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R Crameri

Publications and source records attributed to R Crameri.

At least 37 records · Page 2Linked to original sources
PubMed2001

Recombinant allergens for skin testing.

Skin testing is a basic diagnostic procedure widely used to explore immediate-type reactions to allergen preparations in vivo. Despite their reliability, if standardized extracts are used, skin tests suffer from limited reproducibility due to difficulties in preparing consistently standardized extracts from natural raw material. Starting from allergen-encoding cDNAs, large amounts of highly pure allergens with a high batch-to-batch consistency satisfying the quality requirements of medicinal products manufactured by recombinant DNA technology can be produced. These reagents are expected to be qualitatively superior to the commercially available allergen preparations used for the in vitro and in vivo diagnosis of allergic conditions. In this article the current literature available on skin testing with such recombinant allergens (rAllergens) is reviewed and critically analyzed. To date many different rAllergens of various pollens, moulds, mites, bee venom, latex and celery have been used in skin testing in more than 1,600 allergic and control individuals. Skin prick tests as well as intradermal skin tests with rAllergens prove to be highly specific and safe. The diagnostic sensitivity of single rAllergens is generally lower than those obtained with allergen extracts, but can be considerably increased by using rAllergen panels covering the most important allergenic structures present in a given complex allergenic extract. Moreover, quantitative skin testing with single rAllergens allows interesting insights into correlations between the in vivo and in vitro sensitization to a given allergen. In conclusion, skin testing with rAllergens offers a highly specific and safe additional diagnostic tool to elucidate patient- and disease-specific sensitization patterns which will be needed for the development of patient-tailored immunotherapeutic treatments.

Allergens↗
PubMed2001

Identification of a Hevea brasiliensis latex manganese superoxide dismutase (Hev b 10) as a cross-reactive allergen.

BACKGROUND: Cross-reactive allergens play an increasingly important role in latex allergy in complicating both the diagnosis and time course of allergic symptoms. Manganese superoxide dismutase (MnSOD), a ubiquitous protein of prokaryotic and eukaryotic organisms, was described as a cross-reactive allergen in Aspergillus fumigatus. Little information is available on the importance of this pan-allergen in Hevea brasiliensis latex. The aim of this study was to clone and express MnSOD from H. brasiliensis latex, and to obtain the soluble and immunologically active recombinant allergen for diagnosis of latex allergy and to investigate possible cross-reactivities with the structurally related A. fumigatus and human MnSODs. METHODS: A complementary DNA coding for Hevea latex MnSOD was amplified by PCR. The recombinant protein was produced in Escherichia coli with an N-terminal hexahistidyl tag. Enzymatic activity of the recombinant protein was determined using an enzyme assay for SODs. IgE immunoblotting and IgE inhibition assays were performed to characterize the recombinant allergen and its cross-reactivity. RESULTS: A Hevea latex MnSOD consisting of 206 amino acid residues was cloned and expressed in E. coli. The allergen was designated Hev b 10. The recombinant protein was enzymatically active, indicating the correct folding of the protein. In immunoblots, latex- as well as A. fumigatus-allergic patients revealed IgE binding to recombinant (r)Hev b 10. Cross-reactivity to Asp f 6, the MnSOD from A. fumigatus, and human MnSOD was determined by inhibition of IgE binding to these MnSODs by rHev b 10. CONCLUSIONS: Hev b 10 is a new cross-reactive allergen of H. brasiliensis which belongs to the 'latex-mold' group of latex allergens. Furthermore, it is a candidate for primary sensitization in patients allergic to the pan-allergen MnSOD.

Allergens↗
PubMed2001

The powerful combination of phage surface display of cDNA libraries and high throughput screening.

