Validation of an ELISA method for quantification of the major Timothy grass pollen allergen Phl p 5a (BSP090).

Progress towards standardisation of allergen products has been made in recent years. Nevertheless, no standardised test method to quantify the allergen content of grass pollen allergen products is available at present. One aim of the BSP090 project was to validate a quantitative assay for a major Timothy grass (Phleum pratense) pollen allergen, Phl p 5.

Standardisation of allergen products: 4. Validation of a candidate European Pharmacopoeia standard method for quantification of major grass pollen allergen Phl p 5.

Background: The aim of the BSP090 project is the establishment of European Pharmacopoeia Chemical Reference Substances (CRSs) in combination with corresponding standard ELISA methods for quantification of major allergens in allergen products. Here, we present data of a Phl p 5-specific sandwich ELISA that proved suitable for the quantification of Phl p 5, one of the major Timothy grass (Phleum pratense) pollen allergens.

Methods: A Phl p 5-specific ELISA system was assessed with respect to accuracy, precision, inter-assay (within laboratory) and inter-laboratory variations, in a ring trial including 14 laboratories in Europe and the USA.

Validation of ELISA methods for quantification of the major birch allergen Bet v 1 (BSP090).

To date, the potency of allergen products in Europe is expressed in manufacturer-specific units relative to a product-specific in-house reference. Consequently, cross-product comparability of allergen products from different manufacturers with respect to strength and efficacy is impossible. The Biological Standardisation Programme (BSP) project BSP090 addresses this issue via the establishment of reference standards in conjunction with ELISA methods for the quantification of major allergens in allergen products.

RItA: The Italian severe/uncontrolled asthma registry.

Background: The Italian severe/uncontrolled asthma (SUA) web-based registry encompasses demographic, clinical, functional, and inflammatory data; it aims to raise SUA awareness, identifying specific phenotypes and promoting optimal care.

Methods: Four hundred and ninety three adult patients from 27 Italian centers (recruited in 2011-2014) were analyzed.

Results: Mean age was 53.

Exposure to indoor allergens and association with allergy symptoms of employees in a work environment.

Exposure to indoor allergens is an important risk factor for sensitisation and respiratory allergy. The aim of this paper was to evaluate the levels of mite, cat and latex allergens in dust collected from an indoor workplace and to assess whether the exposure to these allergens was associated with the allergy symptoms reported by employees. Sixty dust samples were collected.

Characterization and comparison of commercially available mite extracts for in vivo diagnosis.

Background: Assessment of sensitization by allergen-specific IgE testing and skin prick testing (SPT) are primary tools in routine clinical diagnosis of allergies. To perform a correct diagnosis, it is critical that the allergen reagent used contains an adequate amount of all relevant components. This study aimed at evaluating commercially available mite extracts for in vivo diagnosis from eight manufacturers.

Evaluation of allergenicity of genetically modified soybean protein extract in a murine model of oral allergen-specific sensitization.

Background: With the development of genetically modified crop plants there has been a growing interest in the approaches available to assess the potential allergenicity of novel gene products. For additional assessment of the potential allergenicity of expressed proteins, informative data can be generated using animal models. Soybean is one of the major source of protein in human and animal nutrition, and has also been well characterized as a major allergenic source.

Immunological characterization of a recombinant tropomyosin from a new indoor source, Lepisma saccharina.

Background: The presence of specific IgE antibodies to invertebrates is common among patients with rhinitis and asthma. Tropomyosin has been described as an invertebrate cross-reactive allergen. We have recently characterized an allergenic extract from silverfish (Lepisma saccharina).

Cloning and Expression of the Olea europaea allergen Ole e 5, the pollen Cu/Zn superoxide dismutase.

Background: Recombinant DNA technology does provide pure, well-defined and reproducible products to be used for clinical purposes, by cloning and expressing the cDNA of allergens present in a specific extract. Ole e 5 is a pollen allergen of Olea europaea with an IgE-binding frequency of about 35%, which has been identified as a superoxide dismutase (SOD). The aim of this study was to clone the cDNA of Ole e 5, to express Ole e 5 in Escherichia coli and to characterize its immunoreactivity.

Refolding of the Cupressus arizonica major pollen allergen Cup a1.02 overexpressed in Escherichia coli.

The cDNA encoding an isoform of the cypress major pollen allergen, Cup a1.02, has been cloned and expressed in Escherichia coli as a N-terminal 6x His-tagged protein. To increase recovery, Cup a1.

Localisation of a carbohydrate epitope recognised by human IgE in pollen of Cupressaceae.

The objectives of the present study were: (1) to localise, at the subcellular level, the allergens in pollen of Cupressaceae species, using a monoclonal antibody (mAb 5E6) that is specific for carbohydrate epitopes of allergenic components of Cupressus arizonica pollen extract; (2) to determine whether the glycidic epitope recognised by mAb 5E6 was present in pollen of allergenic species taxonomically unrelated to Cupressaceae; and (3) to determine whether human IgE purified from monosensitive patients recognises the same epitope as mAb 5E6 in Cupressaceae pollen. Immunogold labelling of mAb 5E6 showed a high density of gold particles on the orbicules, supporting the hypothesis that they are important vectors of allergens. A high density was also found on the exine and in the cytoplasm, with the latter finding confirming that fragments of pollen ruptured under humid conditions can represent a vector.

