IgE Reactivity Profiles Research Articles

Bos d 13, A Novel Heat-Stable Beef Allergen.

Red meat, a staple food of Western diets, can also induce IgE-mediated allergic reactions. Yet, apart from the heat-labile protein serum albumin and the carbohydrate α-Gal, the molecules causing allergic reactions to red meat remain unknown. IgE reactivity profiles of beef-sensitized individuals are analyzed by IgE-immunoblotting with protein extracts from raw and cooked beef. Two IgE-reactive proteins are identified by peptide mass fingerprinting as myosinlight chain 1 (MYL1) and myosin light chain 3 (MYL3) in cooked beef extract and are designated Bos d 13 isoallergens. MYL1 and MYL3 are produced recombinantly in Escherichia coli. ELISAs proved their IgE reactivity and circular dichroism analysis showed that they represent folded molecules with remarkable thermal stability. In vitro gastrointestinal digestion experiments showed the higher stability of rMYL1 as compared to rMYL3. Exposure of a monolayer of Caco-2 cells to rMYL1 indicated that the molecule is able to cross intestinal epithelial cells without disturbing the integrity of the tight junctions, suggesting the sensitizing capacity of MYL1. MYLs are identified as novel heat-stable bovine meat allergens.

A molecular sensitization map of European children reveals exposome- and climate-dependent sensitization profiles.

Understanding differences in sensitization profiles at the molecular allergen level is important for diagnosis, personalized treatment and prevention strategies in allergy. Immunoglobulin E (IgE) sensitization profiles were determined in more than 2800 sera from children in nine population-based cohorts in different geographical regions of Europe; north [BAMSE (Sweden), ECA (Norway)], west/central [PIAMA (the Netherlands), BiB (the United Kingdom), GINIplus (Germany)], and south [INMA Sabadell and Gipuzkoa (Spain) and ROBBIC Rome and Bologna (Italy)] using the MeDALL-allergen chip. Sensitization to grass pollen allergen, Phl p 1, and to major cat allergen, Fel d 1, dominated in most European regions whereas sensitization to house dust mite allergens Der p 1, 2 and 23 varied considerably between regions and were lowest in the north. Less than half of children from Sabadell which has a hot and dry climate were sensitized to respiratory allergens, in particular house dust mite allergens as compared to Gipuzkoa nearby with a more humid climate. Peanut allergen Ara h 1 was the most frequently recognized class 1 food allergen in Northern/Western Europe, while the fruit allergens Pru p 3, Act d 1 and 2 were prominent in Southern and Western/Central Europe. Ves v 5-sensitization dominated in North and West/Central Europe. We show regional, exposome- and climate-dependent differences in molecular IgE-reactivity profiles in Northern, Western/Central and Southern Europe which may form a molecular basis for precision medicine-based approaches for treatment and prevention of allergy.

Relationship between IgE Levels Specific for Ragweed Pollen Extract, Amb a 1 and Cross-Reactive Allergen Molecules

Ragweed (Ambrosia artemisiifolia) pollen is a major endemic allergen source responsible for severe allergic manifestations in IgE-sensitized allergic patients. It contains the major allergen Amb a 1 and cross-reactive allergen molecules, such as the cytoskeletal protein profilin, Amb a 8 and calcium-binding allergens Amb a 9 and Amb a 10. To assess the importance of Amb a 1, profilin and calcium-binding allergen, the IgE reactivity profiles of clinically well-characterized 150 ragweed pollen-allergic patients were analysed regarding specific IgE levels for Amb a 1 and cross-reactive allergen molecules by quantitative ImmunoCAP measurements, IgE ELISA and by basophil activation experiments. By quantifying allergen-specific IgE levels we found that Amb a 1-specific IgE levels accounted for more than 50% of ragweed pollen-specific IgE in the majority of ragweed pollen-allergic patients. However, approximately 20% of patients were sensitized to profilin and the calcium-binding allergens, Amb a 9 and Amb a 10, respectively. As shown by IgE inhibition experiments, Amb a 8 showed extensive cross-reactivity with profilins from birch (Bet v 2), timothy grass (Phl p 12) and mugwort pollen (Art v 4) and was identified as a highly allergenic molecule by basophil activation testing. Our study indicates that molecular diagnosis performed by the quantification of specific IgE to Amb a 1, Amb a 8, Amb a 9 and Amb a 10 is useful to diagnose genuine sensitization to ragweed pollen and to identify patients who are sensitized to highly cross-reactive allergen molecules present in pollen from unrelated plants, in order to enable precision medicine-based approaches for the treatment and prevention of pollen allergy in areas with complex pollen sensitization.