Phage surface display of cDNA libraries facilitates cloning, expression and rapid selection of functional gene products physically linked to their genetic information through gene product-ligand interactions. Efficient screening technologies based on selective enrichment of clones expressing desired gene products allows, within a short time, the isolation of all ligand-specific clones that are present in a library. Manual identification of clones by restriction analysis and random sequencing is unlike to be successful for the isolation of gene products derived from rare mRNA species resulting from selection of the libraries using polyvalent ligands like serum from patients. Here we describe rapid handling of large numbers of individual clones selected from molecular libraries displayed on phage surface using the power of robotics-based high throughput screening. The potential of the combination of cDNA-phage surface display, with selection for specific interactions by functional screening and robotic technology is illustrated by the isolation of more sequences potentially encoding IgE-binding proteins than postulated from Western blot analyses using extracts derived from raw material of complex allergenic sources. The subsequent application of functional enrichment and robotics-based screening will facilitate the rapid generation of information about the repertoire of protein structures involved in allergic diseases.

Bacteriophages↗
PubMed2001

Influence of environmental and genetic factors on allergen-specific immunoglobulin-E levels in sera from Lipizzan horses.

To investigate whether allergen-specific IgE production is influenced by environmental and genetic factors, IgE levels against 2 mould extracts (Alternaria alternata [Alt a] and Aspergillus fumigatus [Asp f]) and against recombinant (r) rAlt a 1, rAsp f 7 and rAsp f 8 were determined by ELISA in sera from 448 Lipizzan horses living in 6 studfarms. Statistical evaluation showed a significant effect of studfarm-specific environment on IgE levels against the different allergens, but genetic factors also influenced allergen-specific IgE production: an heritability of 0.33 was found for IgE levels against the 2 mould extracts and of 0.21 for rAsp f 8-specific IgE. Heritability estimates for rAlt a 1- and rAsp f 7-specific IgE were negligible. Investigations for a possible association between Major Histocompatibility Complex (MHC) class I antigens and specific IgE levels were carried out. The most consistent significant association was found between the equine leucocyte antigen (ELA) A8 and undetectable IgE titres against rAsp f 7 and rAsp f 8. Significant ELA associations were also demonstrated between ELA A1 and higher specific IgE levels, between ELA A14 and lower IgE levels against the mould extracts and in one studfarm between ELA Be27 and lower Aspergillus-specific IgE levels.

Age Factors↗
PubMed2001

Phage display as a tool for rapid cloning of allergenic proteins.

Allergic diseases represent an immune disorder associated with the production of immunoglobulin E (IgE) against normally innocuous antigens (allergens). Almost 20% of the population in industrialized countries suffer from type I allergic symptoms such as allergic rhinitis, conjunctivitis, urticaria or asthma. Although the mechanisms responsible for these allergic reactions are quite well understood, knowledge about the repertoire of molecules able to elicit type I symptoms is still limited. To clone and characterize entire allergen repertoires from complex allergenic sources in a fast and efficient way, new technologies are required. The phage surface display of cDNA libraries described here has proven to be a versatile cloning system to selectively isolate allergens physically linked to their genetic information. The screening of cDNA libraries displayed on phage surfaces with immobilised serum IgE from allergic patients reduces the time required for the selection of candidate clones to a few weeks. Robot-assisted high-throughput screening of the enriched library provides a fast and cost-effective way to isolate complete allergen repertoires. The biotechnological production of recombinant allergens derived from these sequences bears a high potential for the improvement of the diagnosis of allergic diseases.

Allergens↗
PubMed2000

Allergen-specific IgE levels against crude mould and storage mite extracts and recombinant mould allergens in sera from horses affected with chronic bronchitis.

