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Background: Chestnut and natural rubber latex (NRL) allergy are often associated in the latex-fruit syndrome. Aim of the study: To establish whether the concurrent NRL and chestnut IgE antibody reactivity are the results of co-sensitization or cross-reactivity. Methods: Sera from 19 patients with chestnut- and NRL-specific IgE were selected and tested for reactivity with recombinant (r) latex allergens. Cross-reactivity was explored by IgE-inhibition experiments using chestnut or NRL allergens as solid phase on ImmunoCAP. Results: IgE-antibodies were detected to rHev b 6.01 (prohevein) in 58% of the sera, to rHev b 5 in 32%, to rHev b 12 in four of 13 sera, to rHev b 7.02 and rHev b 11 in four, and to rHev b 1 in two of 19 sera. rHev b 8-IgE antibodies were found in nine sera (47%), whereas six displayed mono-sensitization to rHev b 8 with regard to our test panel. Three of 16 sera showed IgE to cross-reactive carbohydrate determinants. In most sera recognizing rHev b 5 and/or rHev b 6.01 as major allergens the IgE-reactivity to NRL remained unaffected by chestnut extract and chestnut-IgE remained unaffected by NRL extract. Conversely, in sera with rHev b 8 as dominant allergen IgE-binding to NRL was nearly completely inhibited by chestnut and vice versa. IgE-binding to rHev b 8 was abolished by chestnut extract. Conclusions: Although patients have concomitant IgE antibody reactivity to chestnut and NRL, cross-reactivity could be demonstrated mainly in those patients with IgE to Hev b 8 (profilin) from NRL. [ABSTRACT FROM AUTHOR]

Background Characterized native and recombinant Hevea brasiliensis (rHev b) natural rubber latex (NRL) allergens are available to assess patient allergen sensitization profiles. Objective Quantification of individual IgE responses to the spectrum of documented NRL allergens and evaluation of cross-reactive carbohydrate determinants (CCDs) for more definitive diagnosis. Methods Sera of 104 healthcare workers (HCW; 51 German, 21 Portuguese, 32 American), 31 spina bifida patients (SB; 11 German, 20 Portuguese) and 10 Portuguese with multiple surgeries (MS) were analysed for allergen-specific IgE antibody (sIgE) to NRL, single Hev b allergens and CCDs with ImmunoCAP™ technology. Results In all patient groups rHev b 5-sIgE concentrations were the most pronounced. Hev b 2, 5, 6.01 and 13 were identified as the major allergens in HCW and combined with Hev b 1 and Hev b 3 in SB. In MS Hev b 1 displayed an intermediate relevance. Different sIgE antibody levels to native Hevea brasiliensis (nHev b) 2 and rHev b 6.01 allowed discrimination of SB with clinical relevant latex allergy vs. those with latex sensitization. Sensitization profiles of German, Portuguese and American patients were equivalent. rHev b 5, 6.01 and nHev b 13 combined detected 100% of the latex-allergic HCW and 80.1% of the SB. Only 8.3% of the sera showed sIgE response to CCDs. Conclusions Hev b 1, 2, 5, 6.01 and 13 were identified as the major Hev b allergens and they should be present in standardized latex extracts and in vitro allergosorbents. CCDs are only of minor relevance in patients with clinical relevant latex allergy. Component-resolved diagnostic analyses for latex allergy set the stage for an allergen-directed immunotherapy strategy. [ABSTRACT FROM AUTHOR]

Background: New IgE sensitizations to proteins in allergen extracts have been shown to occur during allergen-specific immunotherapy (IT). Methods: Twenty-four healthcare workers (HCWs) – patients included in a latex IT study – were analysed, 16 in active treatment and eight in placebo. Sera were obtained at baseline and after 6 months of IT and analysed with immunoblotting and CAP System with eight single recombinant latex allergens (rHev b 1, 3, 5, 6.01, 8, 9, 10, 11, and a mix of rHev b1, 5, 6.01 and 8). Results: After IT with latex, three patients in the active treatment group had new IgE sensitizations, one to Hev b 5, one to Hev b 11 and another to Hev b 6.01. No other significant variation in mean of specific IgE to latex or recombinant allergens were observed in patients who received placebo or active treatment. A significant ( P = 0.012) negative correlation (−0.72) was observed between maximal tolerated dose and specific IgE to Hev b 6.01 at baseline. After IT, immunoblot analysis demonstrated a significant increase in IgE binding in a band of approximately 22 kDa ( P = 0.032) that may correspond to Hev b 6.01. New or more intense bands appeared in seven patients of the active group, while in three subjects a reduction was observed. Conclusions: Hev b 6.01 seems to be the most relevant latex allergen in HCWs. New or more intense IgE binding to latex allergenic components occurs during latex immunotherapy. However, the levels of specific IgE against these new components are low and do not seem to have clinical relevance. [ABSTRACT FROM AUTHOR]

