Biomedical subjects

Stefan Vieths

Publications and source records attributed to Stefan Vieths.

At least 19 recordsLinked to original sources

PubMed2007

IgE-mediated food allergy diagnosis: Current status and new perspectives.

In June 2005, the work of the EU Integrated Project EuroPrevall was started. EuroPrevall is the largest research project on food allergy ever performed in Europe. Major aims of the project are to generate for the first time reliable data on the prevalence of food allergies across Europe and on the natural course of food allergy development in infants. Improvement of in vitro diagnosis of food allergies is another important aim of the project. The present review summarizes current knowledge about the clinical presentation of food allergy and critically reviews available diagnostic tools at the beginning of the project period. A major problem in diagnosis is a relatively poor 'clinical specificity', i. e. both positive skin tests and in vitro tests for specific IgE are frequent in sensitized subjects without food allergy symptoms. So far, no in vitro test reliably predicts clinical food allergy. EuroPrevall aims at improving the predictive value of such tests by proceeding from diagnosis based on allergen extracts to purified allergen molecules, taking into account the affinity of the IgE-allergen interaction, and evaluating the potential of biological in vitro tests such as histamine release tests or basophil activation tests including assays performed with permanently growing cell lines.

Allergens↗

PubMed2006

Reduced allergenicity of tomato fruits harvested from Lyc e 1-silenced transgenic tomato plants.

BACKGROUND: Profilin is a small actin-binding protein that contributes to the allergenic potency of many fruits and vegetables, including tomato. Two highly similar genes encoding tomato profilin have been isolated and designated as allergen Lyc e 1.01 and Lyc e 1.02. OBJECTIVE: The aim of the study was to generate profilin-reduced hypoallergenic tomato fruits by silencing of both genes in transgenic tomato plants by means of RNA interference (RNAi). METHODS: The efficiency of gene silencing was documented by means of Northern blotting, immunoblotting, and skin prick testing. RESULTS: Quantification of the remaining protein revealed that profilin accumulation in transgenic fruits was decreased 10-fold compared with that seen in untransformed controls. This decrease was sufficient to cause a reduced allergenic reactivity in patients with tomato allergy, as determined with skin prick tests. Because most patients with tomato allergy are not monosensitized to profilin, the IgE reactivity to the profilin-silenced tomato fruits in vivo varied widely between individuals tested. CONCLUSION: We could demonstrate the efficient silencing of both profilin genes in transgenic tomato plants using RNAi. This resulted in Lyc e 1-diminished tomato fruits, providing proof of concept and demonstrating that RNAi can be used to design allergen-reduced food. However, simultaneous silencing of multiple allergens will be required to design hypoallergenic tomatoes. CLINICAL IMPLICATIONS: Our findings demonstrate the feasibility of creating low-allergenic food by using RNAi. This concept constitutes a novel approach to allergen avoidance.

Adult↗

PubMed2006

Skin prick tests reveal stable and heritable reduction of allergenic potency of gene-silenced tomato fruits.

BACKGROUND: Today, for patients with food allergy, the only possibility to prevent allergic reactions is avoidance of the allergenic food. Genetic engineering of hypoallergenic plants by means of RNA interference (RNAi) could be an approach to improve the quality of life of subjects with food allergy. OBJECTIVES: We sought to achieve stable inhibition of expression of the allergenic nonspecific lipid transfer protein Lyc e 3 in tomato and to analyze the reduction of allergenicity in vitro by using histamine release assays and in vivo by using skin prick tests with transgenic tomato fruits. METHODS: Gene silencing was performed by means of RNAi and monitored by using Western blotting with nonspecific lipid transfer protein-specific antibodies and sera from patients with tomato allergy. Dose-dependent basophil histamine release assays, prick-to-prick skin testing, and determination of endogenous histamine content were performed with fruits harvested from plants of the first and second generation to assess the allergenic potency compared with that of wild-type fruits. RESULTS: We demonstrated that silencing of Lyc e 3 by means of RNAi contributes to reduced skin reactivity and is passed on to the next generation of fruits. A significant reduction of allergenic potency was determined in vitro and confirmed by using skin prick tests. CONCLUSION: Taken together, these results indicate that RNAi technology is an effective tool to generate foods with reduced allergenicity. CLINICAL IMPLICATIONS: Allergen-reduced plant foods might allow reduction of dietary restrictions for patients allergic to panallergen families.

