part to government support and the strong demand that developed in Japanese society.

  18. Trace element water improves the antioxidant activity of buckwheat (Fagopyrum esculentum Moench) sprouts.

    PubMed

    Liu, Chia-Ling; Chen, Yih-Shyuan; Yang, Joan-Hwa; Chiang, Been-Huang; Hsu, Cheng-Kuang

    2007-10-31

    Buckwheat (Fagopyrum esculentum Moench) was grown in trace element water (TEW) (100, 200, 300, 400, and 500 ppm) and deionized water (DIW) to evaluate whether the beneficial effects of trace elements on the antioxidant activity could be accomplished with the supplement of TEW. At 300 ppm, TEW significantly increased the Cu, Zn, Mn, and Fe contents in buckwheat sprout but not the Se content. However, the levels of rutin, isoorientin, vitexin, and isovitexin did not differ between buckwheat sprouts grown in TEW and DIW. The ethanolic extract from buckwheat sprout grown in 300 ppm of TEW showed higher ferrous ion chelating activity and inhibitory activity toward lipid peroxidation than that grown in DIW. The extract in the TEW group also enhanced intracellular superoxide dismutase activity and lowered reactive oxygen species and superoxide anion in the human Hep G2 cell. It was concluded that TEW could increase the antioxidant activities of buckwheat sprouts.

  19. Antioxidant Capacity, Mineral Content and Sensory Properties of Gluten-Free Rice and Buckwheat Cookies

    PubMed Central

    Pestorić, Mladenka; Mišan, Aleksandra; Nedeljković, Nataša; Jambrec, Dubravka; Jovanov, Pavle; Banjac, Vojislav; Torbica, Aleksandra; Hadnađev, Miroslav; Mandić, Anamarija

    2015-01-01

    Summary Light buckwheat flour was used to substitute rice flour at the level of 10, 20 and 30% to produce gluten-free cookies. The substitution of gluten-free cookie formulation with light buckwheat flour contributed to the significantly higher mineral content, especially magnesium, potassium, iron and copper, in comparison with the control rice cookies (p<0.05). Gluten-free cookies made with rice flour and buckwheat flour exhibited significantly higher total phenolic and rutin content, scavenging activity against 1,1-diphenyl-2-picrylhydrazyl radicals (DPPH•), antioxidant activity and reducing power than the control cookies (p<0.05). Comparing all evaluated sensory properties, cookies containing 20% of light buckwheat flour had the most acceptable sensory properties. The obtained results of principal component analysis showed that the cookies with 20 and 30% buckwheat flour had better antioxidant and sensory properties in comparison with other two cookie samples. PMID:27904330

  20. The impact of protein cross-linking induced by alkali on the quality of buckwheat noodles.

    PubMed

    Guo, Xiao-Na; Wei, Xiao-Ming; Zhu, Ke-Xue

    2017-04-15

    The impact of alkali addition on the dough rheological properties and quality of buckwheat noodles was investigated. Farinograph measurements showed that the addition of alkali increased the water absorption and development time of the dough. Dynamic rheological properties analysis showed that alkali addition enhanced G' and G″ of dough. In addition, the texture properties of buckwheat noodles improved by the increase of the hardness and tensile force. Furthermore, an obvious decrease in the intensity of the protein bands with lower molecular weights was observed in SDS-PAGE patterns and the extractability of protein in sodium dodecyl sulfate containing medium (SDSEP) decreased, which demonstrated that alkali addition promoted the degree of protein polymerization in the buckwheat noodles. CLSM analysis showed alkali addition produced a tight and continuous protein network in buckwheat noodles. The protein cross-linking induced by alkali improved rheological properties of dough and texture properties of buckwheat noodles.

  1. Pepino mosaic virus infection of tomato affects allergen expression, but not the allergenic potential of fruits.

    PubMed

    Welter, Saskia; Dölle, Sabine; Lehmann, Karola; Schwarz, Dietmar; Weckwerth, Wolfram; Worm, Margitta; Franken, Philipp

    2013-01-01

    The plant pathogen Pepino mosaic virus (PepMV) is a major disease of greenhouse tomato crops worldwide. Plant pathogens can induce expression of defence- or pathogenesis-related proteins, including identified allergens. Therefore we hypothesised that PepMV infection results in the expression of allergens leading to a higher allergenic potential of tomato fruits. Transcript level analyses showed differential expression of 17 known and putative tomato fruit allergen encoding genes at early and late time points after PepMV inoculation, but no general induction was detected. Immunoblot analyses were conducted and IgEs from a serum pool of tomato allergic subjects reacted with 20 proteins, of which ten have not yet been described. In parallel, skin prick tests with a group of tomato allergic subjects did not show a general difference between PepMV infected and non-infected tomato fruits and basophil activation tests confirmed these results. In summary, PepMV infection of tomato plants can lead to long-lasting up-regulation of particular allergens in fruits, but the hypothesis that this results in a higher allergenic potential of the fruits proved invalid.

  2. Pepino mosaic virus Infection of Tomato Affects Allergen Expression, but Not the Allergenic Potential of Fruits

    PubMed Central

    Welter, Saskia; Dölle, Sabine; Lehmann, Karola; Schwarz, Dietmar; Weckwerth, Wolfram; Worm, Margitta; Franken, Philipp

    2013-01-01

    The plant pathogen Pepino mosaic virus (PepMV) is a major disease of greenhouse tomato crops worldwide. Plant pathogens can induce expression of defence- or pathogenesis-related proteins, including identified allergens. Therefore we hypothesised that PepMV infection results in the expression of allergens leading to a higher allergenic potential of tomato fruits. Transcript level analyses showed differential expression of 17 known and putative tomato fruit allergen encoding genes at early and late time points after PepMV inoculation, but no general induction was detected. Immunoblot analyses were conducted and IgEs from a serum pool of tomato allergic subjects reacted with 20 proteins, of which ten have not yet been described. In parallel, skin prick tests with a group of tomato allergic subjects did not show a general difference between PepMV infected and non-infected tomato fruits and basophil activation tests confirmed these results. In summary, PepMV infection of tomato plants can lead to long-lasting up-regulation of particular allergens in fruits, but the hypothesis that this results in a higher allergenic potential of the fruits proved invalid. PMID:23762294

  3. Allergenic proteins in Urtica dioica, a member of the Urticaceae allergenic family.

    PubMed

    Vega-Maray, Ana María; Fernández-González, Delia; Valencia-Barrera, Rosa; Suárez-Cervera, María

    2006-09-01

    Allergy to the pollen of flowering plant species significantly affects the health of people in many parts of the world. Pollens of related genera usually share common antigens and are often, but not always, cross-reactive. Several studies have shown that Parietaria pollen is one of the most common causes of pollinosis in the Mediterranean area, whereas Urtica has no allergenic significance. To report on the localization of Parietaria judaica major allergen in Urtica dioica pollen grains and on the detection of allergenic proteins in U. dioica pollen grains during the hydration-activation process. A combination of transmission electron microscopy and immunocytochemical methods was used to locate allergenic proteins in U. dioica pollen grains after different periods of hydration-activation using the anti-Par j 1 (4.1.3.) monoclonal antibody and serum samples from allergic patients. No significant labeling was noted for Parj 1 allergen after 10, 15, and 20 minutes in the walls and cytoplasm. Slight labeling was observed for allergic proteins in the walls of U. dioica after 10 minutes of hydration, and no significant labeling was found after 15 and 20 minutes of hydration. Immunocytochemical methods confirmed the absence of cross-reactivity between 2 related genera, Parietaria and Urtica, and the lowest allergenic potential of U. dioica.

  4. Paprika rhinoconjunctivitis case reveals new occupational Capsicum allergens.

    PubMed

    Airaksinen, Liisa; Riekki, Riitta; Vuokko, Aki; Puustinen, Anne

    2015-07-01

    No allergens related to paprika or cayenne respiratory allergy have been identified thus far. We describe a previously healthy 28-year woman who developed work-related rhinoconjunctivitis after four years of kebab-restaurant work. The allergy was studied using skin prick tests, serum specific IgE and nasal provocation tests. Specific IgE protein reactions were studied by Western blot analysis. Paprika, cayenne and curry allergens were identified from the strongest immunoblot bands using tandem mass spectrometry. A positive skin prick test, high specific IgE and positive nasal provocation test confirmed occupational rhinoconjunctivitis from Capsicum spices. Defensin J1 and Vicilin were identified as major paprika and cayenne allergens in this case. Vicilin was detected also from the curry ingredients. Two new occupational respiratory allergens from the Capsicum species were identified. These differ from previously reported bell pepper allergens. We emphasize that substantial spice handling at work poses an allergy risk.