IgE reactivity patterns in Asian and central European cockroach-sensitized patients reveal differences in primary sensitizing allergen sources.

The prevalence of cockroach (CR) sensitization and its relevance as a trigger of allergy symptoms differs greatly in different geographic areas. This study aimed to compare molecular IgE reactivity profiles in CR-sensitized patients with perennial allergy symptoms from Hong Kong (HK) and Austria and identify the main primary sensitizers. IgE sensitization was assessed by skin prick test and/or IgE reactivity with CR extract. Molecular IgE reactivity profiles were analyzed via multiplex assay for sensitization to allergens and extracts from CR, house dust mite (HDM), shellfish, and 3 additional insect species. HDM was the main primary sensitizer in both cohorts. In the HK group, genuine sensitization to CR was found in 45%, but none of the patients in the Austrian cohort was truly sensitized to that allergen source. Most patients from HK were cross-sensitized to other insects and/or shellfish, presumably by broad reactivity to tropomyosin and arginine kinase. About half of Austrian subjects lacked IgE to these pan-allergens, indicating co- but not cross-sensitization to insects and/or shellfish. Regarding IgE recognition frequencies, arginine kinases (64% HK, 10% Austria) and tropomyosins (42% HK, 15% Austria) were most frequently recognized; Bla g 4 (lipocalin) was detected in HK patients only (42%). Tropomyosin (Per a 7) was significantly more frequently recognized in patients with asthma. Sera from HDM-sensitized subjects from HK showed a higher proportion of sensitization to minor mite allergens. Molecular profiling identified differences between CR-sensitized allergic patients from HK and Austria in terms of primary sensitizers and molecular IgE reactivity patterns. Tropomyosin from American cockroach (Per a 7) was shown to be significantly associated with asthma symptoms and might be suitable as biomarker for more severe respiratory allergy symptoms.

Complex IgE sensitization patterns in ragweed allergic patients: Implications for diagnosis and specific immunotherapy.

BackgroundRagweed (Ambrosia artemisiifolia) is one of the most important allergen sources, worldwide, causing severe respiratory allergic reactions in late summer and fall, in sensitized patients. Amb a 1 has been considered as the most important allergen in ragweed but 12 ragweed pollen allergens are known. The aim of our study was to investigate IgE reactivity profiles of ragweed allergic patients and to associate them with clinical symptoms.MethodsIgE sensitization profiles from clinically well‐characterized ragweed allergic patients (n = 150) were analyzed using immunoblotted ragweed pollen extract. Immunoblot inhibition experiments were performed with two Amb a 1 isoforms and CCD markers and basophil activation experiments were performed with IgE serum before and after depletion of Amb a 1‐specific IgE.ResultsBy IgE‐immunoblotting 19 different IgE reactivity patterns with and without Amb a 1‐sensitization were found. The majority of patients (>95%) suffered from rhino‐conjunctivitis, around 60% reported asthma‐like symptoms and about 25% had skin reactions. Patients with complex IgE sensitization profiles tended to have more clinical symptoms. Serum with and without Amb a 1‐specific IgE induced basophil activation.ConclusionsRagweed pollen allergic patients exhibit complex IgE reactivity profiles to ragweed allergens including Amb a 1 isoforms and cross‐reactive carbohydrates indicating the importance of Amb a 1 isoforms and additional allergens for diagnosis and allergen‐specific immunotherapy of ragweed allergy.

Molecular reactivity profiling upon immunotherapy with a 300 IR sublingual house dust mite tablet reveals marked humoral changes towards major allergens.