Adult↗

PubMed2006

Structure and stability of 2S albumin-type peanut allergens: implications for the severity of peanut allergic reactions.

Resistance to proteolytic enzymes and heat is thought to be a prerequisite property of food allergens. Allergens from peanut (Arachis hypogaea) are the most frequent cause of fatal food allergic reactions. The allergenic 2S albumin Ara h 2 and the homologous minor allergen Ara h 6 were studied at the molecular level with regard to allergenic potency of native and protease-treated allergen. A high-resolution solution structure of the protease-resistant core of Ara h 6 was determined by NMR spectroscopy, and homology modelling was applied to generate an Ara h 2 structure. Ara h 2 appeared to be the more potent allergen, even though the two peanut allergens share substantial cross-reactivity. Both allergens contain cores that are highly resistant to proteolytic digestion and to temperatures of up to 100 degrees C. Even though IgE antibody-binding capacity was reduced by protease treatment, the mediator release from a functional equivalent of a mast cell or basophil, the humanized RBL (rat basophilic leukaemia) cell, demonstrated that this reduction in IgE antibody-binding capacity does not necessarily translate into reduced allergenic potency. Native Ara h 2 and Ara h 6 have virtually identical allergenic potency as compared with the allergens that were treated with digestive enzymes. The folds of the allergenic cores are virtually identical with each other and with the fold of the corresponding regions in the undigested proteins. The extreme immunological stability of the core structures of Ara h 2 and Ara h 6 provides an explanation for the persistence of the allergenic potency even after food processing.

2S Albumins, Plant↗

PubMed2006

Cloning of oleosin, a putative new hazelnut allergen, using a hazelnut cDNA library.

The clinical presentation of non-pollen related allergy to hazelnut can be severe and systemic. So far, only a limited number of non-pollen related hazelnut allergens have been identified and characterized. The aim of this study was to identify and clone new hazelnut allergens. A lambda ZAP cDNA library of hazelnut was constructed. The library was screened with serum of six hazelnut allergic patients displaying different IgE-binding patterns on hazelnut immunoblot. Rapid amplification of cDNA ends (RACE) protocols were applied to obtain full-length clones. Expression experiments were carried out in Eschericchia coli. Expression was monitored by SDS-PAGE, protein staining and immunoblotting. A hazelnut cDNA library was constructed. IgE screening resulted in the cloning of two isoforms of a novel putative hazelnut allergen. The clones were identified as oleosins, with theoretical molecular masses of 16.7 and 14.7 kDa and pI of 10.5 and 10.0, respectively. The isoforms demonstrated only 37% amino acid sequence identity but contained the typical hydrophobic stretch in the middle of the protein (53% identity) with the characteristic oleosin proline knot region (11/12 amino acids identical). Expression in E. coli of the longer isoform resulted in a clear band on SDS-PAGE. The expressed protein was recognized on an immunodot blot by IgE from serum that was used for screening the cDNA library. Hazelnut contains multiple isoforms of oleosin. IgE binding of a hazelnut-allergic patient to a recombinant version suggest that hazelnut oleosin is an allergen, as has been described for peanut and sesame.

Amino Acid Sequence↗

PubMed2006

Germin-like protein Cit s 1 and profilin Cit s 2 are major allergens in orange (Citrus sinensis) fruits.

Oranges are clinically relevant allergenic foods. To date, orange allergens have not been characterized in detail. The study is aimed at analyzing the sensitization profile in orange-sensitized subjects with and without clinical allergy, and to identify orange allergens. Fifty-six sensitized subjects with self-reported reactions to orange were grouped into reactors (anaphylaxis or multiple episodes of immediate reactions and/or positive challenge tests) and non-reactors (negative open food challenge tests). Allergens were characterized by IgE immunoblotting, N-terminal sequencing, IgE-inhibition assays, and mediator release assays were performed to determine the allergenic potency of orange profilin. Of 56 subjects, 23 were classified as orange allergic showing mainly an oral allergy syndrome. Of 23 subjects classified as orange allergic, 22 were sensitized to profilin, Cit s 2. In patients with mono-sensitization to profilin in vitro histamine releases up to 75% from basophils were induced using orange extract and purified plant profilins. Of the allergic patients 78% were sensitized to germin-like protein, Cit s 1. Both allergens showed retained IgE reactivity in heat-processed orange juice. Interestingly, subjects with and without clinical allergy showed a comparable sensitization profile. Profilin and germin-like proteins are major orange allergens. The potential clinical relevance of orange profilin was indicated by its strong capacity to release histamine from basophils. However, a predominant sensitization to both allergens in subjects without symptoms also indicates a high frequency of clinically insignificant sensitization.

