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. Since the initiation of BSP090, the recombinant major allergen Bet v 1 has been adopted by the European Pharmacopoeia Commission as a Chemical Reference Substance (CRS). In parallel, two sandwich ELISA systems for quantification of Bet v 1 were found suitable in preliminary phases of BSP090 to be validated in a large collaborative study. In this study, the candidate ELISA systems were compared with respect to accuracy, precision and variability. Thirteen participating laboratories tested model samples containing the CRS as well as spiked and unspiked birch pollen extracts. Both in pre-testing and in the collaborative study, the 2 candidate ELISA systems confirmed their suitability to quantify recombinant and native Bet v 1. As no clear-cut decision for one of the ELISA systems could be made based on the results of the collaborative study, a post-study testing was performed. Bet v 1 content of 30 birch pollen allergen products was determined in parallel in both ELISA systems. Consequently, 1 candidate ELISA system was selected to be proposed as the future European Pharmacopoeia standard method for Bet v 1 quantification.

Advances in the quantification of relevant allergens in allergenic extracts.

Relevant allergens are major contributors to the safety and efficacy of allergenic extracts used in allergen immunotherapy (AIT). As such, they should be accurately quantified, as recommended by the 2008 European guidelines on allergen products. Until now, the quantification of relevant allergens was mainly performed by using immunoassays (e.g. ELISA) that relying upon specific antibodies. Although antibody-based quantification is commonly used to assess the concentration of relevant allergens in allergenic extracts, results must be taken with caution in the light of the inherent limitations of such techniques. In the present study, we discuss how those limitations can be overcome by using comprehensive mass spectrometry-based techniques.

Establishment of recombinant major allergens Bet v 1 and Phl p 5a as Ph. Eur. reference standards and validation of ELISA methods for their measurement. Results from feasibility studies.

The potency of allergen extracts is determined as total allergenic activity without consideration of their composition and the units differ from one manufacturer to another, making it very difficult to compare the different products. Recently, purified major allergens have been obtained by recombinant DNA technology and produced under Good Manufacturing Practice (GMP) conditions. In principle, such recombinant allergens could be established as reference standards and could help for the standardisation of the major allergen content of allergen extracts. Two recombinant major allergens, one from birch pollen, rBet v 1, and one from Timothy grass pollen, Phl p 5a, have been selected at the end of the CREATE programme as a potential starting point for the establishment as European Pharmacopoeia (Ph. Eur.) Reference Standards through a project run by the Biological Standardisation Programme (BSP) of the European Directorate for the Quality of Medicines & HealthCare (EDQM). To this end, bulk candidate recombinant materials, produced under GMP conditions, were procured from two European manufacturers and subsequently formulated and lyophilised. Four ELISA systems from three different manufacturers were included in the project, two for Bet v 1 and two for Phl p 5a with the aim of establishing reference methods for determination of the respective major antigens both in natural allergen extracts as well as in recombinant allergen products. The project was run in 3 phases: a preparatory and preliminary testing phase (feasibility phase or Phase 1), an extended feasibility phase carried out in 3 laboratories (Phase 2) to confirm the transferability of the methods and an international collaborative study with a large number of participating laboratories (Phase 3). This article describes the work done in Phase 1 and Phase 2, i.e. the physico-chemical and biological characterisation of the recombinant candidate reference standards, the assessment of their suitability for the intended purpose as well as the evaluation of the candidate ELISA systems. The results show that both candidate reference standards are suitable for the intended purpose. In addition, three out of the four ELISA systems that were included in the preliminary phase were found to be appropriate for further evaluation in the collaborative study which was organised in 2011. The results of the collaborative study will be published separately.

Molecular variability of group 1 and 5 grass pollen allergens between Pooideae species: implications for immunotherapy.

BACKGROUND: Differences between major allergens from distinct grass species remain to be investigated, both in terms of structure and antigenicity. METHODS: Group 1 and 5 allergens purified from five common Pooideae species were analysed by mass spectrometry (MS). Major histocompatibility complex (MHC) class II-restricted T cell epitopes were identified using predictive algorithms and human leucocyte antigen (HLA)-binding assays. CD4+ T cell reactivity and IgE binding were assessed based on the induction of CD154 expression in peripheral blood mononuclear cells and using competitive ELISA assays, respectively. RESULTS: MS analysis of group 5 pollen allergens reveals considerable intra- and inter-species variability in amino acid sequence, with 30-50 predominant isoforms found for each species. Differences in the amino acid sequence as well as N- and O-glycosylation contribute to the variability of group 1 allergens, yielding 5-10 main isoforms, depending on the species. Out of 14 MHC class II-restricted T cell epitopes identified within group 1, only one is conserved among the five grass species. Significant differences in binding affinities for HLA-DR molecules result in variable CD4+ T cell recognition of group 1 and 5 allergens purified from the various species. Up to 38% and 85% of patients exhibit seric IgE responses to species-restricted (or semi-restricted) epitopes associated with group 1 or 5 allergens, respectively. CONCLUSION: Major pollen allergens from distinct grass species bear both shared and species-restricted T and B cell immune epitopes. When compared with single extracts, a five grass pollen extract is thus more suitable for specific immunotherapy, as it contains a broader repertoire of the IgE epitopes to which patients are sensitized.