Immunoglobulin E antibody (IgE) levels against four recombinant (r) mould allergens (r-Aspergillus fumigatus [rAsp f] 7, 8 and 9; r-Alternaria alternata 1 [rAlta1]) and crude mould (Aspergillus fumigatus, Alternaria alternata, Penicillium notatum) and storage mite extracts were determined by ELISA in sera from 24 pulmonary sound control horses and 26 horses suffering from chronic bronchitis/bronchiolitis (CB), also called chronic obstructive pulmonary disease (COPD). Serum IgG and IgA titres were also determined against Aspergillus fumigatus extract and rAsp f 8.IgE against the crude extracts could be measured in all sera, but there was no significant difference between CB-affected and control horses. In contrast, only 8-30% of the horses, depending on the r-allergen tested, had detectable IgE levels in serum against the r-allergens. Horses with CB had significantly more often detectable IgE levels than controls against rAlt a 1 (10/26 and 3/24, respectively, p=0. 054), rAsp f 7 (13/26 and 2/24, respectively, p<0.01) and rAsp f 8 (11/26 and 1/24, respectively, p<0.01). Only four horses (three CB-affected and one healthy, p0.05) had detectable IgE levels against rAsp f 9. Furthermore, CB-affected horses were often sensitised against two or more r-allergens (13/26 of the CB-affected horses) while only one of the 24 healthy horses had positive IgE levels against more than one r-allergens. Similarly to IgE levels, no significant differences between CB-affected and healthy horses were found for IgG titres against the Aspergillus fumigatus extract. However, horses with CB had significantly higher serum IgG titres against rAsp f 8 than healthy controls (median=28 versus 10 relative ELISA units [REU], p<0.01). Additionally, horses with detectable IgE titres against rAsp f 8 had significantly higher IgG titres against this r-allergen than horses with undetectable IgE titres (median IgG titres=46 and 13 REU, respectively; p<0.01). For serum IgA titres, neither differences between healthy and CB-affected animals nor correlations between IgA and IgG or IgE titres could be found. These results show that horses suffering from CB are more often sensitised to some Aspergillus fumigatus and Alternaria alternata allergens than control horses and that they are partly sensitised to the same fungal proteins as mould-allergic human patients. Furthermore, this study shows that r-allergens allow a much more sensitive determination of specific serum antibody levels by ELISA than crude mould extracts.

Allergens↗
PubMed2000

Selected recombinant Aspergillus fumigatus allergens bind specifically to IgE in ABPA.

BACKGROUND: Allergic bronchopulmonary aspergillosis (ABPA) is a hypersensitivity lung disease resulting from exposure to Aspergillus fumigatus allergens. Patients with ABPA show elevated Aspergillus-specific serum IgE, a major criterion used in the diagnosis of the disease. Crude culture filtrate and mycelial antigens have been used widely to demonstrate IgE antibody to Aspergillus in the sera of patients. While these antigens have been useful in the diagnosis of ABPA, occasionally they present inconsistency in their reactivity and lack of specificity. Although in recent years, a number of purified A. fumigatus allergens have been produced by molecular cloning, no attempt was made to evaluate them systematically. OBJECTIVE: To evaluate the recombinant proteins from A. fumigatus for their IgE antibody binding, we studied sera from ABPA patients and controls by antigen specific enzyme linked immunosorbent assay (ELISA). METHODS: Recombinant Aspergillus allergens Asp f 1, f 2, f 3, f 4, and f 6 were studied for their specific binding to IgE in the sera of ABPA patients, A. fumigatus skin prick test positive asthmatics, and normal controls from the USA and Switzerland. The sera were blinded and studied by ELISA in two different laboratories. RESULTS: All the recombinant allergens showed IgE antibody binding with sera from patients with ABPA, whereas only fewer asthmatics and normal sera showed significant binding. The three selected recombinant allergens together reacted with all the ABPA patients studied. CONCLUSIONS: The results demonstrate that Asp f 2, f 4, and f 6 can be used in the serodiagnosis of ABPA, while IgE antibody binding to Asp f 1 and f 3 was not specific.

Allergens↗
PubMed2000

[Allergic bronchopulmonary aspergillosis (ABPA)].

ABPA is the most common allergic bronchopulmonary mycosis in humans. The diagnosis of the complex disease is based on defined criteria. Five clinical stages of ABPA were proposed. The extend of the defined criteria varies in the different stages, thus making diagnosis difficult. Particularly the discrimination of ABPA in remission stage (stage II) and allergic asthma with A--sensitisation may be an important problem. Early diagnosis in stages without persistent changes of bronchial wall and lung parenchyma is needed to prevent severe end stages of ABPA. The up to now widely used commercial (crude) allergen extracts for in vitro and in vivo diagnosis show batch to batch variation, insufficient standardization and lack of reproducibility. Potentials and limitations of routine diagnostic procedures in ABPA are described. The production of a panel of recombinant allergens of A. fumigatus and their evaluation for in vivo and particularly in vitro use has brought an important step forward in the early and precise diagnosis of ABPA. A panel of recombinant allergens is now available for routine assay in CAP-System.