Allergens↗

PubMed2006

Allergen-specific IgE testing in the diagnosis of food allergy and the event of a positive match in the bioinformatics search.

Current documents on risk assessment of genetically modified foods recommend including IgE-binding tests on sera from allergic patients. However, there is no generally accepted recommendation on technical aspects of the testing procedures or on the interpretation of the results, despite that fact that both false positive and false-negative results may be caused by variability of the test procedures. The present article discusses the state-of-the-art of serological test procedures for qualitative and quantitative determination of specific IgE and interpretation of test results. It is emphasized that the use of sera from clinically well-characterized subjects is of high importance. In the case of a positive test result, the biological activity of the detected IgE antibodies, i. e., the potential to trigger mediator release from basophils or mast cells in an allergen-specific manner, should be taken into account. However, present data also indicate that validation of such mediator release tests is required, both in terms of experimental protocols and with respect to correlation of the test results with the clinical situation. Further studies are also required to prove the usefulness of targeted serum screening, i. e., the testing of gene products from organisms not known to be allergenic with sera from subjects allergic to related species.

Allergens↗

PubMed2006

A novel approach for investigation of specific and cross-reactive IgE epitopes on Bet v 1 and homologous food allergens in individual patients.

BACKGROUND: A clinically relevant allergic reaction requires recognition of at least two different epitopes on the surface of the allergen by IgE. These epitopes may be specific or cross-reactive. Moreover, patterns of IgE reactivity may be patient-specific. The aim of our study was to compare specific and cross-reactive IgE epitopes and epitope patterns between individual patients. We used Bet v 1-related food allergy as a model. METHODS: Five patients were investigated by cross-competitive ELISA for specific and cross-reacting IgE to Bet v 1, and its homologues Gly m 4 (soybean), Ara h 8 (peanut), and Pru av 1 (cherry). Allergen-specific as well as cross-reactive IgE epitopes were assessed by competitive immunoscreening of a phage-displayed random 7-mer peptide library using polyclonal purified IgE from individual sera. The resulting peptide mimics were mapped on the surface of the 3D-structure of the allergens using a computer-based algorithm. RESULTS: Competitive immunoscreening and epitope mapping identified patient-specific IgE epitope patterns. However, one IgE-binding surface area that was recognized by all patients and two recognized by three patients were identified on all four proteins. These results are consistent with the determination of IgE cross-reactivity of the individual patients' sera against the four recombinant allergens by cross-competitive ELISA. CONCLUSIONS: Selection of phage-displayed peptide mimics with serum IgE from allergic patients in combination with computer-based mapping of the peptide mimics onto the surface of the three-dimensional allergen structure is a promising novel tool to investigate IgE epitope specificity in individual patients. Such basic information on epitope structure may be used for prediction of cross-reactivity and potential allergenicity of novel foods.

2S Albumins, Plant↗

PubMed2006

Component-resolved diagnostics in food allergy.

PURPOSE OF REVIEW: The purpose of this paper is to review and discuss recent studies on component-resolved diagnostics in food allergy, involving panels of pure allergen molecules or arrays of peptides derived from allergen sequences, and to summarize the reporting of new food allergens during the past 2 years. RECENT FINDINGS: Several component-resolved diagnostic studies in food allergy suggest that the use of panels of allergen molecules may allow refined clinical information to be obtained on the likelihood or severity of an allergic food reaction and regarding diagnostic specificity. Further, in some studies the use of pure allergen molecules has led to a clearly higher sensitivity of the immunoglobulin E immunoassay compared with conventional allergen extracts. SUMMARY: While common diagnostic methods in allergy assess the presence or absence of allergen-specific sensitization, to date, no in-vitro or in-vivo test exists which exhibits full correlation with clinical food allergy. A multitude of recently reported findings and observations indicate that molecular analysis of allergen sensitization pattern may serve to enhance the clinical utility of immunoglobulin E antibody-based allergy diagnostics. Pure natural and recombinant allergen molecules as well as panels of synthetic peptides have been used for this purpose.