  5. Analysis of U.S. Food and Drug Administration food allergen recalls after implementation of the food allergen labeling and consumer protection act.

    PubMed

    Gendel, Steven M; Zhu, Jianmei

    2013-11-01

    To avoid potentially life-threatening reactions, food allergic consumers rely on information on food labels to help them avoid exposure to a food or ingredient that could trigger a reaction. To help consumers in the United States obtain the information that they need, the Food Allergen Labeling and Consumer Protection Act of 2004 defined a major food allergen as being one of eight foods or food groups and any ingredient that contains protein from one of these foods or food groups. A food that contains an undeclared major food allergen is misbranded under the U.S. Food, Drug, and Cosmetic Act and is subject to recall. Food allergen labeling problems are the most common cause of recalls for U.S. Food and Drug Administration (FDA)-regulated food products. To help understand why food allergen recalls continue to occur at a high rate, information on each food allergen recall that occurred in fiscal years 2007 through 2012 was obtained from the FDA recall database. This information was analyzed to identify the food, allergen, root cause, and mode of discovery for each food allergen recall. Bakery products were the most frequently recalled food type, and milk was the most frequently undeclared major food allergen. Use of the wrong package or label was the most frequent problem leading to food allergen recalls. These data are the first reported that indicate the importance of label and package controls as public health measures.

  6. Origin and Functional Prediction of Pollen Allergens in Plants1[OPEN

    PubMed Central

    Chen, Miaolin; Xu, Jie; Ren, Kang; Searle, Iain

    2016-01-01

    Pollen allergies have long been a major pandemic health problem for human. However, the evolutionary events and biological function of pollen allergens in plants remain largely unknown. Here, we report the genome-wide prediction of pollen allergens and their biological function in the dicotyledonous model plant Arabidopsis (Arabidopsis thaliana) and the monocotyledonous model plant rice (Oryza sativa). In total, 145 and 107 pollen allergens were predicted from rice and Arabidopsis, respectively. These pollen allergens are putatively involved in stress responses and metabolic processes such as cell wall metabolism during pollen development. Interestingly, these putative pollen allergen genes were derived from large gene families and became diversified during evolution. Sequence analysis across 25 plant species from green alga to angiosperms suggest that about 40% of putative pollen allergenic proteins existed in both lower and higher plants, while other allergens emerged during evolution. Although a high proportion of gene duplication has been observed among allergen-coding genes, our data show that these genes might have undergone purifying selection during evolution. We also observed that epitopes of an allergen might have a biological function, as revealed by comprehensive analysis of two known allergens, expansin and profilin. This implies a crucial role of conserved amino acid residues in both in planta biological function and allergenicity. Finally, a model explaining how pollen allergens were generated and maintained in plants is proposed. Prediction and systematic analysis of pollen allergens in model plants suggest that pollen allergens were evolved by gene duplication and then functional specification. This study provides insight into the phylogenetic and evolutionary scenario of pollen allergens that will be helpful to future characterization and epitope screening of pollen allergens. PMID:27436829

  7. Stability of food allergens to digestion in vitro.

    PubMed

    Astwood, J D; Leach, J N; Fuchs, R L

    1996-10-01

    An integral part of the safety assessment of genetically modified plants is consideration of possible human health effects, especially food allergy. Prospective testing for allergenicity of proteins obtained from sources with no prior history of causing allergy has been difficult because of the absence of valid methods and models. Food allergens may share physicochemical properties that distinguish them from nonallergens, properties that may be used as a tool to predict the inherent allergenicity of proteins newly introduced into the food supply by genetic engineering. One candidate property is stability to digestion. We have systematically evaluated the stability of food allergens that are active via the gastrointestinal tract in a simple model of gastric digestion, emphasizing the major allergens of plant-derived foods such as legumes (peanuts and soybean). Important food allergens were stable to digestion in the gastric model (simulated gastric fluid). For example, soybean beta-conglycinin was stable for 60 min. In contrast, nonallergenic food proteins, such as spinach ribulose bis-phosphate carboxylase/oxygenase, were digested in simulated gastric fluid within 15 sec. The data are consistent with the hypothesis that food allergens must exhibit sufficient gastric stability to reach the intestinal mucosa where absorption and sensitization (development of atopy) can occur. Thus, the stability to digestion is a significant and valid parameter that distinguishes food allergens from nonallergens.

  8. Current overview of allergens of plant pathogenesis related protein families.

    PubMed

    Sinha, Mau; Singh, Rashmi Prabha; Kushwaha, Gajraj Singh; Iqbal, Naseer; Singh, Avinash; Kaushik, Sanket; Kaur, Punit; Sharma, Sujata; Singh, Tej P

    2014-01-01

    Pathogenesis related (PR) proteins are one of the major sources of plant derived allergens. These proteins are induced by the plants as a defense response system in stress conditions like microbial and insect infections, wounding, exposure to harsh chemicals, and atmospheric conditions. However, some plant tissues that are more exposed to environmental conditions like UV irradiation and insect or fungal attacks express these proteins constitutively. These proteins are mostly resistant to proteases and most of them show considerable stability at low pH. Many of these plant pathogenesis related proteins are found to act as food allergens, latex allergens, and pollen allergens. Proteins having similar amino acid sequences among the members of PR proteins may be responsible for cross-reactivity among allergens from diverse plants. This review analyzes the different pathogenesis related protein families that have been reported as allergens. Proteins of these families have been characterized in regard to their biological functions, amino acid sequence, and cross-reactivity. The three-dimensional structures of some of these allergens have also been evaluated to elucidate the antigenic determinants of these molecules and to explain the cross-reactivity among the various allergens.

  9. Current Overview of Allergens of Plant Pathogenesis Related Protein Families

    PubMed Central

    Sinha, Mau; Singh, Rashmi Prabha; Kushwaha, Gajraj Singh; Iqbal, Naseer; Singh, Avinash; Kaushik, Sanket; Sharma, Sujata; Singh, Tej P.

    2014-01-01

    Pathogenesis related (PR) proteins are one of the major sources of plant derived allergens. These proteins are induced by the plants as a defense response system in stress conditions like microbial and insect infections, wounding, exposure to harsh chemicals, and atmospheric conditions. However, some plant tissues that are more exposed to environmental conditions like UV irradiation and insect or fungal attacks express these proteins constitutively. These proteins are mostly resistant to proteases and most of them show considerable stability at low pH. Many of these plant pathogenesis related proteins are found to act as food allergens, latex allergens, and pollen allergens. Proteins having similar amino acid sequences among the members of PR proteins may be responsible for cross-reactivity among allergens from diverse plants. This review analyzes the different pathogenesis related protein families that have been reported as allergens. Proteins of these families have been characterized in regard to their biological functions, amino acid sequence, and cross-reactivity. The three-dimensional structures of some of these allergens have also been evaluated to elucidate the antigenic determinants of these molecules and to explain the cross-reactivity among the various allergens. PMID:24696647

  10. Positive reaction to allergen (image)

    MedlinePlus

    Allergic reaction is a sensitivity to a specific substance, called an allergen, that is contacted through the skin, inhaled into the lungs, swallowed or injected. The body's reaction to an allergen can be mild, such as ...