Allergens↗
PubMed2000

IgE-binding epitopes of enolases, a class of highly conserved fungal allergens.

BACKGROUND: Cladosporium herbarum and Alternaria alternata are two of the most prominent fungal species inducing type I allergy. Previously, we have demonstrated that enolase (Cla h 6) is the second most important allergen of C herbarum in terms of frequency of sensitization. OBJECTIVE: IgE-reactive B-cell epitopes of C herbarum enolase were analyzed, and cross-reactivity between fungal enolases was investigated. METHODS: Cla h 6 glutathione-S-transferase fusion peptides were constructed by means of PCR cloning. A alternata enolase (Alt a 5) was isolated by screening a complementary (c)DNA expression library with a C herbarum enolase DNA probe. RESULTS: Mapping of Cla h 6 IgE-binding epitopes identified a peptide with a length of 69 amino acids (peptide 9), which bound IgE from 8 of 8 patients. Analysis of the conformation of peptide 9 revealed that it does not form a compact structure but rather spans the whole length of the protein, with side chains exposed to solvent at 3 locations. Peptide 9 in the context of Escherichia coli glutathione-S-transferase not only binds IgE but also competitively inhibits IgE binding to Alt a 5. This result indicates that the epitope or epitopes on peptide 9 constitute a major cross-reacting epitope or epitopes on the enolases from C herbarum and A alternata in the case of the one patient tested. CONCLUSIONS: We demonstrated that the glycolytic enzyme enolase is an allergen not only in C herbarum but also in A alternata. Additionally, enolase was shown to exhibit high cross-reactivity to other fungal enolases. On the basis of the results presented here, we propose the use of recombinant Cla h 6 or maybe even peptide 9 of Cla h 6 for diagnosis and possibly therapy of mold allergy.

Allergens↗
PubMed1999

Humoral and cell-mediated autoimmune reactions to human acidic ribosomal P2 protein in individuals sensitized to Aspergillus fumigatus P2 protein.

A panel of cDNAs encoding allergenic proteins was isolated from an Aspergillus fumigatus cDNA library displayed on the surface of filamentous phage. Solid phase-immobilized serum immunoglobulin E (IgE) from A. fumigatus-allergic individuals was used to enrich phage displaying IgE-binding molecules. One of the cDNAs encoded a 11.1-kD protein that was identified as acidic ribosomal phosphoprotein type 2 (P2 protein). The allergen, formally termed rAsp f 8, shares >62% sequence identity and >84% sequence homology to corresponding eukaryotic P2 proteins, including human P2 protein. The sequences encoding human and fungal P2 protein were subcloned, expressed in Escherichia coli as His6-tagged fusion proteins, and purified by Ni2+-chelate affinity chromatography. Both recombinant P2 proteins were recognized by IgE antibodies from allergic individuals sensitized to the A. fumigatus P2 protein and elicited strong type 1-specific skin reactions in these individuals. Moreover, human and fungal P2 proteins induced proliferative responses in peripheral blood mononuclear cells of A. fumigatus- allergic subjects sensitized to the fungal P2 protein. These data provide strong evidence for in vitro and in vivo humoral and cell-mediated autoreactivity to human P2 protein in patients suffering from chronic A. fumigatus allergy.

Allergens↗
PubMed1999

In situ measurements of skeletal muscle power output using new capacitive strain gauge.