Multiple immunoglobulin E (IgE)-binding proteins in natural rubber latex extracts have been identified. In the case of Hev b 6 a differentiation was made between the precursor protein prohevein (Hev b 6.01) and its two post-transcriptionally formed proteins, the N-terminal hevein (Hev b 6.02) and the C-terminal domain (Hev b 6.03). All three components act as independent allergens. The aim of this study was a detailed analysis of the T-cell responses and the IgE-binding capacity of Hev b 6.01, Hev b 6.02 and Hev b 6.03 by using these allergens as recombinant maltose-binding fusion (MBP) proteins and the usage of synthetic modified hevein peptides. Latex-allergic health care workers (HCWs) suffering from rhinitis, conjunctivitis, contact urticaria and/or asthma with increased specific IgE-antibodies to latex were tested for their IgE-binding capacity and T-cell reactivity (by proliferation response) to the recombinant MBP-rHev b 6.01, MBP-rHev b 6.02, MBP-rHev b 6.03, to native Hev b 6.02, to modified hevein peptides and wheat germ agglutinin (WGA). For testing of the human leucocyte antigen (HLA) class II restriction of MBP-rHev b 6.01 induced peripheral blood mononuclear cell (PBMC) responses, monoclonal antibodies against HLA-DR, HLA-DP or HLA-DQ were added. Seventeen of 18 (94%) serum samples from latex-allergic HCWs had increased levels of specific IgE to MBP-rHev b 6.01, 16 (89%) to MBP-rHev b 6.02 and 13 (72%) to MBP-rHev b 6.03. A significant difference existed between the specific IgE-values of MBP-rHev b 6.02 and MBP-rHev b 6.03 ( P < 0.01). Proliferation responses of PBMC of the same 18 latex-allergic patients were positive for MBP-rHev b 6.01 and MBP-rHev b 6.03 in 83 and 67% of the tested PBMC suspension, whereas the proliferation responses induced with MBP-rHev b 6.02 or native Hev b 6.02 were very low (5.6 and 22.2%). Sera from nine additional latex-allergic patients showed specific IgE binding to the native Hev b 6.02, but none of these sera showed specific IgE binding to the modified Hev b 6.02-peptides [whereby all eight cysteine residues were substituted by serine (C → S) or by alanine (C → A)]. Proliferation responses induced by the modified Hev b 6.02 peptides were not significantly different from that induced by Hev b 6.02. Potential HLA-DR4Dw4(DRB1*0401)-restricted T-cell epitopes of Hev b 6.01 predicted by two computer algorithms were only found in the Hev b 6.03-part of Hev b 6.01. In the Hev b 6.01 precursor the regions responsible for IgE binding and those for inducing the T-cell proliferation responses are settled in different parts of the protein. The Hev b 6.02 domain is responsible for IgE binding and carries discontinuous B-cell epitopes whereas Hev b 6.03 is a better inducer of a proliferation response and contains HLA-DR4-binding motifs. [ABSTRACT FROM AUTHOR]

Abstract: In the last few years many efforts have been made to reduce the incidence of latex allergy. The identification of the major latex allergens and their recombinant production are useful steps towards the understanding of latex allergy and its management. A clear advantage of the recombinant proteins in general is the possibility to produce large-scale quantities at highly reproducible quality. Recombinant allergens also facilitate the study of the molecular bases of the immune reactivity of the proteins. Nevertheless, they have to be validated against native proteins for equivalence in allergenicity before they can be more widely adapted for clinical use. Currently 13 latex allergens have been included in the latest nomenclature list of the International Nomenclature Committee of Allergens and assigned the official names Hev b 1–13. With the use of single native and/or recombinant allergens the determination of specific sensitization profiles for different patient groups dependent on their various routes of exposure is possible. The specific IgE-diagnostic was further improved significantly by addition of recombinant Hev b 5 to natural latex extract, which was underrepresented in the initial test extract. [Copyright &y& Elsevier]

Presents a comprehensive data collection on latex allergens investigated at the molecular level in order to demonstrate their individual importance in latex allergy. Normal latex allergens; Particle-bound proteins; C-serum proteins; Allergens in natural rubber latex products; Co-sensitization and crossreactivity of latex allergens.