  11. Multiple grass mixes as opposed to single grasses for allergen immunotherapy in allergic rhinitis.

    PubMed

    Gangl, K; Niederberger, V; Valenta, R

    2013-11-01

    Grass pollen allergy affects approximately 40% of allergic patients. Subcutaneous allergen immunotherapy (SCIT) is the only allergen-specific and disease-modifying treatment available. Currently available therapeutic vaccines for the treatment of grass pollen allergy are based on natural grass pollen extracts which are either made from pollen of one cross-reactive grass species or from several related grass species. Clinical studies have shown that SCIT performed with timothy grass pollen extract is effective for the treatment of grass pollen allergy. Moreover, it has been demonstrated that recombinant timothy grass pollen allergens contain the majority of relevant epitopes and can be used for SCIT in clinical trials. However, recent in vitro studies have suggested that mixes consisting of allergen extracts from several related grass species may have advantages for SCIT over single allergen extracts. Here, we review current knowledge regarding the disease-relevant allergens in grass pollen allergy, available clinical studies comparing SCIT with allergen extracts from timothy grass or from mixes of several related grass species of the Pooideae subfamily, in vitro cross-reactivity studies performed with natural allergen extracts and recombinant allergens and SCIT studies performed with recombinant timothy grass pollen allergens. In vitro and clinical studies performed with natural allergen extracts reveal no relevant advantages of using multiple grass mixes as opposed to single grass pollen extracts. Several studies analysing the molecular composition of natural allergen extracts and the molecular profile of patients' immune responses after SCIT with allergen extracts indicate that the major limitation for the production of a high quality grass pollen vaccine resides in intrinsic features of natural allergen extracts which can only be overcome with recombinant allergen-based technologies. © 2013 John Wiley & Sons Ltd.

  12. Genes Outside the S Supergene Suppress S Functions in Buckwheat (Fagopyrum esculentum)

    PubMed Central

    MATSUI, K.; NISHIO, T.; TETSUKA, T.

    2004-01-01

    • Background and Aims Common buckwheat (Fagopyrum esculentum) is a dimorphic self-incompatible plant with either pin or thrum flowers. The S supergene is thought to govern self-incompatibility, flower morphology and pollen size in buckwheat. Two major types of self-fertile lines have been reported. One is a type with long-homostyle flowers, Kyukei SC2 (KSC2), and the other is a type with short-homostyle flowers, Pennline 10. To clarify whether the locus controlling flower morphology and self-fertility of Pennline 10 is the same as that of KSC2, pollen tube tests and genetic analysis have been performed. • Methods Pollen tube growth was assessed in the styles and flower morphology of KSC2, Pennline 10, F1 and F2 plants that were produced by the crosses between plants with pin or thrum and Pennline 10. • Key Results Pollen tubes of Pennline 10 reached ovules of all flower types. The flower morphology of F1 plants produced by the cross between thrum and Pennline 10 were thrum or pin, and when pin plants were used as maternal plants, all the F1 plants were pin. Both plants with pin or short-pin flowers, whose ratio of style length to anther height was smaller than that of pin, appeared in F2 populations of thrum × Pennline 10 as well as in those of pin × Pennline 10. • Conclusion The results suggest that Pennline 10 possesses the s allele as pin does, not an allele produced by the recombination in the S supergene, and that the short style length of Pennline 10 is controlled by multiple genes outside the S supergene. PMID:15465964

  13. Identification of grape and wine allergens as an endochitinase 4, a lipid-transfer protein, and a thaumatin.

    PubMed

    Pastorello, Elide A; Farioli, Laura; Pravettoni, Valerio; Ortolani, Claudio; Fortunato, Donatella; Giuffrida, Maria Gabriella; Perono Garoffo, Lorenza; Calamari, Ambra Marianna; Brenna, Oreste; Conti, Amedeo

    2003-02-01

    Few allergic reactions to grape are reported in the literature. In some cases an association with peach and cherry allergy was observed. No IgE-mediated reactions to wine have been described, and no grape major allergens have yet been identified. We describe several severe reactions to grape or wine. We characterized the grape major allergens and tried to identify the allergen in wine. We collected documented histories of allergic reactions to grape and wine. Grape allergens were identified by means of SDS-PAGE and immunoblotting and purified by means of HPLC. Using amino acid sequencing and mass spectrometry, we identified the family of proteins to which the allergens belong. Cross-reactivity with peach and cherry was evaluated by means of cross-wise inhibition experiments. Eleven patients with reactions to grape and 3 with anaphylactic reactions to wine were recruited. The major allergens were an endochitinase 4A and a lipid-transfer protein (LTP) that was homologous to and cross-reactive with peach LTP. A 24-kd protein homologous to the cherry thaumatin-like allergen was a minor allergen. Endochitinase 4A is very likely the allergen in vino novello and in vino Fragolino. Grape and wine might cause severe allergic reactions in sensitive patients. The major allergens of grape are endochitinase 4A, which is also the allergen of wine, and an LTP cross-reacting with the peach major allergen.

  14. Perilla frutescens leaf extract inhibits mite major allergen Der p 2-induced gene expression of pro-allergic and pro-inflammatory cytokines in human bronchial epithelial cell BEAS-2B.

    PubMed

    Liu, Jer-Yuh; Chen, Yi-Ching; Lin, Chun-Hsiang; Kao, Shao-Hsuan

    2013-01-01

    Perilla frutescens has been used in traditional medicine for respiratory diseases due to its anti-bacterial and anti-inflammatory activity. This study aimed to investigate effects of Perilla frutescens leaf extract (PFE) on expression of pro-allergic and pro-inflammatory cytokines in airway epithelial cells exposed to mite major allergen Der p 2 (DP2) and the underlying mechanisms. Our results showed that PFE up to 100 µg/mL had no cytotoxic effect on human bronchial epithelial cell BEAS-2B. Further investigations revealed that PFE dose-dependently diminished mRNA expression of pro-allergic cytokine IL-4, IL-5, IL-13 and GM-CSF, as well as pro-inflammatory cytokine IL-6, IL-8 and MCP-1 in BEAS-2B cells treated with DP2. In parallel to mRNA, the DP-2-elevated levels of the tested cytokines were decreased. Further investigation showed that DP2-indued phosphorylation of p38 MAPK (P38) and JNK, but not Erk1/2, was also suppressed by PFE. In addition, PFE elevated cytosolic IκBα level and decreased nuclear NF-κB level in DP2-stimulated BEAS-2B cells. Taken together, these findings revealed that PFE significantly diminished both mRNA expression and protein levels of pro-allergic and pro-inflammatory cytokines in response to DP2 through inhibition of P38/JNK and NK-κB activation. These findings suggest that PFE should be beneficial to alleviate both allergic and inflammatory responses on airway epithelium in response to aeroallergens.

  15. Perilla frutescens Leaf Extract Inhibits Mite Major Allergen Der p 2-induced Gene Expression of Pro-Allergic and Pro-Inflammatory Cytokines in Human Bronchial Epithelial Cell BEAS-2B

    PubMed Central

    Liu, Jer-Yuh; Chen, Yi-Ching; Lin, Chun-Hsiang; Kao, Shao-Hsuan

    2013-01-01

    Perilla frutescens has been used in traditional medicine for respiratory diseases due to its anti-bacterial and anti-inflammatory activity. This study aimed to investigate effects of Perilla frutescens leaf extract (PFE) on expression of pro-allergic and pro-inflammatory cytokines in airway epithelial cells exposed to mite major allergen Der p 2 (DP2) and the underlying mechanisms. Our results showed that PFE up to 100 µg/mL had no cytotoxic effect on human bronchial epithelial cell BEAS-2B. Further investigations revealed that PFE dose-dependently diminished mRNA expression of pro-allergic cytokine IL-4, IL-5, IL-13 and GM-CSF, as well as pro-inflammatory cytokine IL-6, IL-8 and MCP-1 in BEAS-2B cells treated with DP2. In parallel to mRNA, the DP-2-elevated levels of the tested cytokines were decreased. Further investigation showed that DP2-indued phosphorylation of p38 MAPK (P38) and JNK, but not Erk1/2, was also suppressed by PFE. In addition, PFE elevated cytosolic IκBα level and decreased nuclear NF-κB level in DP2-stimulated BEAS-2B cells. Taken together, these findings revealed that PFE significantly diminished both mRNA expression and protein levels of pro-allergic and pro-inflammatory cytokines in response to DP2 through inhibition of P38/JNK and NK-κB activation. These findings suggest that PFE should be beneficial to alleviate both allergic and inflammatory responses on airway epithelium in response to aeroallergens. PMID:24204835

  16. Challenges in testing genetically modified crops for potential increases in endogenous allergen expression for safety.