Experiments are described in which a fatigue index is determined for the latissimus dorsi muscle of sheep in situ, using capacitive strain gauges. Parallel experiments for invasive and non-invasive measurements are conducted, measuring global contraction and relaxation rates and shortening duration for paced muscle. The results show that, above one pulse per burst (5 V, 100 microseconds pulsewidth), contraction rates (62 +/- 11 mm s-1) and relaxation rates (50 +/- 7 mm s-1) are constant for unloaded muscle. For one animal, fatigue testing with a 2.5 kg load at six pulses per burst shows shortening rates increasing to a maximum (80 mm s-1) after 30 s and reducing to 5 mm s-1 after 150 s. The decrease in shortening amplitude is used as a fatigue index, log displacement against time. Power output is load dependent, measuring 4.7 W kg-1 with a 2.5 kg load. There is good agreement between the invasive and non-invasive measurements, thus providing a method for monitoring changes in muscle parameters non-invasively during future pacing transformation.

Animals↗
PubMed1999

Reduced in vivo allergenicity of Bet v 1d isoform, a natural component of birch pollen.

BACKGROUND: The major allergen of birch pollen, Bet v 1, is present in structurally slightly different isoforms. It has been postulated that certain isoforms show a distinct ability to bind birch pollen-specific IgE, although the T-cell response remains similar. OBJECTIVE: We verified the hypothesis of a distinct allergenicity but similar T-cell immunogenicity of 2 isoforms in birch pollen-allergic subjects by in vivo tests and an in vitro assay for T-cell stimulation. METHODS: Forty-eight birch pollen-allergic, 11 grass pollen-allergic, and 10 nonatopic control individuals were tested with 10-fold increasing concentrations (0.01 to 10.0 microg/mL) of recombinant (r) Bet v 1a and rBet v 1d by skin prick test (SPT), intradermal test (IDT), and conjunctival provocation test (CPT). An allergen-specific proliferation assay was performed on 21 patients with the 2 recombinant and the natural birch pollen allergens. RESULTS: In each test system only birch pollen-allergic subjects but no controls reacted to the recombinant allergens. A positive in vivo response to 10 microg/mL of rBet v 1a was observed in 21 of 48 by SPT, in 48 of 48 by IDT, and in 33 of 48 by CPT. In contrast, the IDT response to 10 microg/mL of rBet v 1d was reduced by a factor of 100 because it was equivalent to the response to 0.1 microg/mL of rBet v 1a. rBet v 1d failed to elicit a positive reaction in SPT and CPT. The proliferative response of T cells was similar for both recombinant isoforms because 8 of 21 individuals reacted to rBet v 1a and 6 of 21 to rBet v 1d. Only 1 subject had a positive reaction to rBet v 1d alone. CONCLUSION: The natural isoforms rBet v 1a and rBet v 1d differ in their ability to bind IgE but are similar in their immunogenicity for T cells. Thus rBet v 1d might be a promising candidate for use in immunotherapy of birch pollen-allergic individuals.

Adult↗
PubMed1999

Skin test reactivity to 2 recombinant Aspergillus fumigatus allergens in A fumigatus-sensitized asthmatic subjects allows diagnostic separation of allergic bronchopulmonary aspergillosis from fungal sensitization.

BACKGROUND: Aspergillus fumigatus, an opportunistic pathogen, is associated with an impressive list of pulmonary complications. Among these, allergic bronchopulmonary aspergillosis (ABPA) represents a complex clinical syndrome that is difficult to diagnose. A clear distinction between allergic sensitization to A fumigatus and ABPA is essential for therapy to prevent deterioration of pulmonary function in subjects with ABPA. OBJECTIVE: This study was carried out to determine the specificity and sensitivity of 2 A fumigatus allergens for the in vivo diagnosis of ABPA. METHODS: Serologic investigations with recombinant A fumigatus allergens indicated the existence of disease-specific allergens that are useful for discrimination between ABPA and fungal sensitization. However, serologic studies fail to indicate the allergen-specific IgE levels required to elicit an allergic reaction in vivo. RESULTS: We show that the recombinant A fumigatus allergens rAsp f 4, a protein with unknown biologic function, and rAsp f 6 (manganese superoxide dismutase) are able to provoke immediate skin reactions exclusively in patients with ABPA. The reactions, which are elicited by a few nonograms of the allergens, strictly depend on the presence of allergen-specific serum IgE. The IgE cut-off values for positive skin reactions to rAsp f 4 and rAsp f 6 of 0.9 and 1.2 kU(A)/L correspond to allergen-specific serum concentrations of 2 to 3 microg/L and allow a sensitive, highly specific diagnosis of ABPA. CONCLUSIONS: In contrast to fungal extracts, rAsp f 4 and rAsp f 6 allow discrimination between ABPA and sensitization to A fumigatus. Moreover, the allergens are suitable for an automated serologic diagnosis of ABPA, facilitating their introduction in clinical practice.