Molecular antibody reactivity profiles have not yet been studied in depth in patients treated by sublingual house dust mite (HDM) tablet immunotherapy. Humoral immune responses to a large panel of HDM mite allergens were studied using allergen microarray technology in a subset of clinically defined high and low responder patients from a double-blind placebo-controlled allergen-specific immunotherapy (AIT) trial using sublingual 300IR HDM tablets. Serum levels of IgE, IgG and IgG4 to 13 Dermatophagoides pteronyssinus molecules were measured at baseline and after 1-year AIT, using allergen microarrays in 100subjects exhibiting high or low clinical benefit. Derp1, Derp2 and Derp23 were the most frequently recognized allergens in the study population. Patients with HDM-related asthma had significantly higher allergen-specific IgE levels to Derp1 and Derp23. No significant difference in the distribution of allergen sensitization pattern was observed between high and low responders. An increase in serum allergen-specific IgG and IgG4 occurred upon AIT, in particular to allergens Derp1, Derp2 and Derp23 (p<0.0001). We confirm for our study population that Derp1- and Derp23-specific IgE levels are associated with asthma. IgE reactivity profiles were not predicitive of sublingual AIT outcomes, with 300 IR tablets as efficacious in pauci- and multi-sensitized subjects. Our study is the first to demonstrate the induction of IgG and IgG4 specific for the HDM allergens Derp1, Derp2 and Derp23 by sublingual AIT.

Acid Hydrolysis of Gluten Enhances the Skin Sensitizing Potential and Drives Diversification of IgE Reactivity to Unmodified Gluten Proteins.

Personal care products containing hydrolyzed gluten have been linked to spontaneous sensitization through the skin, however the impact of the hydrolysate characteristics on the sensitizing capacity is generally unknown. The physicochemical properties of five different wheat-derived gluten products (one unmodified, one enzyme hydrolyzed, and three acid hydrolyzed) are investigated, and the skin sensitizing capacity is determined in allergy-prone Brown Norway rats. Acid hydrolyzed gluten products exhibited the strongest intrinsic sensitizing capacity via the skin. All hydrolyzed gluten products induced cross-reactivity to unmodified gluten in the absence of oral tolerance to wheat, but were unable to break tolerance in animals on a wheat-containing diet. Still, the degree of deamidation in acid hydrolyzed products is associated with product-specific sensitization in wheat tolerant rats. Sensitization to acid hydrolyzed gluten products is associated with a more diverse IgE reactivity profile to unmodified gluten proteins compared to sensitization induced by unmodified gluten or enzyme hydrolyzed gluten. Acid hydrolysis enhances the skin sensitizing capacity of gluten and drives IgE reactivity to more gluten proteins. This property of acid hydrolyzed gluten may be related to the degree of product deamidation, and could be a strong trigger of wheat allergy in susceptible individuals.

Hevea latex-associated allergies: piecing together the puzzle of the latex IgE reactivity profile

ABSTRACTIntroduction: IgE-mediated Hevea latex allergy and associated food-allergies constitute a significant health issue with serious consequences of diagnostic error. Hence, there is a need for more reliable confirmatory diagnostics.Areas covered: Here, we summarize the major limitations of conventional tests using native extracts and describe how piecing together the IgE reactivity profile can benefit correct diagnosis in difficult cases in whom conventional tests yield equivocal or negative results. A diagnostic algorithm integrating traditional sIgE and component-resolved diagnosis (CRD) is presented.Expert opinion: Moreover, it is clear that the discoveries in the field of the Hevea latex proteome will contribute to our understandings and accurate approach of sometimes complex cross-reactivity phenomena that extend beyond the ‘latex-fruit syndrome.’

Clinical and immunological differences between asymptomatic HDM-sensitized and HDM-allergic rhinitis patients.