    PubMed

    Panda, R; Ariyarathna, H; Amnuaycheewa, P; Tetteh, A; Pramod, S N; Taylor, S L; Ballmer-Weber, B K; Goodman, R E

    2013-02-01

    Premarket, genetically modified (GM) plants are assessed for potential risks of food allergy. The major risk would be transfer of a gene encoding an allergen or protein nearly identical to an allergen into a different food source, which can be assessed by specific serum testing. The potential that a newly expressed protein might become an allergen is evaluated based on resistance to digestion in pepsin and abundance in food fractions. If the modified plant is a common allergenic source (e.g. soybean), regulatory guidelines suggest testing for increases in the expression of endogenous allergens. Some regulators request evaluating endogenous allergens for rarely allergenic plants (e.g. maize and rice). Since allergic individuals must avoid foods containing their allergen (e.g. peanut, soybean, maize, or rice), the relevance of the tests is unclear. Furthermore, no acceptance criteria are established and little is known about the natural variation in allergen concentrations in these crops. Our results demonstrate a 15-fold difference in the major maize allergen, lipid transfer protein between nine varieties, and complex variation in IgE binding to various soybean varieties. We question the value of evaluating endogenous allergens in GM plants unless the intent of the modification was production of a hypoallergenic crop.

  17. Plantlet Regeneration of Tartary Buckwheat (Fagopyrum tataricum Gaertn.) in Vitro Tissue Cultures.

    PubMed

    Wang, Cheng-Long; Dong, Xue-Ni; Ding, Meng-Qi; Tang, Yi-Xiong; Zhu, Xue-Mei; Wu, Yan-Min; Zhou, Mei-Liang; Shao, Ji-Rong

    2016-01-01

    Tartary buckwheat is an ancient annual dicotyledonous herb, which is widely distributed around the world, specifically in the high altitude area of southwestern China and in the hill region of Himalayan. The plantlet regeneration of tartary buckwheat via somatic embryogenesis or multiple shoot induction was investigated in two different tartary buckwheats, Yuanzi and Xichang. The regeneration ability of Yuanzi was better than Xichang tartary buckwheat, and the hypocotyls were better than cotyledons as tartary buckwheat plantlet regeneration explants via somatic embryogenesis. The most suitable medium for callus induction was Murashige and Skoog basal medium added 2 mg/L 2, 4- dichlorophenoxyacetic acid and 1 mg/L Kinetin, which could reach up to 98.96% callus induction percentage. The plantlet regeneration percentage from callus of tartary buckwheat could reach up to 55.77%, which induced on 2.0 mg/L Benzyladenine and 1.0 mg/L KT in MS basal medium. In addition, maximum of multiple shoot induction percentage was 69.05%, which was observed in case of Yuanzi tartary buckwheat in MS basal medium with added 3.0 mg/L 6-BA and 1.0 mg/L Thidiazuron. Roots induction of regenerated plants were achieved on 1/2 MS basal medium with added 1mg/L Indole-3-Butytric acid, which has 75% survival after transferred regenerated plants to soil under field conditions.

  18. Physiological characterization of aluminum tolerance and accumulation in tartary and wild buckwheat.

    PubMed

    Wang, Hua; Chen, Rong Fu; Iwashita, Takashi; Shen, Ren Fang; Ma, Jian Feng

    2015-01-01

    Ionic aluminum (Al) is toxic for plant growth, but some plant species are able to accumulate Al at high concentrations without showing toxicity symptoms. In order to determine whether other species in the genus Fagopyrum are able to accumulate Al like common buckwheat (Fagopyrum esculentum), we investigated the external and internal detoxification mechanisms of Al in two self-compatible species: tartary (Fagopyrum tataricum) and wild buckwheat (Fagopyrum homotropicum). Both tartary and wild buckwheat showed high Al tolerance comparable to common buckwheat. Furthermore, these two species also secreted oxalate rapidly from the roots in response to Al in a time-dependent manner. Both tartary and wild buckwheat accumulated > 1 mg g(-1) Al in the leaves after short-term exposure to Al. Analysis with (27) Al-nuclear magnetic resonance (NMR) revealed that Al was present in the form of Al-oxalate (1 : 3 ratio) in the roots and leaves, but in the form of Al-citrate (1 : 1 ratio) in the xylem sap in both species. These results indicate that similar to common buckwheat, both tartary and wild buckwheat detoxify Al externally and internally, respectively, by secreting oxalate from the roots and by forming the Al-oxalate complex, which is a nonphytotoxic form. These features of Al response and accumulation may be conserved in genus Fagopyrum. © 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.

  19. Identification of tartary buckwheat tea aroma compounds with gas chromatography-mass spectrometry.

    PubMed

    Qin, Peiyou; Ma, Tingjun; Wu, Li; Shan, Fang; Ren, Guixing

    2011-08-01

    Tartary buckwheat tea, which is an important and healthy product, has a distinct malty aroma. However, its characteristic aroma compounds have not been elucidated. The aims of present study were identification and quantification of its aroma compounds. The analyses were performed by gas chromatography-mass spectrometry (GC-MS) after 3 different isolation techniques. Seventy-seven compounds were identified. Among these compounds, 35 were quantified by available standards. The compounds with a high probability of contribution to the tartary buckwheat tea aroma (OAV ≥ 10) were as follows: 2,5-dimethyl-4-hydroxy-3(2H)-furanone, nonanal, 2,3-diethyl-5-methylpyrazine, benzeneacetaldehyde, maltol, 2,5-dimethylpyrazine, 2-ethyl-5-methylpyrazine, trimethylpyrazine. Some nutritional and bioactive compounds were also identified in this study, such as linoleic acid, niacin, vanillic acid, 7-hydroxycoumarin, butylated hydroxytoluene. Practical Application: Tartary buckwheat, one type of buckwheat, has gained much attention from nutritionists and medical doctors in recent years. It is rich in rutin, quercetin, and other nutrients that are good for health. Tartary buckwheat-based product such as tartary buckwheat tea is an important and popular healthy product in China, Japan,South Korea, European countries as well as in American countries. It has a distinct malty aroma. The present study first identified and quantified of its aroma compounds. The results will draw attention to other researchers in food flavor and buckwheat filed.

  20. Sensory analysis and aroma compounds of buckwheat containing products-a review.

    PubMed

    Starowicz, Małgorzata; Koutsidis, Georgios; Zieliński, Henryk

    2017-07-07

    Buckwheat is a rich source of starch, proteins, minerals and antioxidants, and as such has become a popular functional ingredient incorporated in diverse recipes/products with particular use in the gluten free market. Due to the absence of gluten, application of buckwheat or buckwheat derived ingredients in this particular food sector has increased significantly over recent years with many buckwheat-based products appearing globally. Sensory analysis is an integral part of the development of products that fulfill consumer expectations. Therefore, investigations on the incorporation of health promoting functional ingredients such as buckwheat into traditional recipes are often complemented by the evaluation of appearance, aroma, taste and texture as well as overall quality through standardized procedures involving trained judges or consumer panels. Aroma is of particular importance in driving consumer preference and its sensory assessment is often complemented with analytical workflows aiming to isolate and determine the concentration of volatile compounds in food and understand the effect of food components on the overall aroma intensity and/or perception of the final product. The present manuscript provides a review of recent advances and knowledge on the sensory characteristics, consumer preference and volatile compound analysis of buckwheat and buckwheat based products.

  1. Persistence of peanut allergen on a table surface.

    PubMed

    Watson, Wade Ta; Woodrow, Annmarie; Stadnyk, Andrew W

    2013-02-18

    A diagnosis of peanut allergy has a major impact on an individual's quality of life. Exposure to even small amounts of peanut can trigger serious reactions. Common cleaning agents can easily remove peanut allergen from surfaces such as table tops. Parents of children with peanut allergy frequently ask if peanut allergen can persist on surfaces if they have not been cleaned. The purpose of this study was to determine the persistence of peanut allergen on a typical table surface over time. Five mL of peanut butter was evenly smeared on a 12 inch by 12 inch (30.5 by 30.5 cm) square on a nonporous (laminated plastic) table surface. Five squares were prepared in the same manner. The table was kept in a regular hospital office at room temperature and ambient lighting. No cleaning occurred for 110 days. Samples were taken at regular intervals from different areas each time. A monoclonal-based ELISA for arachis hypogaea allergen 1 (Ara h 1), range of detection 1.95-2000 ng/mL, was used to assess peanut allergen on the table surface. At baseline, there was no detectable Ara h 1 allergen. Immediately post application and for 110 days of collecting, detectable Ara h 1 was found each time a sample was taken. There was no obvious allergen degradation over time. Active cleaning of the contaminated surface with a commercial cleaning wipe resulted in no detectable Ara h 1 allergen. Peanut allergen is very robust. Detectable Ara h 1 was present on the table surface for 110 days. Active cleaning of peanut contaminated surfaces easily removed peanut residue and allergen. Regular cleaning of surfaces before and after eating should be reinforced as a safety measure for all individuals with peanut allergy.