Adult↗
PubMed1999

IgE-binding proliferative responses and skin test reactivity to Cop c 1, the first recombinant allergen from the basidiomycete Coprinus comatus.

BACKGROUND: Basidiomycetes spores are ubiquitously distributed, found throughout the year in outdoor and indoor air, and represent relevant sources of aeroallergens associated with allergy and asthma. OBJECTIVE: Cloning and characterization of Coprinus comatus (shaggy cap mushroom) allergens is essential to elucidate their molecular characteristics and to improve the diagnosis of allergy. METHODS: A complementary DNA (cDNA) library of C comatus displayed on phage surface was screened with sera of basidiomycete-sensitized individuals. Subcloning and high-level expression of one of the enriched cDNAs allowed the isolation of a [His](6)-tagged recombinant protein formally termed rCop c 1. The allergenic properties of rCop c 1 were investigated in vitro by ELISA, inhibition experiments, immunoblots, and proliferation assays and in vivo by skin tests. RESULTS: The rCop c 1-encoding cDNA spans 435 bp and contains an open reading frame of 246 bp, predicting a protein of 8.96 kd without significant sequence homology to known proteins. Immunoblots with [His](6)-rCop c 1 fusion protein show a background free IgE-binding band of the expected size that can be completely inhibited by crude C comatus extracts in ELISA. rCop c 1 induced specific proliferative responses in PBMCs of C comatus-sensitized individuals. The incidence of sensitization to rCop c 1 among 92 sera of basidiomycete-sensitized individuals tested in ELISA was 25%, indicating that Cop c 1 is an intermediate allergen. However, prick tests showed that less than 2 pmol of the rCop c 1 protein was able to induce strong specific skin reactions in sensitized individuals. CONCLUSIONS: rCop c 1, the first cloned allergen from the genus Coprinus, fulfills all the criteria required to be classified as a clinically relevant allergen. The data demonstrate at the molecular level the presence of sensitizing molecules among Basidiomycetes, the most important source contributing to the total spore load in the outdoor air.

Allergens↗
PubMed1999

IgE reactivity to Api g 1, a major celery allergen, in a Central European population is based on primary sensitization by Bet v 1.

BACKGROUND: Up to 70% of patients with tree pollen allergy display allergic symptoms when eating certain fruits and vegetables. Homologous proteins with allergenic features are present in a wide range of plant species and can cause allergic reactions. OBJECTIVE: The aim of this study was to evaluate recombinant Api g 1, a major celery allergen, for in vivo and in vitro diagnosis of celery allergy in populations from Davos, Switzerland, and Montpellier, France. METHODS: A group of patients with celery and birch pollen allergy from Davos was tested, and the results from those tests were compared with results from a group of patients allergic to celery from Montpellier. Skin prick tests were performed with a commercial celery extract, crude celery, and purified recombinant Api g 1. Quantitative and qualitative serology was done with natural and recombinant allergens by means of RASTs and immunoblotting. RESULTS: Recombinant Api g 1 allowed accurate in vivo diagnosis of celery allergy in all patients from the Swiss group. RAST results with celery extract were negative in 8 of 24 patients; results of immunoblotting with celery extract were negative in 4 of 24 patients, and results of immunoblotting with recombinant (r)Api g 1 were negative in 8 of 24 patients. In the French group 11 of 12 patients had a positive skin reaction with crude celery extract, but only 2 patients reacted with rApi g 1. RAST results for celery were positive in 8 of 12 patients. In immunoblotting experiments 8 patient sera displayed IgE directed against various celery allergens, whereas no patients sera had rApi g 1-specific IgE. CONCLUSION: Our results document that rApi g 1 allows accurate in vivo diagnosis only in areas where birch trees are common. In areas where no birch trees grow, primary sensitization takes place through different pollen allergens (eg, mugwort pollen). Moreover, it became evident that birch pollen and celery allergy are highly related in Central Europe, whereas in Southern Europe the mugwort-celery type is predominant.