Confirmation of the clinical relevance of sensitisation is important for the diagnosis of allergic rhinitis. To investigate the usefulness of an in vitro basophil activation test and component-resolved diagnosis in distinguishing between symptomatic allergic rhinitis patients and asymptomatic sensitization to house dust mites (HDMs). Thirty-six subjects with a positive skin prick test (SPT) for HDM were divided into a symptomatic (n=17) and an asymptomatic (n=19) group on the basis of their clinical history and a nasal provocation test. A basophil CD63 response to in vitro stimulation with Dermatophagoidespteronyssinus whole allergen extract and the IgE reactivity profiles for Der p 1, 2, 4, 5, 7, 10, 11, 14, 15, 18, 21, 23 were evaluated. Serum IgE and IgG specific to Dpteronyssinus whole allergen extract and total IgE were measured. There were no statistically significant differences in the levels of IgE (IgE levels were higher in symptomatic patients with P=0.055) and IgG specific to Dpteronyssinus and total IgE. Symptomatic patients showed a lower threshold for in vitro basophil activation (3.33ng/mL vs 33.3ng/mL), a higher area under the curve (AUC) of basophil activation (171 vs 127) (P=0.017), a higher response to positive control with anti-FcεRI stimulation (97% vs 79%) (P<0.001), a recognition of more HDM allergens (4 vs 2) and more frequent sensitization to rDer p 7 (P=0.016) and rDer p 23 compared to asymptomatic subjects (P=0.018). There was a positive correlation (r=0.63; P<0.001) between the number of recognized allergens and the AUC of basophil activation. In the subjects studied, the differences in the basophil response to Dpteronyssinus allergen extract, number of recognized HDM allergens and reactivity to rDer p 7 and rDer p 23 distinguish symptomatic from asymptomatic HDM sensitisation better than SPT or allergen extract-specific IgE. Information regarding the clinical relevance of sensitization is important for the prescription of allergen-specific immunotherapy.

Identification of polcalcin as a novel allergen of Amaranthus retroflexus pollen

IntroductionAmaranthus retroflexus (Redroot Pigweed) is one of the main sources of allergenic pollens in temperate areas. Polcalcin is a well-known panallergen involved in cross-reactivity between different plants. The aim of this study was the molecular cloning and expression of polcalcin, as well as evaluating its IgE-reactivity with A. retroflexus sensitive patients’ sera. MethodsAllergenic extract was prepared from A. retroflexus pollen and the IgE-reactivity profile was determined by ELISA and immunoblotting using sera from twenty A. retroflexus sensitive patients. Polcalcin-coding sequence was amplified by conventional PCR method and the product was inserted into pET-21b(+) vector. The recombinant protein was expressed in E. coli BL21 and purified by metal affinity chromatography. The IgE-binding capability of the recombinant protein was analyzed by ELISA and immunoblotting assays, and compared with crude extract. ResultsOf 20 skin prick test positive patients, 17 patients were positive in IgE-specific ELISA. Western blotting confirmed that approximately 53% of ELISA positive patients reacted with 10kDa protein in crude extract. The A. retroflexus polcalcin gene, encoding to 80 amino acid residues was cloned and expressed as a soluble protein and designated as Ama r 3. The recombinant polcalcin showed rather identical IgE-reactivity in ELISA and western blotting with 10kDa protein in crude extract. These results were confirmed by inhibition methods, too. ConclusionThe recombinant form of A. retroflexus polcalcin (Ama r 3) could be easily produced in E. coli in a soluble form and shows rather similar IgE-reactivity with its natural counterpart.

Piecing Together the IgE-Reactivity Profile of Cannabis sativa in a North-Western European Region

Piecing Together the IgE-Reactivity Profile of Cannabis sativa in a North-Western European Region

Underestimation of house dust mite–specific IgE with extract-based ImmunoCAPs compared with molecular ImmunoCAPs

Underestimation of house dust mite–specific IgE with extract-based ImmunoCAPs compared with molecular ImmunoCAPs

Do Plantago lanceolata Skin Prick Test-Positive Patients Display IgE to Genuine Plantain Pollen Allergens? Investigation of Pollen Allergic Patients from the North-East of France

Background: English plantain (Plantago lanceolata) is an important weed pollen allergen source triggering allergic symptoms during summer. To elucidate genuine versus cross-reactive sensitization, we investigated IgE reactivity patterns and inhibition capacities of plantain-sensitized patients. Methods: Sera of 35 rhinoconjunctivitis patients from the north-east of France with positive skin prick tests (SPT) to Plantago lanceolata pollen were tested with clinically relevant allergen sources using ELISA, ImmunoCAP, and immunoblot inhibition. Results: The patients were multisensitized with additional reactivity to grass (94.3%), ash (74.3%), birch (71.4%), and mugwort (55.2%) pollen in SPT. Sensitization prevalence to allergen molecules was 34.3% (Pla l 1), 94.3% (Phl p 1/5), 60.0% (Ole e 1), 65.7% (Bet v 1), 37.1% (profilin), and 40.0% (CCD). In immunoblot, IgE reactivity to plantain pollen was inhibited with relevant pollen extracts and purified rPla l 1. Two sera did not reveal any IgE cross-reactivity, while reactivity to plantain was efficiently inhibited by grass pollen in the sera of 10 patients. The sera from 17 different patients could be inhibited by grass, birch, or ash pollen to varying degrees. Thus, only 37.1% of our patients demonstrated true plantain pollen sensitization, while 62.9% were solely positive due to IgE cross-reactive molecules from other clinically relevant pollen. Conclusions: Plantain pollen-sensitized patients are multi-reactors demonstrating varying and complex IgE-reactivity profiles. In vivo and in vitro tests using extracts are typically blurred due to the presence of homologous allergens or CCD in grass, birch, or ash pollen. So far, Pla l 1 represents the only indicative marker allergen for the diagnosis of genuine plantain pollen sensitization.