Background The rubber elongation factor in Hevea rubber (Hey b 1) is one of the most important latex allergen and is leading cause of latex type I hypersensitivity in children with spina bifida. Objective The aim of this study was to define the allergenic and antigenic epitopes of Hey b 1. Methods The immunoglobulin- (Ig)E and IgG antibody binding rites on Hey b I allergen were delineated by enzyme linked immunosorbent assay (ELISA) using synthetic overlapping peptides covering the whole Hey b 1 sequence. In order to improve the binding capacity and specificity all peptides were biotinylated at the N-terminal end via a 6-aminohexanoic acid as spacer and then adsorbed to streptavidin pre-coated microtitre plates. Fine mapping to define the essential amino acid residues for the antibody binding was achieved by using overlapping peptides with one amino acid offset. Results It was demonstrated that the IgE epitopes were located in different regions of Hey b I including the C-terminal segment (121–137) and the segments with amino acid residues of 30–49 and 46–64. Two monoclonal antibodies (MoAbs) II2F3 and II4G9 raised against purified Hey b 1 recognized the C-terminal segment only. The results of epitope mapping with three rabbit antisera revealed that five positive peptides, including the epitope peptides 31–49, 46–64 and 121–137, were involved in the antibody-binding sites. Fine mapping on the scgments 46–64 and 121–137 showed that the two MoAbs reacted with the peptide 125–134 in the C-terminal region, whereas the peptide with amino acids 124–134 was essential for recognition by human IgE antibodies. Epitopes to rabbit polyclonal IgG and human IgE were also found to be involved in the amino acid residues of 47–59. Conclusion Our results indicate that the most allergenic/antigenic portions of Hey b 1 allergen are the C-terminal region and the region with amino acid residues of 31–64. In both regions, the minimal IgE-binding epitope is almost identical with the IgG-binding epitope. [ABSTRACT FROM AUTHOR]

The IUIS Allergen Nomenclature Sub- Committee, under the auspices of the World Health Organization and the International Union of Immunological Societies, maintains the systematic nomenclature of allergenic proteins and publishes a database of approved allergen names on its Web site, . In this paper, we summarize updates of allergen names approved at the meetings of the committee in 2011 through 2013. These changes reflect recent progress in identification, cloning, and sequencing of allergens. The goals of this update were to increase consistency in the classification of allergens, isoallergens, and variants and in the incorporation of the evolutionary classification of proteins into allergen nomenclature, while keeping changes of established names to a minimum in the interest of continuity. Allergens for which names have been updated include respiratory allergens from birch and ragweed pollen, midge larvae, and horse dander; food allergens from peanut, cow's milk, and tomato; and cereal grain allergens. The IUIS Allergen Nomenclature Sub- Committee encourages researchers to use these updated allergen names in future publications. [ABSTRACT FROM AUTHOR]

Skin prick testing (SPT) in combination with the clinical history of the patient is one important step in the diagnosis of IgE-mediated occupational allergies. However, skin test performance is related to the quality of allergen extracts. The present consensus document was prepared by an EAACI Task Force consisting of an expert panel of allergologists and occupational physicians from Germany, Italy, Spain, France, Austria, and Poland. All members of the panel were also involved in the data collection within the European multicentre study STADOCA ( Standard diagnosis for occupational allergy). The aim of this Task Force was the assessment of the quality of commercially available SPT solutions for selected occupational allergens under standardized procedure conditions in different European centres and institutes of Occupational Medicine. The data evaluation shows a wide variability among SPT solutions and also indicates that the sensitivity of several SPT solutions is low. Therefore, improvement and standardization of SPT solutions for occupational allergens is highly recommended. Clinical practitioners should also not presume that their SPT solutions are fully reliable. The main objective of the document is to issue consensus suggestions for the use of SPT with occupational allergens based on the European multicentre study STADOCA, on existing scientific evidence and the expertise of a panel of allergologists. [ABSTRACT FROM AUTHOR]