Allergens↗

PubMed2006

Standardization of allergen extracts for immunotherapy: where do we stand?

PURPOSE OF REVIEW: The current state of the art in allergen standardization and recent progress in the field is summarized, and future developments are discussed. RECENT FINDINGS: The main focus of recent research in allergen standardization was on sandwich enzyme-linked immunosorbent assays or competitive tests for the quantification of individual allergens in extracts. New assays for quantifying major or minor allergens have been developed for tree and weed pollens from the Mediterranean area, grass pollens, and foods such as peanut and apple. In several cases, a good correlation with allergenic activity, measured by inhibition tests, was obtained. In addition, the potential of cellular mediator release assays in allergen standardization was evaluated in one study. SUMMARY: Several new tests have been developed to make more major and minor allergens from various allergen sources accessible to allergen standardization programmes such as the CREATE project. It is expected that assays to determine the majority of all clinically relevant major allergens from aeroallergen sources will be available in the near future. Standardized and validated mediator release assays may be a complementary tool for evaluating the biological potency of reference allergens and for correlating allergen concentrations to biological potency.

Allergens↗

PubMed2006

A critical assessment of allergen component-based in vitro diagnosis in cherry allergy across Europe.

BACKGROUND: Food allergy to cherry occurs throughout Europe, typically with restricted oral reactions in the central and northern parts but with frequent systemic reactions in the Mediterranean region. Previous studies have demonstrated insufficient sensitivity of commercially available cherry extract reagents in the diagnosis of cherry allergy. OBJECTIVE: To assess the diagnostic performance of specific IgE tests based on recombinant cherry allergens in comparison with an extract-based assay and to skin prick test (SPT). A secondary objective was to analyse the frequency of systemic reactions in cherry-allergic subjects across Europe, including the largest population of LTP-sensitized subjects from central Europe studied to date. METHODS: A total of 186 subjects from central Europe and Spain were studied. Serum IgE was analysed with ImmunoCAP tests carrying rPru av 1, 3 and 4, combined and separately, and cherry extract. RESULTS: Among the central European cherry allergics, the mix of rPru av 1, 3 and 4 had a sensitivity of 95%, compared with 65% for cherry extract, and the IgE binding capacity of the recombinant mix was considerably higher. The sensitivity of the two tests was more comparable in the Spanish population, 95% and 86%, respectively. The recombinant allergen ImmunoCAP equalled SPT in terms of sensitivity and specificity. Consistent with previous reports, major geographic differences in sensitization pattern and prevalence of systemic reactions were found. A significantly higher rate of systemic reactions was found in Spanish patients sensitized to Pru av 3 whereas German patients sensitized to LTP only had oral allergy syndrome. CONCLUSIONS: The recombinant cherry allergen ImmunoCAP is a highly sensitive diagnostic tool, clearly superior to any diagnostic method based on cherry extract. Three cherry allergens are sufficient for detecting sensitization in 95% of cherry-allergic subjects. Systemic reactions are common in LTP-sensitized individuals but seem to require at least one additional causative factor.

Adolescent↗

PubMed2006

Design of tomato fruits with reduced allergenicity by dsRNAi-mediated inhibition of ns-LTP (Lyc e 3) expression.

Plant genetic engineering has the potential to introduce new allergenic proteins into foods but, at the same time, it can be used to remove established allergens. Here, we report the molecular characterization of Lyc e 3, a new tomato (Lycopersicon esculentum) allergen, and the efficient down-regulation of its expression in transgenic tomato plants. Following the identification of an immunoglobulin E (IgE)-binding 9-kDa polypeptide in tomato peel, designated Lyc e 3, its partial amino acid sequence was determined by N-terminal protein sequencing. Sequence comparison revealed that Lyc e 3 encodes a nonspecific lipid transfer protein (ns-LTP). In plants, ns-LTPs are encoded by large gene families which differ in primary amino acid sequence, expression and proposed cellular function. To identify Lyc e 3 encoding complementary DNAs (cDNAs), public tomato expressed sequence tag (EST) databases were screened for ns-LTP sequences. Following this strategy, two cDNAs, LTPG1 and LTPG2, with high homology to the N-terminal sequence of Lyc e 3, were identified. Ectopic expression of LTPG1 and LTPG2 in Escherichia coli, followed by immunoblotting, verified their IgE reactivity. Subsequently, transgenic tomato plants constitutively expressing LTPG1- or LTPG2-specific double-stranded RNA interference (dsRNAi) constructs were created and tested for the suppression of Lyc e 3 accumulation. Efficient silencing of Lyc e 3 was documented by Northern and Western blotting. In both cases, Lyc e 3 accumulation was decreased to levels below the detection limit (less than 0.5% of the wild-type protein). The allergenic potential of Lyc e 3-deficient tomato fruits was tested by measuring histamine release from sensitized human basophils stimulated with transgenic and parental lines. These assays revealed a strong (10- to 100-fold) decrease in histamine release of human basophils challenged with transgenic fruit extracts when compared with control extracts. These results demonstrate the feasibility of creating low allergenic tomato fruits by means of dsRNAi inhibition.