Molecular allergen profiling in horses by microarray reveals Fag e 2 from buckwheat as a frequent sensitizer.

BackgroundCompanion animals are also affected by IgE‐mediated allergies, but the eliciting molecules are largely unknown. We aimed at refining an allergen microarray to explore sensitization in horses and compare it to the human IgE reactivity profiles.MethodsCustom‐designed allergen microarray was produced on the basis of the ImmunoCAP ISAC technology containing 131 allergens. Sera from 51 horses derived from Europe or Japan were tested for specific IgE reactivity. The included horse patients were diagnosed for eczema due to insect bite hypersensitivity, chronic coughing, recurrent airway obstruction and urticaria or were clinically asymptomatic.ResultsHorses showed individual IgE‐binding patterns irrespective of their health status, indicating sensitization. In contrast to European and Japanese human sensitization patterns, frequently recognized allergens were Aln g 1 from alder and Cyn d 1 from Bermuda grass, likely due to specific respiratory exposure around paddocks and near the ground. The most prevalent allergen for 72.5% of the tested horses (37/51) was the 2S‐albumin Fag e 2 from buckwheat, which recently gained importance not only in human but also in horse diet.ConclusionIn line with the One Health concept, covering human health, animal health and environmental health, allergen microarrays provide novel information on the allergen sensitization patterns of the companion animals around us, which may form a basis for allergen‐specific preventive and therapeutic concepts.

Cross-sectional study on allergic sensitization of Austrian adolescents using molecule-based IgE profiling.

Allergen-specific IgE antibodies are a hallmark of type I allergy. The aim of this cross-sectional study was to analyze the sensitization profiles of an Austrian adolescent population utilizing molecule-based IgE diagnosis. Serum samples of 501 nonselected pupils from Salzburg, Austria, were tested in ImmunoCAP ISAC® for IgE reactivity to 112 single allergens. Sensitization profiles were assessed and statistically coordinated with reported allergies. In the population aged 12-21 years, 53.5% showed IgE reactivity to at least one allergen tested. The highest prevalence was found for Phl p 1 from grass pollen (26.5%), group 2 mite allergens (18.2%), Bet v 1 from birch pollen (16.3%) and Fel d 1 from cat (14.4%). The majority of participants showed a complex sensitization profile and reacted on average to 9 allergens. Pollen sensitization was highly prevalent (41.7%) and mainly driven by group I grass and PR-10 allergens of the Betulaceae family, while Pla l 1 represented the most relevant weed. Diagnosed and self-reported allergies were noted in 21.9% and 45.5% of participants, respectively, and correlated well with in vitro results. Among atopic individuals, 71.4% reported to suffer from at least one allergy; concordance was found for grass and cat sensitization, while venom- and weed pollen-positive individuals were frequently asymptomatic. More than half of the tested adolescent population had already established an atopic status presenting a complex IgE reactivity profile dominated by pollen sensitization. Detailed molecule-based analysis allows determining relevant biomarkers and monitoring of the atopic status in populations.