Objective: Cross-reactivity between grass pollen and grain flour allergens is well known, but their significance in apprentice bakers with primary sensitization to grass pollen is not known. Methods: Twenty-five subjects with mild asthma (most of them with hay fever), but without prior occupational flour exposure, underwent standardized experimental inhalation challenges with placebo and wheat and rye flours in randomized order on three consecutive days. Sensitization to flours and environmental allergens was assessed by skin prick tests and specific IgE antibodies in sera. IgE inhibition experiments were performed with wheat and rye flours as solid phases, and grass and tree pollen as inhibitors. Results: Five subjects experienced a positive reaction after flour inhalation (responders), whereas 20 subjects did not show such a reaction (non-responders). All responders showed sensitizations to flours, whereas only 25% of the non-responders demonstrated the same sensitization. Specific IgE concentrations to flours, but also to grass and tree pollen, were higher in responders. Inhibition experiments demonstrated cross-reactivity between flours and grass or tree pollen, with higher inhibition rates in responders. Conclusions: Subjects with pronounced sensitizations to grass or tree pollen, but without prior occupational exposure to flours, may experience both sensitization and allergic asthmatic reactions to flours due to cross-reactive grass and tree pollen allergens. [ABSTRACT FROM AUTHOR]

Background: Several wheat flour allergens relevant to baker's asthma have been identified in the last 25 years. The aim of this study was to determine the frequency of sensitization to these allergens in German bakers. Methods: Using recombinant DNA technology, the following wheat flour allergens were cloned, expressed in Escherichia coli and purified: five subunits of the wheat α-amylase inhibitors (WTAI-CM1, WTAI-CM2, WTAI-CM3, WDAI-0.19 and WMAI-0.28), thioredoxin, thiol reductase or 1-cys-peroxiredoxin homologues, triosephosphate-isomerase, αβ-gliadin, serpin, glyceraldehyde-3-phosphate-dehydrogenase, a nonspecific lipid transfer protein (nsLTP), dehydrin, profilin and peroxidase. In addition, ImmunoCAPs with the recombinant allergen ω-5-gliadin and two cross-reactive carbohydrate determinants (CCDs), horse radish peroxidase (HRP) and the N-glycan of bromelain (MUXF), were used. Specific IgE was measured in wheat flour-positive sera from 40 German bakers with work-related asthma/rhinitis and 10 controls with pollinosis. Results: Thirty bakers (75%) had IgE to at least one of the 19 single allergens. Most frequent was IgE to WDAI-0.19, HRP and MUXF (25% each), followed by WTAI-CM1 (20%), thiol reductase (16%), WTAI-CM3 (15%), WTAI-CM2 and thioredoxin (12.5%), WMAI-28, triosephosphate-isomerase, αβ-gliadin (10%), 1-cys-peroxiredoxin (7.5%), dehydrin, serpin, glyceraldehyde-3-phosphate-dehydrogenase (5%), ω-5-gliadin, nsLTP and profilin (2.5%). Fifteen bakers (38%) had IgE to any α-amylase inhibitor and 12 (30%) to at least one CCD. The controls reacted exclusively to CCDs (80%), profilin (60%), thioredoxin (30%), triosephosphate isomerase and nsLTP (10%). Conclusions: The single allergen sensitization profiles obtained with 17 recombinant wheat flour allergens and two CCDs revealed no major allergen for German bakers. The highest frequencies were found for α-amylase inhibitors and CCDs. [ABSTRACT FROM AUTHOR]

