Allergen microarray detects high prevalence of asymptomatic IgE sensitizations to tropical pollen-derived carbohydrates.
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ABSTRACT:
SUBMITTER: Cabauatan CR
PROVIDER: S-EPMC6597356 | biostudies-literature | 2014 Mar
REPOSITORIES: biostudies-literature
Publications
Allergen microarray detects high prevalence of asymptomatic IgE sensitizations to tropical pollen-derived carbohydrates.
Cabauatan Clarissa R CR Lupinek Christian C Scheiblhofer Sandra S Weiss Richard R Focke-Tejkl Margarete M Bhalla Prem L PL Singh Mohan B MB Knight Paul A PA van Hage Marianne M Ramos John Donnie A JD Valenta Rudolf R
The Journal of allergy and clinical immunology 20131206 3
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Project description:BackgroundCurrent diagnostics for allergies, such as skin prick and radioallergosorbent tests, do not allow for inexpensive, high-throughput screening of patients. Additionally, extracts used in these methods are made from washed pollen that lacks pollen surface materials that may contain allergens.Methodology/principal findingsWe sought to develop a high-throughput assay to rapidly measure allergen-specific IgE in sera and to explore the relative allergenicity of different pollen fractions (i.e. surface, cytoplasmic, commercial extracts). To do this, we generated a protein microarray containing surface, cytoplasmic, and commercial extracts from 22 pollen species, commercial extracts from nine non-pollen allergens, and five recombinant allergenic proteins. Pollen surface and cytoplasmic fractions were prepared by extraction into organic solvents and aqueous buffers, respectively. Arrays were incubated with <25 uL of serum from 176 individuals and bound IgE was detected by indirect immunofluorescence, providing a high-throughput measurement of IgE. We demonstrated that the allergen microarray is a reproducible method to measure allergen-specific IgE in small amounts of sera. Using this tool, we demonstrated that specific IgE clusters according to the phylogeny of the allergen source. We also showed that the pollen surface, which has been largely overlooked in the past, contained potent allergens. Although, as a class, cytoplasmic fractions obtained by our pulverization/precipitation method were comparable to commercial extracts, many individual allergens showed significant differences.Conclusions/significanceThese results support the hypothesis that protein microarray technology is a useful tool for both research and in the clinic. It could provide a more efficient and less painful alternative to traditionally used skin prick tests, making it economically feasible to compare allergen sensitivity of different populations, monitor individual responses over time, and facilitate genetic studies on pollen allergy.
| S-EPMC2856625 | biostudies-literature
Project description:BackgroundAffinity and clonality of allergen-specific IgE antibodies are important determinants for the magnitude of IgE-mediated allergic inflammation.ObjectiveWe sought to analyze the contribution of heavy and light chains of human allergen-specific IgE antibodies for allergen specificity and to test whether promiscuous pairing of heavy and light chains with different allergen specificity allows binding and might affect affinity.MethodsTen IgE Fabs specific for 3 non-cross-reactive major timothy grass pollen allergens (Phl p 1, Phl p 2, and Phl p 5) obtained by means of combinatorial cloning from patients with grass pollen allergy were used to construct stable recombinant single chain variable fragments (ScFvs) representing the original Fabs and shuffled ScFvs in which heavy chains were recombined with light chains from IgE Fabs with specificity for other allergens by using the pCANTAB 5 E expression system. Possible ancestor genes for the heavy chain and light chain variable region-encoding genes were determined by using sequence comparison with the ImMunoGeneTics database, and their chromosomal locations were determined. Recombinant ScFvs were tested for allergen specificity and epitope recognition by means of direct and sandwich ELISA, and affinity by using surface plasmon resonance experiments.ResultsThe shuffling experiments demonstrate that promiscuous pairing of heavy and light chains is possible and maintains allergen specificity, which is mainly determined by the heavy chains. ScFvs consisting of different heavy and light chains exhibited different affinities and even epitope specificity for the corresponding allergen.ConclusionOur results indicate that allergen specificity of allergen-specific IgE is mainly determined by the heavy chains. Different heavy and light chain pairings in allergen-specific IgE antibodies affect affinity and epitope specificity and thus might influence clinical reactivity to allergens.