Comparison between the native glycosylated and the recombinant Cup a1 allergen: role of carbohydrates in the histamine release from basophils.

Background: Cypress pollinosis is an important cause of respiratory allergies. Recently, the Cupressus arizonica major allergen, Cup a1, has been cloned and expressed. The native counterpart of this allergen has been purified and characterized by our group.

Preparation and characterization of silverfish (Lepisma saccharina) extract and identification of allergenic components.

Background: Airborne insect antigens represent important aeroallergens which have been widely investigated. Although it has been demonstrated that house dust contains significant silverfish (Lepisma saccharina) levels, none of the extracts obtained so far has been extensively characterized. Thus, we have prepared and characterized a silverfish extract and investigated its IgE-reactive components by testing the reactivity of sera from patients allergic to inhalant insect allergens.

Molecular, structural, and immunologic relationships between different families of recombinant calcium-binding pollen allergens.

Background: Calcium-binding plant allergens can be grouped in different families according to the number of calcium-binding domains (EF hands).

Objective: We sought to identify pollens containing crossreactive calcium-binding allergens and to investigate structural and immunologic similarities of members belonging to different families of calcium-binding allergens.

Methods: By means of multiple sequence alignment and molecular modeling, we searched for structural similarities among pollen allergens with 2 (Phl p 7, timothy grass; Aln g 4, alder), 3 (Bet v 3, birch) and 4 EF hands (Jun o 4, prickly juniper).

Rapid isolation, characterization, and glycan analysis of Cup a 1, the major allergen of Arizona cypress (Cupressus arizonica) pollen.

Background: A rapid method for the purification of the major 43-kDa allergen of Cupressus arizonica pollen, Cup a 1, was developed.

Methods: The salient feature was a wash of the pollen in acidic buffer, followed by an extraction of the proteins and their purification by chromatography. Immunoblotting, ELISA, and lectin binding were tested on both the crude extract and the purified Cup a 1.

A monoclonal antibody specific for a carbohydrate epitope recognizes an IgE-binding determinant shared by taxonomically unrelated allergenic pollens.

Carbohydrate epitopes are capable of binding human IgE from allergic subjects and these epitopes play a role in the cross-reactivity between allergens from unrelated sources. A monoclonal antibody (5E6), specific for a carbohydrate epitope detectable on components of Cupressus arizonica pollen extract, has been produced and characterized. To study the relationship between the epitopes recognized by the monoclonal antibody and by IgE from allergic subjects.

Allergenicity of mare's milk in children with cow's milk allergy.

Background: Cow's milk allergy is a common disease of infancy and early childhood. If the baby is not breast-fed, a substitute for cow's milk formula is necessary.

Objective: The aim of this study was to investigate, in vitro and in vivo, the allergenicity of mare's milk in a population of selected children with severe IgE-mediated cow's milk allergy.

Role of carbohydrate moieties in IgE binding to allergenic components of Cupressus arizonica pollen extract.

Background: A reduction of IgE immunoreactivity after periodate-treatment has been previously reported for various glycoprotein allergens.

Objective: The aim of this study was to investigate the role of glycan moiety of a C. arizonica extract in the binding of patients' IgE and to identify the carbohydrates possibly involved.

Specific IgE to cross-reactive carbohydrate determinants strongly affect the in vitro diagnosis of allergic diseases.

Background: Cross-reacting carbohydrate determinants (CCDs) are antigenic structures shared by allergenic components from taxonomically distant sources. The case history of a patient with a great discrepancy between skin test and specific IgE results led us to investigate the role of these determinants in his specific case and in an allergic population.

Objective: We sought to determine the role of CCDs in causing false-positive and clinically irrelevant results in in vitro tests.

Arizona cypress (Cupressus arizonica) pollen allergens. Identification of cross-reactive periodate-resistant and -sensitive epitopes with monoclonal antibodies.

Species of the Cupressaceae family are a worldwide cause of respiratory allergies. We used monoclonal antibodies (mAbs) to investigate the presence and the nature of cross-reacting epitopes shared by various components within Cupressus arizonica pollen extract (CaE) or by CaE and pollen extract from C. sempervirens (CsE).

Molecular characterization of a cross-reactive Juniperus oxycedrus pollen allergen, Jun o 2: a novel calcium-binding allergen.

Background: Species belonging to the Cupressaceae family are a relevant source of allergens that are present in a wide number of countries.

Objective: We sought to identify, purify, and characterize recombinant allergens from Juniperus oxycedrus, a species belonging to the Cupressaceae family.

Methods: Double-stranded cDNA was synthesized from mRNA and cloned into the lambda-ZAP expression vector.

Juniperus oxycedrus: a new allergenic pollen from the Cupressaceae family.

Background: Cupressaceae allergy is a worldwide pollinosis caused by several species. Some species in limited geographic areas pollinate in fall and winter. Juniperus oxycedrus matches these features.