  2. Sequence conservation predicts T cell reactivity against ragweed allergens

    PubMed Central

    Pham, John; Oseroff, Carla; Hinz, Denise; Sidney, John; Paul, Sinu; Greenbaum, Jason; Vita, Randi; Phillips, Elizabeth; Mallal, Simon; Peters, Bjoern; Sette, Alessandro

    2016-01-01

    Background Ragweed is a major cause of seasonal allergy, affecting millions of people worldwide. Several allergens have been defined based on IgE reactivity, but their relative immunogenicity in terms of T cell responses has not been studied. Objective We comprehensively characterized T cell responses from atopic, ragweed-allergic subjects to Amb a 1, Amb a 3, Amb a 4, Amb a 5, Amb a 6, Amb a 8, Amb a 9, Amb a 10, Amb a 11, and Amb p 5, and examined their correlation with serological reactivity and sequence conservation in other allergens. Methods Peripheral blood mononuclear cells (PBMCs) from donors positive for IgE toward ragweed extracts after in vitro expansion for secretion of IL-5 (a representative Th2 cytokine) and IFNγ (Th1) in response to a panel of overlapping peptides spanning the above listed allergens. Results Three previously identified dominant T cell epitopes (Amb a 1 176–191, 200–215, and 344–359) were confirmed and three novel dominant epitopes (Amb a 1 280–295, 304–319, and 320–335) were identified. Amb a 1, the dominant IgE allergen, was also the dominant T cell allergen, but dominance patterns for T cell and IgE responses for the other ragweed allergens did not correlate. Dominance for T cell responses correlated with conservation of ragweed epitopes with sequences of other well-known allergens. Conclusion and clinical relevance These results provide the first assessment of the hierarchy of T cell reactivity in ragweed allergens, which is distinct from that observed for IgE reactivity and influenced by T cell epitope sequence conservation. The results suggest that ragweed allergens associated with lesser IgE reactivity and significant T cell reactivity may be targeted for T cell immunotherapy, and further support the development of immunotherapies against epitopes conserved across species to generate broad reactivity against many common allergens. PMID:27359111

  3. Development of a sandwich enzyme-linked immunosorbent assay (ELISA) for detection of buckwheat residues in food.

    PubMed

    Panda, Rakhi; Taylor, Steve L; Goodman, Richard E

    2010-08-01

    Buckwheat is a pseudocereal (an eudicot with seed qualities and uses similar to those of monocot cereals, family Poaceae) that is consumed in some Asian countries as a staple, and in some western countries as a health food. Allergic reactions to buckwheat are common in some countries. The objective was to develop a specific and sensitive sandwich enzyme-linked immunosorbent assay (ELISA) to detect traces of buckwheat that might inadvertently contaminate other foods in order to assure accurate labeling and consumer protection. Buckwheat-specific antibodies produced in 3 species of animals were tested for specificity and titer by direct ELISA and immunoblot. A sandwich ELISA was developed utilizing pooled rabbit antibuckwheat sera to capture buckwheat proteins and pooled goat antibuckwheat sera, followed by enzyme-labeled rabbit antigoat immunoglobulin G (IgG), to detect bound buckwheat proteins. The lower limit of quantification (LOQ) of the sandwich ELISA was 2 parts per million (ppm) of buckwheat in the presence of complex food matrices. The ELISA is highly specific with no cross-reactivity to any of 80 food ingredients and matrices tested. Validation studies conducted with buckwheat processed into noodles and muffins showed greater than 90% and 60% recovery, respectively. The percent recovery of buckwheat from noodles was similar to that achieved with a commercial buckwheat ELISA kit (ELISA Systems Pty. Ltd., Windsor, Queensland, Australia) at high buckwheat concentrations. However, the sensitivity of this ELISA was greater than the commercial ELISA. This newly developed ELISA is sufficiently specific and sensitive to detect buckwheat residues in processed foods to protect buckwheat-allergic subjects from potential harm. Practical Application: Buckwheat is becoming a common food ingredient in a number of processed foods due to potentially beneficial nutritional properties, without the celiac disease inducing glutenin proteins of wheat and related cereals. However

  4. Anaphylaxis to hidden potato allergens in a peach and egg allergic boy.

    PubMed

    Martín-Muñoz, M F; Diaz-Perales, A; Cannabal, J; Quirce, S

    2017-01-01

    More than 170 foods have been identified as being potentially allergenic. However, a minority of these foods cause the majority of reactions. Sweets are frequently implicated in allergic reactions in children with cow's milk, egg, nuts or fruits allergy, and they are the most relevant foods investigated as responsible allergens. We report an anaphylactic reaction to candies in an egg and peach allergic boy. We performed a study to identify responsible allergens for the reaction. We investigated hidden egg and peach allergens in the candies, but they were not found. Finally, the causative allergen resulted to be a vegetable protein from potato peel. We diagnosed a new allergy in our patient and Sol t 4 was identified as the responsible allergen of the anaphylactic reaction. We conclude that responsible allergens should always be studied and identified in whatever allergic reaction in order to prevent new reactions.

  5. Economic Factors Impacting Food Allergen Management: Perspectives from the Food Industry.

    PubMed

    Gupta, Ruchi S; Taylor, Steve L; Baumert, Joseph L; Kao, Lauren M; Schuster, Erik; Smith, Bridget M

    2017-10-01

    Food allergies affect up to 8% of children in the United States and may occasionally lead to severe life-threatening reactions. Because there is currently no cure for food allergies, strict avoidance of the allergen-containing foods is the only means of preventing an allergic reaction. Consumers rely on food manufacturers to reliably track and declare the presence of food allergens in products. Over the past 10 to 20 years, the food industry has increasingly adopted allergen control approaches in its processing facilities. However, the major industry costs related to food allergen management have not been fully described. The objective of this study was to characterize the factors that contribute to the economic impact of food allergen control practices on the food industry. A focus group (n = 100) was conducted with food industry professionals to identify key areas of cost for food allergen management. A survey based on the domains identified was then developed and disseminated to a convenience sample (n = 50) of quality control food industry specialists with knowledge of their company's food allergen management practices. Nearly all companies (92%) produced food products containing one or more of the top eight allergenic foods recognized by the U.S. Food and Drug Administration or sesame seeds. Cleaning procedures, employee training, and the potential for a recall due to allergen cross-contact were most frequently rated as the important factors in food allergen management. Recalls due to food allergen cross-contact, cleaning procedures, equipment and premises design, and employee training were ranked as the greatest allergen management expenses. Although 96% of companies had a food allergen control plan in place, nearly half (42%) had at least one food allergen-related recall within the past 5 years. The industry appears to endorse a willingness to unify precautionary allergen labeling to communicate a clear message more effectively to consumers.

  6. New Insights into Cockroach Allergens.

    PubMed

    Pomés, Anna; Mueller, Geoffrey A; Randall, Thomas A; Chapman, Martin D; Arruda, L Karla

    2017-04-01

    This review addresses the most recent developments on cockroach allergen research in relation to allergic diseases, especially asthma. The number of allergens relevant to cockroach allergy has recently expanded considerably up to 12 groups. New X-ray crystal structures of allergens from groups 1, 2, and 5 revealed interesting features with implications for allergen standardization, sensitization, diagnosis, and therapy. Cockroach allergy is strongly associated with asthma particularly among children and young adults living in inner-city environments, posing challenges for disease control. Environmental interventions targeted at reducing cockroach allergen exposure have provided conflicting results. Immunotherapy may be a way to modify the natural history of cockroach allergy and decrease symptoms and asthma severity among sensitized and exposed individuals. The new information on cockroach allergens is important for the assessment of allergen markers of exposure and disease, and for the design of immunotherapy trials.