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Project description:BackgroundBecause of a high risk to develop fatal anaphylaxis, early detection of immunoglobulin E (IgE)-dependent allergy is of particular importance in patients with mastocytosis.ObjectiveWe examined whether microarray-based screening for allergen-reactive IgE (allergen-chip) is a sensitive and robust approach to detect specific IgE in patients with mastocytosis.MethodsSera for 42 patients were analyzed, including 4 with cutaneous mastocytosis, 2 with mastocytosis in the skin, and 36 with systemic mastocytosis. In addition, sera from an age- and sex-matched control cohort (n = 42) were analyzed.ResultsIn 15 of 42 patients with mastocytosis (35.7%), specific IgE was detected by allergen-chip profiling. Ves v 5 and Bet v 1 were the most frequently detected allergens (Ves v 5: 16.7% of patients; Bet v 1: 11.9% of patients). Allergen reactivity was confirmed by demonstrating upregulation of CD203c on blood basophils upon exposure to the respective allergen(s) in these patients. Specific IgE was identified by chip studies in 11 of 26 patients with mastocytosis with mediator-related symptoms (42.3%) and in 4 of 14 patients with mastocytosis without symptoms (28.6%). In the cohort with known allergy, 9 of 9 patients (100%) had a positive allergen-chip result. In patients with mastocytosis without a known allergy (n = 31), the chip identified 6 positive cases (19.5%). The prevalence of chip-positive patients was slightly lower in the mastocytosis group (35.7%) compared with age- and sex-matched controls (40.5%).ConclusionsAlthough specific IgE may not be detectable in all sensitized patients with mastocytosis, allergy chip-profiling is a reliable screening approach for the identification of patients with mastocytosis suffering from IgE-dependent allergies.
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Project description:BackgroundGroup 5 allergens are small proteins that consist of two domains. They belong to the most potent respiratory allergens.ObjectiveTo determine the binding sites and to study allergic patients' IgE recognition of the group 5 allergen (Phl p 5) from timothy grass pollen using human monoclonal IgE antibodies that have been isolated from grass pollen allergic patients.MethodsUsing recombinant isoallergens, fragments, mutants and synthetic peptides of Phl p 5, as well as peptide-specific antibodies, the interaction of recombinant human monoclonal IgE and Phl p 5 was studied using direct binding and blocking assays. Cross-reactivity of monoclonal IgE with group 5 allergens in several grasses was studied and inhibition experiments with patients' polyclonal IgE were performed.ResultsMonoclonal human IgE showed extensive cross-reactivity with group 5 allergens in several grasses. Despite its small size of 29 kDa, four independent epitope clusters on isoallergen Phl p 5.0101, two in each domain, were recognized by human IgE. Isoallergen Phl p 5.0201 carried two of these epitopes. Inhibition studies with allergic patients' polyclonal IgE suggest the presence of additional IgE epitopes on Phl p 5.Conclusions & clinical relevanceOur results reveal the presence of a large number of independent IgE epitopes on the Phl p 5 allergen explaining the high allergenic activity of this protein and its ability to induce severe allergic symptoms. High-density IgE recognition may be a general feature of many potent allergens and form a basis for the development of improved diagnostic and therapeutic procedures in allergic disease.
| S-EPMC4278554 | biostudies-literature
Project description:Identifying allergen distribution is meaningful and significant for effective diagnosis and treatment of allergic diseases. This study compared the allergen sensitivity in four southern China cities. We enrolled 55,432 participants (27,408 male, 28,024 female) between 2007 and 2019. The allergen-specific IgE levels were compared by the χ2 test. The five prevalent sensitivities were for mite mix (10,985, 19.82%), cockroach (4,860, 8.77%), crab (4,450, 8.03%), fish mix (3,874, 6.99%), and house dust (3,486, 6.29%). Almost all allergen sensitivities decreased with age, particularly from infant to middle aged participants (p < 0.05). An exception was Shenzhen, where food allergen positive rates remained constant in all age groups studied. The proportion of male sensitive to at least one food allergen (OR 1.130; 95% CI 1.088-1.174, p < 0.0025) or aeroallergen (OR, 1.117; 95% CI, 1.078-1.158, p < 0.0025) was higher than female in all four cities. Except for dog dander and tree mix, all aeroallergens differed significantly between seasons (p < 0.05). Liuzhou had the highest rates of food allergen- and aeroallergen-positive participants. The allergen-specific IgE distribution differed among the studied cities, with significant seasonal differences. Young age, male sex, and aeroallergens were risk factors for allergic disease.
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Project description:BackgroundAllergic diseases are some of the most common diseases worldwide. Genome-wide association studies (GWASs) have been conducted to elucidate the genetic factors of allergic diseases. However, no GWASs for allergen component sensitization have been performed.ObjectiveWe sought to detect genetic variants associated with differences in immune responsiveness against allergen components.MethodsThe participants of the present study were recruited from the Tokyo Children's Health, Illness, and Development study, and allergen component-specific IgE level at age 9 years was measured by means of allergen microarray immunoassays. We performed GWASs for allergen component sensitization against each allergen (single allergen component sensitization, number of allergen components analyzed, n = 31), as well as against allergen protein families (allergen protein group sensitization, number of protein groups analyzed, n = 16).ResultsWe performed GWAS on 564 participants of the Tokyo Children's Health, Illness, and Development study and found associations between Amb a 1 sensitization and the immunoglobulin heavy-chain variable gene on chromosome 14 and between Phl p 1 sensitization and the HLA class II region on chromosome 6 (P < 5.0 × 10-8). A GWAS-significant association was also observed between the HLA class II region and profilin sensitization (P < 5.0 × 10-8).ConclusionsOur data provide the first demonstration of genetic risk for allergen component sensitization and show that this genetic risk is related to immune response genes including immunoglobulin heavy-chain variable gene and HLA.