Allergens↗

PubMed2005

Reduced allergenic potency of VR9-1, a mutant of the major shrimp allergen Pen a 1 (tropomyosin).

The major shrimp allergen, tropomyosin, is an excellent model allergen for studying the influence of mutations within the primary structure on the allergenic potency of an allergen; Pen a 1 allows systematic evaluation and comparison of Ab-binding epitopes, because amino acid sequences of both allergenic and nonallergenic tropomyosins are known. Individually recognized IgE Ab-binding epitopes, amino acid positions, and substitutions critical for IgE Ab binding were identified by combinatorial substitution analysis, and 12 positions deemed critical were mutated in the eight major epitopes. The mutant VR9-1 was characterized with regard to allergenic potency by mediator release assays using sera from shrimp-allergic subjects and sera from BALB/c, C57BL/6J, C3H/HeJ, and CBA/J mice sensitized with shrimp extract using alum, cholera toxin, and Bordetella pertussis, as adjuvants. The secondary structure of VR9-1 was not altered; however, the allergenic potency was reduced by 90-98% measuring allergen-specific mediator release from humanized rat basophilic leukemia (RBL) cells, RBL 30/25. Reduced mediator release of RBL-2H3 cells sensitized with sera from mice that were immunized with shrimp extract indicated that mice produced IgE Abs to Pen a 1 and to the same epitopes as humans did. In conclusion, data obtained by mapping sequential epitopes were used to generate a Pen a 1 mutant with significantly reduced allergenic potency. Epitopes that are relevant for human IgE Ab binding are also major binding sites for murine IgE Abs. These results indicate that the murine model might be used to optimize the Pen a 1 mutant for future therapeutic use.

Allergens↗

PubMed2005

Gastrointestinal digestion of Bet v 1-homologous food allergens destroys their mediator-releasing, but not T cell-activating, capacity.

BACKGROUND: Food allergy to apples, hazelnuts, and celery is frequent in individuals with birch pollen allergy because IgE antibodies specific for the major birch pollen allergen, Bet v 1, cross-react with structurally related allergens in these foods. In addition, T lymphocytes specific for Bet v 1 also cross-react with these dietary proteins. OBJECTIVE: We sought to evaluate the effects of simulated gastrointestinal degradation of Bet v 1-related food allergens on their mediator-releasing and T cell-activating capacity. METHODS: Recombinant Mal d 1, Cor a 1.04, and Api g 1 were incubated separately with pepsin and trypsin. Binding of IgE was tested in immunoblots. After successive incubation with both enzymes, allergens were tested in mast cell mediator release assays and used to stimulate PBMCs and Bet v 1-specific T-cell lines and clones. Proteolytic fragments of allergens were analyzed and sequenced by means of mass spectrometry. RESULTS: Pepsin completely destroyed IgE binding of all allergens within 1 second, and trypsin completely destroyed IgE binding of all allergens within 15 minutes, except for the major hazelnut allergen, which remained intact for 2 hours of trypsinolysis. Allergens after gastrointestinal digestion did not induce basophil activation but induced proliferation in PBMCs from allergic and nonallergic individuals. Digested Mal d 1 and Cor a 1.04 still activated Bet v 1-specific T cells, whereas digested Api g 1 did not. Different proteolytic fragments of Mal d 1 and Cor a 1.04 matching relevant Bet v 1 T-cell epitopes were found. CONCLUSION: Gastrointestinal degradation of Bet v 1-related food allergens destroys their histamine-releasing, but not T cell-activating, property. Our data emphasize that birch pollen-related foods are relevant activators of pollen-specific T cells.