IgE-reactivity profiles to nonspecific lipid transfer proteins in a northwestern European country

IgE-reactivity profiles to nonspecific lipid transfer proteins in a northwestern European country

Real-Life Study for the Diagnosis of House Dust Mite Allergy - The Value of Recombinant Allergen-Based IgE Serology

Background:Dermatophagoides pteronyssinus is one of the most important perennial allergen sources worldwide. Molecular diagnostics using the commercially available major allergens (Der p 1 and Der p 2) in combination with Der p 10 do not detect house dust mite (HDM) sensitization in a number of cases when used alone. The objective was to evaluate the IgE reactivity profiles of these patients using an experimental immunoassay biochip. Methods: Sera of HDM-allergic patients (positive skin prick test, CAP class ≥1 for allergen extract, and positive intranasal provocation) were tested for IgE antibodies against Der p 1, Der p 2, and Der p 10 by ImmunoCAP fluorescence enzyme immunoassay. Negatively tested sera were examined by an experimental chip containing 13 microarrayed HDM allergens. Results: Of 97 patients tested, 16 showed negative results to Der p 1, Der p 2, and Der p 10. MeDALL chip evaluation revealed 5 patients monosensitized to Der p 23, and 11 patients were negative for all HDM MeDALL chip components. Seven sera were available for further testing, and 3 of them showed IgE reactivity to dot-blotted nDer p 1, and 2 reacted with high-molecular weight components (>100 kDa) in nitrocellulose-blotted HDM extract when tested with 125I-labeled anti-IgE in a RAST-based assay. The HDM extract-specific IgE levels of the 11 patients were <3.9 kU/l. Conclusions: Recombinant allergen-based IgE serology is of great value when conventional IgE diagnostics fails. Der p 23 is an important HDM allergen, especially when major allergens are negative. Therefore, it would be desirable to have Der p 23 commercially available. Further research concerning the prevalence and clinical significance of different HDM allergens is needed.

Different IgE recognition of mite allergen components in asthmatic and nonasthmatic children

BackgroundHouse dust mites (HDMs) represent one of the most important inducers of respiratory allergies worldwide.ObjectiveWe sought to investigate the IgE and IgG reactivity profiles to a comprehensive panel of HDM allergens in children with allergic asthma and to compare them with those of nonasthmatic atopic children.MethodsSera from clinically well-characterized asthmatic children with HDM allergy (n = 105), nonasthmatic children (n = 53), and nonatopic nonasthmatic children (n = 53) were analyzed for IgE and IgG reactivity to a panel of 7 HDM allergens (nDer p 1, rDer p 2, rDer p 5, rDer p 7, rDer p 10, rDer p 21, and rDer p 23) by means of allergen microarray technology.ResultsAsthmatic children with HDM allergy more frequently showed an IgE response to each of the HDM allergens and recognized more allergens than nonasthmatic children with HDM allergy. Furthermore, IgE levels to certain HDM allergens (nDer p 1, P = .002; rDer p 2, P = .007; rDer p 5, P = .031; and rDer p 23, P < .001) were significantly higher in asthmatic children than in children without asthma. By contrast, fewer asthmatic children showed IgG reactivity to HDM allergens than nonasthmatic children, but allergen-specific IgG levels were comparable.ConclusionThe IgE and IgG reactivity profiles to HDM allergens, as well as IgE levels to certain allergen components, differed considerably between children with and without asthmatic symptoms caused by HDM allergy. In fact, asthmatic children were characterized by an expanded IgE repertoire regarding the numbers of recognized allergen components and by increased specific IgE levels.

Transmission of allergen-specific IgG and IgE from maternal blood into breast milk visualized with microarray technology

Transmission of allergen-specific IgG and IgE from maternal blood into breast milk visualized with microarray technology

Analysis of the IgE- and IgG-reactivity profiles of asthmatic and non-asthmatic HDM-allergic patients using the ISAC microarray system

Results HDM-allergic individuals suffering from asthma showed striking differences regarding their IgE reactivity profiles compared to the non-asthmatic group. First, the frequency of IgE reactivity to the tested HDM allergens was up to 3fold higher in the asthmatic than in the non-asthmatic group. Seventy percent of the asthmatics reacted with 3 to 6 of the tested allergens compared to only 45% in the nonasthmatic group. Furthermore, IgE-levels to nDer p 1, rDer p 2, rDer p 5 and rDer p 23 were significantly higher in the asthmatic group. In contrast, HDM-allergic asthma patients showed a lower IgG-binding frequency to the seven HDM allergens than HDM-allergic patients without asthma. Conclusion The IgE and IgG reactivity profiles to HDM allergens differ considerably in patients with mild (i.e., rhinitis) and severe (i.e., asthma) respiratory symptoms due to HDM allergy.