Adult↗
PubMed1999

Selective cloning of allergens from the skin colonizing yeast Malassezia furfur by phage surface display technology.

The yeast Malassezia furfur, also known as Pityrosporum orbiculare (ovale), is part of the normal microflora of the human skin but has also been associated with different skin diseases including atopic dermatitis. More than 50% of atopic dermatitis patients have positive skin test and specific IgE to M. furfur extracts; however, the pathophysiologic role of these IgE-mediated reactions in the development of the disease remains unknown. The yeast is able to produce a wide panel of IgE-binding proteins, variably recognized by sera of individual patients. In order to assess the contribution of individual components to the disease, highly pure allergen preparations are required. We have cloned M. furfur allergens from a cDNA library displayed on the phage surface, sequenced the inserts and produced recombinant proteins in Escherichia coli. Phage displaying IgE-binding proteins were selectively enriched from the library using IgE from a M. furfur-sensitized atopic dermatitis patient as a ligand. We were able to identify five different inserts coding for IgE-binding polypeptides. Three of the sequenced cDNA encode incomplete gene products with molecular masses of 21.3 kDa (MF 7), 14.4 kDa (MF 8), and 9.7 kDa (MF 9), respectively, having no sequence similarity to known proteins. The other two cDNA encode allergens of 18.2 kDa (Mal f 5) and 17.2 kDa (Mal f 6). Mal f 5 shows significant homology to M. furfur allergens Mal f 2, Mal f 3 and an Aspergillus fumigatus allergen Asp f 3. Mal f 6 has significant homology with cyclophilin. All of the recombinant polypeptides were capable of binding serum IgE from atopic dermatitis patients in immunoblotting experiments. The availability of pure recombinant M. furfur allergens will allow the careful investigation of the role of IgE-binding proteins in atopic dermatitis.

Allergens↗
PubMed1999

Selective cloning of peanut allergens, including profilin and 2S albumins, by phage display technology.

BACKGROUND: Peanut kernels contain many allergens able to elicit IgE-mediated type 1 allergic reactions in sensitized individuals. Sera from sensitized patients recognize variable patterns of IgE-binding proteins. The identification of the IgE-binding proteins of peanut extract would faciliate improvement of diagnostic and immunotherapeutic approaches as well as development of sensitive test systems for the detection of hidden peanut allergens present as additives in various industrial food products and the investigation of their stability during processing of food products. METHODS: We applied the pJuFo cloning system based on the phage surface display of functional cDNA expression products to clone cDNAs encoding peanut allergens. Sera (n = 40) of peanut-allergic individuals were selected according to case history, radioallergosorbent test and immunoblot analysis to demonstrate IgE binding towards the newly identified recombinant allergens. RESULTS: In addition to the known allergens Ara h 1 and Ara h 2 we were able to identify four allergens with estimated molecular weights of 36, 16, 14.5 and 14 kDa. Three of them formally termed Ara h 4, Ara h6 and Ara h 7 show significant sequence similarities to the family of seed storage proteins and the fourth (Ara h 5) corresponds to the well-known plant allergen profilin. Immunoblotting of the six expressed recombinant allergens with 40 patients sera shows 14 individual recognition patterns and the following frequency of specific IgE binding: Ara h 1 was recognized by 65%, Ara h 2 by 85%, Ara h 4 by 53%, Ara h 5 by 13%, Ara h 6 by 38% and Ara h 7 by 43% of the selected sera. CONCLUSIONS: All of the selected peanut-positive sera can detect at least one of the six identified recombinant allergens which can be used to establish individual patients' reactivity profiles. A comparison of these profiles with the clinical data will possibly allow a further insight into the relationship between clinical severity of the symptoms and specific IgE levels towards the six peanut allergens.

2S Albumins, Plant↗