  7. Grass Pollen Allergens

    PubMed Central

    Augustin, Rosa

    1959-01-01

    Grass pollen allergens are shown to remain associated with protein material and a yellow pigment during paper chromatography and during dialyses and ultrafiltrations of various types. Dialysable* allergens comprise only a fraction of 1 per cent of the total activity and the amount of activity extractable by diethylene glycol (DEG) and similar solvents is of the same order. Besides the allergens, the DEG and aqueous extracts contain large amounts of inositol, glucose and fructose, also some yellow pigments and phosphates. Larger amounts of free and combined amino acids are found in the aqueous than in the DEG extracts, but the reverse is true for sucrose. In addition the DEG extracts contain a yellow glucoside different from the dactylen of the aqueous extracts, a glucosan and an arabinose-galactose-pigment complex, only the latter being associated with any activity. The spontaneous release of the crystalline dactylen from originally clear aqueous pollen extracts is found not to be caused by enzymes. The washed crystals are found to be chromatographically and electrophoretically homogeneous and devoid of allergenic activity. ImagesFIG. 1FIG. 2FIG. 3FIG. 4FIG. 5FIG. 6FIG. 7 PMID:13640676

  8. Bleach Neutralizes Mold Allergens

    ERIC Educational Resources Information Center

    Science Teacher, 2005

    2005-01-01

    Researchers at National Jewish Medical and Research Center have demonstrated that dilute bleach not only kills common household mold, but may also neutralize the mold allergens that cause most mold-related health complaints. The study, published in the Journal of Allergy and Clinical Immunology, is the first to test the effect on allergic…

  9. Bleach Neutralizes Mold Allergens

    ERIC Educational Resources Information Center

    Science Teacher, 2005

    2005-01-01

    Researchers at National Jewish Medical and Research Center have demonstrated that dilute bleach not only kills common household mold, but may also neutralize the mold allergens that cause most mold-related health complaints. The study, published in the Journal of Allergy and Clinical Immunology, is the first to test the effect on allergic…

  10. Allergenicity of an enzymatic hydrolysate of soybean 2S protein.

    PubMed

    Sung, Dongeun; Ahn, Kang Mo; Lim, Seung-Yong; Oh, Sangsuk

    2014-09-01

    This study was performed to examine how the characteristics of soybean 2S protein influence allergenicity after enzymatic hydrolysis. Soybean 2S protein was extracted and enzymatic hydrolysis was performed using pepsin and chymotrypsin. Allergenicity was observed using soybean-sensitive patients' sera. Only 13.3% (6/45) of soybean-sensitive patients reacted to soybean Kunitz trypsin inhibitor (SKTI), known as the major allergen of soybean 2S protein. After peptic hydrolysis for 90 min at pH 1.2, the intensity of SKTI decreased to 25% but was still visible on SDS-PAGE. Chymotryptic hydrolysis following peptic hydrolysis at pH 8 for 60 min showed a limited hydrolytic effect on soybean 2S protein. Peptic hydrolysis of soybean 2S protein partially reduced the allergenicity of soybean 2S protein, while chymotryptic hydrolysis following peptic hydrolysis increased slightly the allergenicity. Food allergy caused by soybean 2S protein occurred in part of the soybean-sensitive patients. SKTI was partially digested after peptic hydrolysis for 90 min. The allergenicity was decreased with peptic hydrolysis, while subsequent treatment of chymotrypsin increased slightly the allergenicity. © 2014 Society of Chemical Industry.

  11. Array-based profiling of ragweed and mugwort pollen allergens.

    PubMed

    Gadermaier, G; Wopfner, N; Wallner, M; Egger, M; Didierlaurent, A; Regl, G; Aberger, F; Lang, R; Ferreira, F; Hawranek, T

    2008-11-01

    Ragweed (Ambrosia artemisiifolia) and mugwort (Artemisia vulgaris) pollen is the main cause of allergic reactions in late summer and autumn. The differential diagnosis between ragweed and mugwort pollen allergy is a frequent problem encountered by allergologists in areas where both plants are present due to shared antigenic structures and overlapping flowering seasons. To evaluate the sensitization pattern of weed allergic patients towards a large panel of purified allergens in the microarray format and by enzyme-linked immunosorbent assay (ELISA). Eight ragweed and six mugwort pollen allergens were purified from natural source or expressed as recombinant proteins in Escherichia coli. Allergens were spotted on protein microarray slides or coated onto ELISA plates. Sera from 19 ragweed and/or mugwort allergic individuals were used to determine the reactivity towards single molecules in both assays. All ragweed allergic individuals were sensitized to Amb a 1, among them 30% were monosensitized to the major ragweed allergen. Art v 1 and Art v 3 were recognized by 89% of mugwort pollen-allergic patients. Extensive cross-reactivity was observed for both patient groups mainly involving the pan-allergens profilin and nonspecific lipid transfer proteins. Comparable IgE profiles were obtained with both allergen microarray and ELISA methods. Molecule-based diagnosis provides essential information for the differential diagnosis between ragweed and mugwort pollen allergy and for the selection of the appropriate allergen source for specific immunotherapy.

  12. Contact allergens for armpits--allergenic fragrances specified on deodorants.

    PubMed

    Klaschka, Ursula

    2012-11-01

    According to the so-called "26 allergens rule" 26 supposedly allergenic fragrances must be specified on the containers of cosmetic products if they are present above 0.001% in leave-on products and, 0.01% in rinse-off products. This declaration is meant to inform the consumers of potential risks of skin sensitizers in the products. As many consumers of deodorants suffer from allergic or irritant contact dermatitis in the axillae, the presence of allergens in deodorants deserves special attention. The objective of this study was to find answers to the following questions: Does compulsory labeling lead to omission of strong allergenic fragrances in deodorants? Is there a difference in the use patterns of strong and weak allergens? What is the quantitative exposure to fragrances by deodorants? Is the situation in Germany different from other European countries? Is there a difference between deodorants for men and for women? I tested the implementation of the "26 allergens rule" and compiled which allergenic fragrances are specified on the containers of deodorants. Three market studies were conducted in Germany in 2008, 2010 and 2011. The labels of a total number of 374 deodorants were analyzed as to whether any of the "26 allergens" were listed. The frequency of each allergen in the deodorants was compared with results from previous studies by other authors. It was found that up to 83% of the deodorants contain at least one of the "26 allergens" and that up to 30% of all products contain strong allergens above the threshold for labeling (0.001% in the product). The most frequently listed allergens are medium or weak allergens. In comparison with other authors, the frequency of the "26 allergens" in products is slightly smaller in these recent studies for the German market. There is no significant difference between deodorants for men and women, as far as the labeling of the "26 allergens" is concerned. The results show that the mandatory labeling procedure as designed

  13. Natural Variability of Allergen Levels in Conventional Soybeans: Assessing Variation across North and South America from Five Production Years.

    PubMed

    Geng, Tao; Stojšin, Duška; Liu, Kang; Schaalje, Bruce; Postin, Cody; Ward, Jason; Wang, Yongcheng; Liu, Zi Lucy; Li, Bin; Glenn, Kevin

    2017-01-18

    Soybean (Glycine max L. Merrill) is one of eight major allergenic foods with endogenous proteins identified as allergens. To better understand the natural variability of five soybean allergens (Gly m 4, Gly m 5, Gly m 6, Gly m Bd 28k, and Gly m Bd 30k), validated enzyme-linked immunosorbent assays (ELISAs) were developed. These ELISAs measured allergens in 604 soybean samples collected from locations in North and South America over five growing seasons (2009-2013/2014) and including 37 conventional varieties. Levels of these five allergens varied 5-19-fold. Multivariate statistical analyses and pairwise comparisons show that environmental factors have a larger effect on allergen levels than genetic factors. Therefore, from year to year, consumers are exposed to highly variable levels of allergens in soy-based foods, bringing into question whether quantitative comparison of endogenous allergen levels of new genetically modified soybean adds meaningful information to their overall safety risk assessment.