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Project description:BackgroundGroup 2 and 3 grass pollen allergens are major allergens with high allergenic activity and exhibit structural similarity with the C-terminal portion of major group 1 allergens. In this study, we aimed to determine the crystal structure of timothy grass pollen allergen, Phl p 3, and to study its IgE recognition and cross-reactivity with group 2 and group 1 allergens.MethodsThe three-dimensional structure of Phl p 3 was solved by X-ray crystallography and compared with the structures of group 1 and 2 grass pollen allergens. Cross-reactivity was studied using a human monoclonal antibody which inhibits allergic patients' IgE binding and by IgE inhibition experiments with patients' sera. Conformational Phl p 3 IgE epitopes were predicted with the algorithm SPADE, and Phl p 3 variants containing single point mutations in the predicted IgE binding sites were produced to analyze allergic patients' IgE binding.ResultsPhl p 3 is a globular β-sandwich protein showing structural similarity to Phl p 2 and the Phl p 1-C-terminal domain. Phl p 3 showed IgE cross-reactivity with group 2 allergens but not with group 1 allergens. SPADE identified two conformational IgE epitope-containing areas, of which one overlaps with the epitope defined by the monoclonal antibody. The mutation of arginine 68 to alanine completely abolished binding of the blocking antibody. This mutation and a mutation of D13 in the predicted second IgE epitope area also reduced allergic patients' IgE binding.ConclusionGroup 3 and group 2 grass pollen allergens are cross-reactive allergens containing conformational IgE epitopes. They lack relevant IgE cross-reactivity with group 1 allergens and therefore need to be included in diagnostic tests and allergen-specific treatments in addition to group 1 allergens.
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Project description: Not available
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Project description:The timothy grass pollen allergen Phl p 1 belongs to the group 1 of highly cross-reactive grass pollen allergens with a molecular mass of ∼25-30 kDa. Group 1 allergens are recognized by >95% of grass pollen allergic patients. We investigated the IgE recognition of Phl p 1 using allergen-specific IgE-derived single-chain variable Ab fragments (IgE-ScFvs) isolated from a combinatorial library constructed from PBMCs of a grass pollen-allergic patient. IgE-ScFvs reacted with recombinant Phl p 1 and natural group 1 grass pollen allergens. Using synthetic Phl p 1-derived peptides, the binding sites of two ScFvs were mapped to the N terminus of the allergen. In surface plasmon resonance experiments they showed comparable high-affinity binding to Phl p 1 as a complete human IgE-derived Ab recognizing the allergens' C terminus. In a set of surface plasmon resonance experiments simultaneous allergen recognition of all three binders was demonstrated. Even in the presence of the three binders, allergic patients' polyclonal IgE reacted with Phl p 1, indicating high-density IgE recognition of the Phl p 1 allergen. Our results show that multiple IgE Abs can bind with high density to Phl p 1, which may explain the high allergenic activity and sensitizing capacity of this allergen.
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Project description:BackgroundBermuda grass (Cynodon dactylon; subfamily Chloridoideae) is an important source of seasonal aeroallergens in warm tropical and sub-tropical areas worldwide. Improved approaches to diagnosis and therapy of allergic diseases require a thorough understanding of the structure and epitopes on the allergen molecule that are crucial for the antigen-antibody interaction. This study describes the localization of the human IgE-binding regions of the major group 1 pollen allergen Cyn d 1 from Bermuda grass.MethodsA cDNA library was constructed from Bermuda grass pollen (BGP) using a Lambda gt11 expression vector. The gene encoding the Cyn d 1 allergen was isolated by screening the library with a mouse monoclonal antibody raised against grass group 1 allergen. In order to characterize the IgE epitopes on Cyn d 1, seven overlapping fragments and three deletion mutants were cloned and over-expressed in E. coli. The recombinant fragments and deletion mutants were evaluated for their comparative IgE reactivity with sera of non atopic individuals and grass pollen allergic patients by ELISA and a dot-blot assay.ResultsAnalysis of IgE binding regions by overlapping fragments and deletion mutants identified two major allergenic regions corresponding to amino acids 120-170 and 224-244. Deletion of either or both regions led to a significant reduction in IgE binding, emphasizing the importance of the C-terminal region on Cyn d 1 in epitope-IgE interaction.ConclusionAnti-Cyn d 1 IgE antibodies from allergic human sera recognize two epitopes located at the C-terminal end of the molecule. These data will enable the design of improved diagnostic and therapeutic approaches for BGP hypersensitivity.
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