Allergens↗

PubMed2005

Wine anaphylaxis in a German patient: IgE-mediated allergy against a lipid transfer protein of grapes.

BACKGROUND: IgE-mediated allergy to grapes has very rarely been reported in patients from the Mediterranean area. Recently, endochitinase 4 and a lipid transfer protein (LTP) have been identified as major allergens in grape-allergic patients who do not have an associated pollinosis. The purpose of this case study was to identify the allergens responsible for severe anaphylactic reactions after consumption of wine, fresh grapes and raisins in a German patient. METHODS: Prick-to-prick tests and the basophil activation test (BAT) were performed to confirm allergy. Specific IgE was further analyzed by immunoblotting and inhibition tests for the determination of crossreactivity. The IgE-binding protein was subjected to N-terminal microsequencing. RESULTS: Prick-to-prick tests were positive to fresh and cooked white and blue grapes, to raisins, to white and red wine, and to grape extract. Specific IgE against grapes (f259) was 2.43 kU/l (class 2). The BAT showed specific IgE-mediated activation of basophils after stimulation with grape extract. IgE binding to a 15-kDa protein was completely inhibited by pre-incubation with recombinant cherry LTP Pru av 3. N-terminal sequencing identified this 15-kDa protein as grape LTP Vit v 1. CONCLUSION: Our data show that sensitization to LTP can occur outside the Mediterranean area causing severe fruit allergy without association to pollinosis.

Adult↗

PubMed2005

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.

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.

Allergens↗

PubMed2005

Allergenic characteristics of a modified peanut allergen.

Attempts to treat peanut allergy using traditional methods of allergen desensitization are accompanied by a high risk of anaphylaxis. The aim of this study was to determine if modifications to the IgE-binding epitopes of a major peanut allergen would result in a safer immunotherapeutic agent for the treatment of peanut-allergic patients. IgE-binding epitopes on the Ara h 2 allergen were modified, and modified Ara h 2 (mAra h 2) protein was produced. Wild-type (wAra h 2) and mAra h 2 proteins were analyzed for their ability to interact with T-cells, their ability to bind IgE, and their ability to release mediators from a passively sensitized RBL-2H3 cell line. Multiple T-cell epitopes were identified on the major peanut allergen, Ara h 2. Ara h 2 amino acid regions 11-35, 86-125, and 121-155 contained the majority of peptides that interact with T-cells from most patients. The wAra h 2 and mAra h 2 proteins stimulated proliferation of T-cells from peanut-allergic patients to similar levels. In contrast, the mAra h 2 protein exhibited greatly reduced IgE-binding capacity compared to the wild-type allergen. In addition, the modified allergen released significantly lower amounts of beta-hexosaminidase, a marker for IgE-mediated RBL-2H3 degranulation, compared to the wild-type allergen.

2S Albumins, Plant↗

PubMed2005

Novel isoforms of Pru av 1 with diverging immunoglobulin E binding properties identified by a synergistic combination of molecular biology and proteomics.

Birch pollen-related food allergies are mainly associated to Bet v 1. Little is known about isoforms of Bet v 1 homologous in fruit of the Rosaceae family. We attempted to identify novel isoforms of Pru av 1, the major cherry allergen, at the cDNA and the protein level by a combination of molecular biology and proteomic tools. A cDNA library was screened with patients immunoglobulin E (IgE) and a specific hybridization probe. Edman sequencing, mass spectrometry (MS), and MS/MS were performed after detecting Pru av 1 on 2-D maps by immunoblotting using patients IgE and a monoclonal antibody. Partial amino acid sequences were completed with a polymerase chain reaction (PCR) strategy. The IgE-binding properties of the Pru av 1 spots were analyzed by 2-D blot inhibition. cDNA library analysis revealed a novel Pru av 1 isoform. MS and N-terminal sequencing confirmed the cDNA sequences at the protein level. A series of spots were confirmed as the already known Pru av 1. One spot, exclusively detected with patients sera, was identified as the novel isoform. A partial amino acid sequence detected with MS/MS was completed by PCR-cloning. The 2-D blot inhibition revealed epitope differences between the novel isoform and the previously published Pru av 1. Our data demonstrate that a synergistic combination of molecular biology and proteomics represents a powerful tool for reliable and comprehensive identification of allergen isoforms and variants. The newly identified isoform showed diverging IgE-binding properties and may be relevant for the diagnosis or therapy of cherry allergy.

Allergens↗