  14. Anti-Fatigue Properties of Tartary Buckwheat Extracts in Mice

    PubMed Central

    Jin, Hong-Mei; Wei, Ping

    2011-01-01

    Anti-fatigue properties of tartary buckwheat extracts (TBE) was investigated in male Kunming mice. The animals were divided into four groups. The first group, designated as the control group (control), was administered with distilled water by gavage every day for 28 days. The other three groups, designated as TBE treatment groups, were administered with TBE of 60, 120 and 240 mg/kg body weight, respectively, by gavage every day for 28 days. Exhaustive swimming time, blood lactic acid (BLA), blood urea nitrogen (BUN), tissue glycogen, glutathione peroxidase (GPx) and superoxide dismutase (SOD) of mice after swimming were determined. The results showed that tartary buckwheat extracts had anti-fatigue properties, which extended the exhaustive swimming time of mice, effectively inhibiting the increase of BLA, decreasing the level of BUN, increasing the tissue glycogen content and the activities of SOD and GPx of mice. However, further study is needed to elucidate the exact mechanism of the effect of TBE on fatigue. PMID:21954324

  15. Improving the antioxidant activity of buckwheat (Fagopyrum tataricm Gaertn) sprout with trace element water.

    PubMed

    Hsu, Cheng-Kuang; Chiang, Been-Huang; Chen, Yih-Shyuan; Yang, Joan-Hwa; Liu, Chia-Ling

    2008-05-15

    Trace element water (TEW) (100, 200, 300, 400 and 500ppm) was used to grow buckwheat (Fagopyrum tataricm Gaertn) to evaluate whether the beneficial effects of trace elements on the antioxidant activity could be accomplished with the supplement of TEW. At 300ppm, TEW significantly increased the Cu, Zn and Fe contents in buckwheat sprout, but not the Se and Mn contents. The levels of rutin, quercitrin and quercetin did not differ between buckwheat sprouts grown in TEW and de-ionized water (DIW). The ethanolic extract from buckwheat sprout grown in 300ppm TEW showed higher DPPH radical scavenging activity, ferrous ion chelating activity, superoxide anion scavenging activity and inhibitory activity toward lipid peroxidation than that grown in DIW. The extract of the TEW group also enhanced intracellular superoxide dismutase activity and resulted in lower level of reactive oxygen species in human Hep G2 cells.

  16. Steady and Dynamic Shear Rheological Properties of Buckwheat Starch-galactomannan Mixtures

    PubMed Central

    Choi, Dong Won; Chang, Yoon Hyuk

    2012-01-01

    This study investigated the effects of galacomannans (guar gum, tara gum, and locust bean gum) on the rheological properties of buckwheat starch pastes under steady and dynamic shear conditions. The power law and Casson models were applied to describe the flow behavior of the buckwheat starch and galactomannan mixtures. The values of the apparent viscosity (ηa,100), consistency index (K), and yield stress (σoc) for buckwheat starch-galactomannan mixtures were significantly greater than those for the control, indicating that there was a high synergism of the starch with galactomannans. The magnitudes of storage modulus (G′) and loss modulus (G″) for the starch-galactomannan mixtures increased with increasing frequency (ω). The dynamic moduli (G′, G″), and complex viscosity (η*) for the buckwheat starch-galactomannan mixtures were significantly higher than those for the control. PMID:24471083

  17. Reducing peanut allergens by high pressure combined with polyphenol oxidase

    NASA Astrophysics Data System (ADS)

    Chung, Si-Yin; Houska, Milan; Reed, Shawndrika

    2013-12-01

    Polyphenol oxidase (PPO) has been shown to reduce major peanut allergens. Since high pressure (HP) can increase enzyme activity, we postulated that further reduction of peanut allergens can be achieved through HP combined with PPO. Peanut extracts containing caffeic acid were treated with each of the following: (1) HP; (2) HP+PPO; (3) PPO; and (4) none. HP was conducted at 300 and 500 MPa, each for 3 and 10 min, 37 °C. After treatment, SDS-PAGE was performed and allergenic capacity (IgE binding) was determined colorimetrically in inhibition enzyme-linked immunosorbent assay and Western blots, using a pooled plasma from peanut-allergic patients. Data showed that HP alone had no effect on major peanut allergens. However, HP at 500 MPa combined with PPO (HP500/PPO) induced a higher (approximately twofold) reduction of major peanut allergens and IgE binding than PPO alone or HP300/PPO. There was no difference between treatment times. We concluded that HP500/PPO at 3-min enhanced a twofold reduction of the allergenic capacity of peanut extracts, as compared to PPO itself.

  18. Sublingual allergen immunotherapy: immunological mechanisms and prospects for refined vaccine preparation.

    PubMed

    O'Hehir, R E; Sandrini, A; Anderson, G P; Rolland, J M

    2007-01-01

    Allergic diseases constitute a major health issue worldwide. Mainstay treatment constitutes allergen avoidance and pharmacotherapy for symptom relief, but allergen immunotherapy offers advantages of specific treatment with long lasting efficacy, and being able to modify the course of the disease. Conventional immunotherapy involves the subcutaneous injection of gradually increasing amounts of allergen extract but the use of current whole allergen extracts is restricted by the risk of adverse IgE-mediated events especially for potent allergens such as peanut and latex and for asthmatics. This has lead to interest in alternative routes of immunotherapy. Oral tolerance is a well-documented immune process and the sublingual route of administration of allergen immunotherapy is attracting interest. Recent meta-analyses show that sublingual allergen immunotherapy for grass pollen and house dust mite allergy is clinically effective and safer than injection immunotherapy. Some studies show SLIT induces changes of T cell anergy, immune deviation, blocking antibodies, and induction of regulatory T cells, as described for injection immunotherapy pointing to the need to target allergen-specific T cells, there is emergent evidence that the oral mucosa presents distinct regulatory features. Evidence suggests that oral dendritic cells play a key role in inducing tolerance especially when allergen is taken up via Fc receptor bound IgE. This suggests that although both would target allergen-specific T cells, allergen formulations may differ with respect to IgE epitopes for optimal SLIT compared with SCIT. Identification of the molecular nature of the allergen-DC receptor interaction is required to determine whether short peptides or recombinant allergen preparations and of suitable adjuvants specifically tailored for the sublingual route will allow the development of improved allergen formulations and delivery strategies for efficacy of treatment whilst decreasing Ig

  19. Buckwheat (Fagopyrum esculentum Moench) grain and fractions: antioxidant compounds and activities.

    PubMed

    Sedej, Ivana; Sakač, Marijana; Mandić, Anamarija; Mišan, Aleksandra; Tumbas, Vesna; Čanadanović-Brunet, Jasna

    2012-09-01

    Buckwheat (Fagopyrum esculentum Moench) is an alternative crop belonging to the Polygonaceae family. In comparison to antioxidant activity of frequently used cereals, buckwheat has been reported to possess higher antioxidant activity (AOA), mainly due to high rutin content. The objective of this work was to determine the main antioxidant compounds and AOA of buckwheat grain fractions (whole grain, hull, and groat). Buckwheat grain fractions were extracted with ethanol/water (80/20, v/v), followed by determination of total phenolic and flavonoid content. Quantification of phenolic compounds and tocopherols was performed by high-performance liquid chromatography (HPLC). The AOA was estimated by 2 direct electron spin resonance (ESR) and 4 indirect (spectrophotometric) tests. Significantly higher contents of total phenolics and total flavonoids were found in buckwheat hull than in whole grain and groat. Protocatechuic, syringic, and sinapic acid, rutin, and quercetin were found in all tested fractions, whereas vanilic acid was found in whole grain and hull. The content of total tocopherols in investigated samples ranged from 23.3 mmol/g for hull to 61.8 mmol/g for groat. Hull was superior in scavenging activity on 1,1-diphenyl-2-picrylhydrazyl (DPPH(•)), hydroxyl ((•)OH), and superoxide anion (O(2)(•-)) radicals, reducing activity, AOA by β-carotene bleaching method, and chelating activity on Fe(2+) as evidenced by its lower IC(50) value. Obtained results can broaden the utilization of buckwheat, especially a share of hull in whole grain flour production. Obtained results suggest possibility to supplement the whole grain buckwheat flour with hull, which leads toward better usage of by-products in buckwheat production, and enhancement of antioxidant potential of the final product. © 2012 Institute of Food Technologists®

  20. Allergen Levels in the Hair of Different Cattle Breeds.

    PubMed

    Zahradnik, Eva; Sander, Ingrid; Brüning, Thomas; Raulf, Monika

    2015-01-01

    Cattle are well-known sources of respiratory allergens in agricultural environments. Breed-specific differences in Bos d 2 (a major bovine allergen) levels in cattle hair have been previously suggested but not fully characterized. Therefore, the aim of the current study was to determine whether hair from common cattle breeds differs in protein and allergen content. In total, 80 hair samples from 16 different cattle breeds were analyzed. The protein concentration was determined using the Bradford assay. The allergen content was measured using a sandwich ELISA based on polyclonal antibodies against a bovine hair protein extract and a commercial immunoassay based on monoclonal antibodies against Bos d 2. Results are given in micrograms per gram of hair. Statistical analysis was performed using the Kruskal-Wallis test and Spearman's rank correlation. A wide variability in all 3 tested parameters was observed between the individual samples. The protein content differed by about 35-fold (0.3-12 mg/g), the bovine hair allergen content differed by about 500-fold (37-18,553 µg/g), and the Bos d 2 content differed by about 1,200-fold (5-6,323 µg/g). Protein, bovine hair allergen, and Bod d 2 values correlated strongly and significantly with one another. The median Bos d 2/bovine hair allergen ratio was 0.28. No significant differences were found between the most common breeds in Germany (Simmental, Holstein, and Braunvieh) and a group of rare breeds or between female and male animals. The results confirm a high variability in allergen levels between individual animals but also indicate that allergen production is related neither to the breed nor to gender. © 2015 S. Karger AG, Basel.