Background: The allergen content of diagnostics and immunotherapeutics is crucial for effective diagnosis and treatment. The aim of this study was to quantify and compare the allergen content of different grass pollen preparations for skin prick testing and sublingual immunotherapy (SLIT). Methods: Five skin prick test (SPT) solutions and 10 sublingual immunotherapeutics were analysed for protein and allergen concentration by Bradford assay, inhibition of IgE-binding to Phleum pratense ImmunoCAPs and content of the main allergen Phl p 5 by two-site enzyme immunoassay. In addition, the grass pollen preparations were compared by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting analyses. Results: Protein concentrations of SPT solutions ranged from 15 to 427 μg/ml, and Phl p 5 concentrations ranged from 0.15 to 18.3 μg/ml. The ranking of SPT solutions concerning Phl p 5 content and IgE inhibition capacity was the same, and the ranking of protein and allergen content was closely correlated ( r = 0.9). Protein content of the maintenance doses of the immunotheurapeutics ranged from 5 to 153 μg, Phl p 5 content ranged from 0.2 to 21.6 μg. IgE inhibition capacity of the maintenance doses was closely correlated to their Phl p 5 and protein content. SDS-PAGE and immunoblots confirmed the differences in protein and allergen content. Conclusions: Grass pollen preparations for SPT and SLIT varied greatly concerning protein and allergen content. Whereas this result corresponds to previous analyses results of SPT solutions, it was the first comparison of grass pollen immunotherapeutics. For diagnosis and therapy, these differences should be taken into account. [ABSTRACT FROM AUTHOR]

Background: Exposure to airborne wheat allergens in the bakery trade is associated with a high risk of occupational allergy and asthma. Control and reduction of allergen exposure require relatively simple but reliable monitoring techniques. We developed new rabbit IgG-based enzyme immunoassays (EIA) for wheat allergens, which might be a convenient alternative for the thus far used human IgG4 inhibition assay. Methods: The reactivity and specificity of rabbit antibodies were assessed by EIA and immunoblotting, and compared with those of IgE from wheat-sensitized bakers, and with the antibodies used in the IgG4 inhibition EIA. An IgG inhibition and a sandwich EIA were developed for analysis of airborne dust samples. Results: Human IgG4 and rabbit IgG inhibition EIAs had comparable sensitivities, with limits of detection (LOD) between 18 and 88 ng/mL, while the sandwich EIA was much more sensitive (LOD<0.2 ng/mL). Human IgG4 and rabbit IgG reacted in immunoblotting with most of the IgE-binding wheat proteins, although with quantitative differences. All three assays showed a strong reaction with wheat proteins, and some cross-reactivity with rye and barley, but were further highly specific for cereal flour proteins. Concentrations measured with the three EIAs in 432 airborne dust samples were highly correlated ( r>0.95) and their absolute values showed less than 10–20% differences. Conclusion: The rabbit IgG EIAs are valid substitutes for the human IgG4 inhibition EIA, with important practical advantages. The inhibition EIA is recommended for routine wheat allergen measurements. The sandwich EIA may be used to measure low allergen levels, as in short task-related exposure measurements or in subfractions of airborne dust samples. [ABSTRACT FROM AUTHOR]

Background Hevea brasiliensis latex serum is commonly used as the in vivo and in vitro reference antigen for latex allergy diagnosis as it contains the full complement of latex allergens. Objective This study quantifies the concentrations of the significant allergens in latex serum and examines its suitability as an antigen source in latex allergy diagnosis and immunotherapy. Methods The serum phase was extracted from centrifuged latex that was repeatedly freeze-thawed or glycerinated. Quantitation of latex allergens was performed by two-site immunoenzymetric assays. The abundance of RNA transcripts of the latex allergens was estimated from the number of their clones in an Expressed Sequence Tags library. Results The latex allergens, Hev b 1, 2, 3, 4, 5, 6, 7 and 13, were detected in freeze-thawed and glycerinated latex serum at levels ranging from 75 (Hev b 6) to 0.06 nmol/mg total proteins (Hev b 4). Hev b 6 content in the latex was up to a thousand times higher than the other seven latex allergens, depending on source and/or preparation procedure. Allergen concentration was reflected in the abundance of mRNA transcripts. When used as the antigen, latex serum may bias the outcome of latex allergy diagnostic tests towards sensitization to Hev b 6. Tests that make use of latex serum may fail to detect latex-specific IgE reactivity in subjects who are sensitized only to allergens that are present at low concentrations. Conclusion Latex allergy diagnostics and immunotherapy that use whole latex serum as the antigen source may not be optimal because of the marked imbalance of its constituent allergens. [ABSTRACT FROM AUTHOR]

Wood dust is known to cause allergic occupational asthma and obeche (Triplochiton scleroxylon) is a prominent exponent in this field. However, the knowledge about wood allergens is still limited. The aim of this study was to identify and characterize obeche wood allergens.Obeche extracts were prepared from freshly ground in comparison to 7 years stored wood dust and investigated by Sodium dodecyl sulfate-polyacrylamid gel electrophoresis, enzyme-linked allergosorbent test and immunoglobulin (Ig)E-immunoblot. Allergens were detected by specific IgE of seven obeche allergic patients’ sera and protein analysis was performed by mass spectrometry. Cross-reactivity was demonstrated by ImmunoCAP-inhibition with sera of seven obeche and four latex-allergic patients.Obeche extracts showed different protein pattern and IgE-binding capacities depend on the age of the wood dust. A 38 kDa protein was identified as major obeche wood allergen, detected by six of seven (85%) obeche allergic patients’ sera and was entitled as Trip s 1. Trip s 1 is homologous to plant class I chitinases and exhibited enzyme activity demonstrated by chitinolysis. Co-recognition or cross-reactivity of Trip s 1 according to structural similarity was seen in sera of latex allergic patients. IgE inhibition studies with obeche as solid phase and Trip s 1 and latex hevein as inhibitor demonstrated that Trip s 1 was a more effective inhibitor in obeche as well as in latex allergic patients’ sera.Trip s 1 is a new obeche wood allergen of the plant class I chitinase family. This finding may explain the dominant role of obeche in sensitization against wood dust. [ABSTRACT FROM AUTHOR]

Several studies have shown that allergy to natural rubber latex is associated with cross reactivity to other plants. We have investigated a possible cross reactivity with a tree belonging to the Sterculiaceae family, Tryplochiton scleroxylon, in a patient allergic to obeche wood, that began to suffer symptoms of allergy in contact with latex. Determinations of specific IgE by CAP to obeche, natural rubber latex (NRL) and recombinant allergens of latex were done. Immunoblotting was performed to study allergens detected by serum of our patient. CAP-inhibition methods were used to study cross-reactive between NRL and obeche wood. In obeche extract, 4 bands were found of an apparent molecular weight of 18, 38, 75 and 199 kDa. In NRL extract the allergens have a molecular weight around 18, 21, 32, 38, 60, 70 and 199 kDa. IgE to latex was almost completely inhibited (96%) by the obeche extract in CAP-inhibition studies. Using obeche extract as inhibitor, IgE binding to native hevein (nHevb 6.02) was reduced by 25%. The results obtained in CAP-inhibition demonstrate the existence of cross-reactivity between proteins in NRL and obeche wood. In our patient, a band of apparent molecular weight of 38 kDa could be the most important allergen. [ABSTRACT FROM AUTHOR]

Background: Class I chitinase in natural rubber latex (NRL) has been assumed to be an important allergen, especially concerning its cross-reactivity with fruits like avocado and banana. Objectives: The present study aimed to produce a recombinant latex class I chitinase from Hevea brasiliensis leaves and to study its immunoglobulin (Ig)E-binding reactivity. Methods: A class I chitinase-specific complementary DNA from H. brasiliensis leaves was synthesized, subcloned, sequenced and overexpressed in fusion with the maltose-binding protein (MBP) in Escherichia coli . The IgE-binding reactivity of this protein was studied by the Pharmacia CAP SystemTM and by immunoblot experiments using sera from latex-allergic patients. Results: The rHev b 11.0102 was found to have a length of 295 amino acid residues and contains an N-terminal hevein-like domain with a 56% homology to hevein. Analysis by the CAP method revealed the presence of rHev b 11.0102-specific IgE antibodies in 17 of 58 sera (29%) of IgE-mediated latex-allergic subjects tested. Immunoblot analysis of the MBP–rHev b 11.0102 fusion protein and the MBP carrier protein as a negative control confirmed the IgE-reactivity of rHev b 11.0102. Conclusion: Due to its IgE-reactivity rHev b 11.0102 represents an allergen of intermediate prevalence in NRL. Its property to cross-react with certain fruits makes it an important supplement in the diagnostic panel of recombinant NRL allergens. [ABSTRACT FROM AUTHOR]

Summarizes the important aspects of allergy research work on purified allergens. Denominations of allergens; Relation of everyday life with exposure to an enormous number of potential allergens in the environment; Identification and characterization of allergens.