  1. Reduction in allergenicity of grass pollen by genetic engineering.

    PubMed

    Bhalla, P L; Swoboda, I; Singh, M B

    2001-01-01

    Hay fever and allergic asthma triggered by grass pollen allergens affect approximately 20% of the population in cool temperate climates. Ryegrass is the dominant source of allergens due to its prodigious airborne pollen production. Lol p 5 or group 5 is among the most important and widespread grass pollen allergen because it reacts with IgE antibodies of more than 90% of grass pollen-allergic patients, contains most of the grass pollen-specific IgE epitopes and elicits strong biological responses. Significant efforts have been made in developing diagnostic and therapeutic reagents for designing new and more effective immunotherapeutic strategies for treatment of allergic diseases. An alternative approach to this problem could be to reduce the amount of allergen content in the source plant. High velocity microprojectile bombardment was used to genetically engineer ryegrass. Antisense construct targeted to one of major allergen, Lol p 5, was introduced. The expression of antisense RNA was regulated by a pollen-specific promoter. Pollen was analysed for IgE reactivity. Analysis of proteins with allergen-specific monoclonal and polyclonal antibodies did not detect Lol p 5 in the transgenic pollen. The transgenic pollen showed remarkably reduced allergenicity as reflected by low IgE binding capacity of pollen extract as compared to control pollen. The transgenic ryegrass plants in which Lol p 5 gene expression is perturbed showed normal fertile pollen development. Our studies showed that it is possible to selectively 'switch off' allergen production in pollen of ryegrass demonstrating feasibility of genetic engineering of plants for reduced allergenicity. Copyright 2001 S. Karger AG, Basel

  2. Inhalant Allergens in Portugal.

    PubMed

    Gomes Câmara Camacho, Irene

    2017-02-23

    This review aims to present in a simple manner the work performed in Portugal regarding the identification of the most prevalent aeroallergens in the country and the sensitization levels in Portuguese patients. Much of the data was summarized in tables and illustrated on maps, enabling the community of clinicians, researchers, and patient organizations to access the knowledge about the research performed. This study provides an overview about the distribution of aeroallergens in Portugal, signaling regions and critical periods of exposure of the sensitized population. The illustrated data can help the community of allergy specialists to view the temporal and spatial distribution of aeroallergens across the country. In addition, this information can guide clinicians to select the most appropriate allergens for allergy diagnostic testing, treatment, and allergen avoidance.

  3. Flavonoids, Antioxidant Activity and Aroma Compounds Analysis from Different Kinds of Tartary Buckwheat Tea

    PubMed Central

    Peng, L. X.; Zou, L.; Wang, J. B.; Zhao, J. L.; Xiang, D. B.; Zhao, G.

    2015-01-01

    The rutin, quercetin concentrations, antioxidant activity, and aroma compounds in different commercial tartary buckwheat tea were analyzed in our study. Results revealed that the materials and the processing protocol affected the chemical composition and activity of tartary buckwheat tea. Rutin and quercetin concentrations, antioxidant activity were significantly different in various kinds of tartary buckwheat tea, where the whole bran tea and the whole plant tea had the lower rutin, but higher quercetin concentrations and higher antioxidant activity. The whole embryo tea had the converse results. There was strong correlation between quercetin concentration and antioxidant activity (r>0.98, P<0.05). Meanwhile, Twenty eight different aroma compounds in tartary buckwheat tea were identified by gas chromatography-mass spectrometry. Those compounds were mainly composed of pyrazine, aldehydes, fatty acids and ketones. The main type of aroma compounds in different tartary buckwheat tea were similar, but their relative contents were different. The implications to the quality control of buckwheat tea were extensively discussed. PMID:26997692

  4. Hypolipidemic and antioxidant effects of buckwheat leaf and flower mixture in hyperlipidemic rats.

    PubMed

    Ðurendić-Brenesel, Maja; Popović, Tamara; Pilija, Vladimir; Arsić, Aleksandra; Milić, Miljan; Kojić, Danijela; Jojić, Nikola; Milić, Nataša

    2013-05-01

    As a source of biologically active compounds, buckwheat has beneficial effects in nutrition due to its high content of flavonoids, particularly rutin. Aim of our study was to examine effects of buckwheat on plasma lipid status and phospholipids fatty acids composition, histological and parameters of oxidative stress in Wistar rats fed a high-fat diet. This study showed that buckwheat leaf and flower (BLF) mixture supplementation significantly reduce weight gain, plasma lipid concentrations and atherogenic index in rats fed a high-fat diet. Treatment of the high-fat group of animals with buckwheat significantly increased percentage of n-6 fatty acids as well as eicosapentaenoic acid (EPA) and decreased percentage of saturated fatty acids (SFA) and oleic acid. Buckwheat antioxidant effects diminished negative influence of high-fat diet in hyperlipidemic rats, while pathohistological analysis of liver confirmed changes after high-fat consumption. Our results showed hypolipidemic, antiatherogenic and antioxidative features of buckwheat leaf and flower mixture, and these parts of the plant with the highest rutin content could be beneficial in prevention and curing of hyperlipidemia. © 2013 Association of Basic Medical Sciences of FB&H. All rights reserved.

  5. Hypolipidemic and antioxidant effects of buckwheat leaf and flower mixture in hyperlipidemic rats

    PubMed Central

    Đurendić - Brenesel, Maja; Popović, Tamara; Pilija, Vladimir; Arsić, Aleksandra; Milić, Miljan; Kojić, Danijela; Jojić, Nikola; Milić, Nataša

    2013-01-01

    As a source of biologically active compounds, buckwheat has beneficial effects in nutrition due to its high content of flavonoids, particularly rutin. Aim of our study was to examine effects of buckwheat on plasma lipid status and phospholipids fatty acids composition, histological and parameters of oxidative stress in Wistar rats fed a high-fat diet. This study showed that buckwheat leaf and flower (BLF) mixture supplementation significantly reduce weight gain, plasma lipid concentrations and atherogenic index in rats fed a high-fat diet. Treatment of the high-fat group of animals with buckwheat significantly increased percentage of n-6 fatty acids as well as eicosapentaenoic acid (EPA) and decreased percentage of saturated fatty acids (SFA) and oleic acid. Buckwheat antioxidant effects diminished negative influence of high-fat diet in hyperlipidemic rats, while pathohistological analysis of liver confirmed changes after high-fat consumption. Our results showed hypolipidemic, antiatherogenic and antioxidative features of buckwheat leaf and flower mixture, and these parts of the plant with the highest rutin content could be beneficial in prevention and curing of hyperlipidemia. PMID:23725506

  6. Structure of allergens and structure based epitope predictions☆

    PubMed Central

    Dall’Antonia, Fabio; Pavkov-Keller, Tea; Zangger, Klaus; Keller, Walter

    2014-01-01

    The structure determination of major allergens is a prerequisite for analyzing surface exposed areas of the allergen and for mapping conformational epitopes. These may be determined by experimental methods including crystallographic and NMR-based approaches or predicted by computational methods. In this review we summarize the existing structural information on allergens and their classification in protein fold families. The currently available allergen-antibody complexes are described and the experimentally obtained epitopes compared. Furthermore we discuss established methods for linear and conformational epitope mapping, putting special emphasis on a recently developed approach, which uses the structural similarity of proteins in com