Direct and prolonged exposure to dogs does not influence the degree of skin prick test positivity to dog allergen.

BACKGROUND: The relationship between pet ownership and the risk of developing allergic sensitization to pet allergens is still controversial. We assessed the possible effect of direct exposure to dog allergen on skin reactivity in dog-sensitized patients. METHODS: We studied, in a case-control trial, 116 adults sensitized to dog allergens (55 with a dog at home for at least 10 years and 61 without it). The degree of response was assessed by skin prick test, performed in quadruplicate with three concentrations of allergenic extract: A (1:20 w/v), B (1:200 w/v) and C (1:2000 w/v). The mean diameter of each wheal was assessed using a visilog image analysis software. RESULTS: No significant difference between the two groups in the wheal diameters induced by the three concentrations of dog allergen could be demonstrated. CONCLUSION: The results of this study suggest that direct dog exposure in adults with respiratory allergy is not associated with greater cutaneous response to dog allergens, as compared to non exposed subjects.

Diagnosis of cypress pollen allergy: in vivo and in vitro standardization of a Juniperus ashei pollen extract.

BACKGROUND: Cypress pollen allergy is a major cause of rhinoconjunctivitis and asthma in the Mediterranean area. The nonstandardized cypress allergen extracts currently available for the diagnosis of cypress allergy have a low level of activity. The search for an active material has led to the selection of Juniperus ashei (Ja) pollen because of its very high cross-reactivity with cypress extracts and its superior allergenic activity. The aim of this study was to characterize in vitro and calibrate in vivo an in-house reference extract (IHRS) of J. ashei pollen and determine the specificity and sensitivity of a standardized Ja extract for the prick test diagnosis of cypress allergy. METHODS: Juniperus ashei pollen extract was analysed by 2-D electrophoresis. The IHRS Ja extract was calibrated by skin prick testing in 28 cypress-allergic patients. The sensitivity and specificity of cypress allergy diagnosis using a standardized Ja extract was studied by skin prick test in 42 cypress-allergic patients and 53 nonallergic patients. Jun a 1 content of the IHRS was determined by a monoclonal antibody-based electrophoretic technique. RESULTS: The Jun a 1 content of the 100 IR/ml Ja IHRS extract was 180 microg/ml. For in vivo diagnosis of cypress allergy, Ja pollen extract demonstrated a sensitivity of 95%, a specificity of 100%, a negative predictive value of 96%, and a positive predictive value of 100%. CONCLUSION: Standardized Ja pollen extract is therefore a very appropriate tool for the in vivo diagnosis of cypress pollen allergy and good candidate for specific immunotherapy.

[Juniperus ashei: the gold standard of the Cuppressaceae]. / Juniperus ashei: le golden standard des cupressacées.

The non-standardized Cupressus sempervirens allergen extract currently available for the diagnosis of cypress allergy has a low level of activity. The search for an active material consisted of in vitro and in vivo comparison of three Cupressaceae pollen extracts: Cupressus sempervirens (Cs), Cupressus arizonica (Ca) and Juniperus ashei (Ja) (synonyms: Juniperus sabinoides and Mountain Cedar). These 3 trees belong to the same botanical family of Cupressaceae. While Cs and Ca are commonly encountered in Mediterranean regions, Ja is only present in Europe in the Balkans, but is a major cause of allergy in the USA. In vitro, with a similar protein content, the allergenic properties of Ja extract are 20-Fold higher than those of Cs and 11-fold higher than those of Ca. IgE immunoblotting revealed 14, 42 and 70 kDa allergens common to all 3 extracts. The inhibition curves of the 3 extracts were more than 88% parallel. A significant correlation was observed between serum specific IgE titres for Ja and Cs in 23 patients (r = 0.916; p < 0.001). In vivo, in 23 patients with cypress allergy, the mean diameter of the prick test papule at 1/20 W/V of Ja (8.3 mm) was greater than that of the Cs papule (6.3 mm) (p = 0.001) and the Ca papule (6.7 mm) (p < 0.001). Correlations between cutaneous responses to Cs and Ja (r = 0.629; p = 0.002), and to Cs and Ca (r = 0.75; p = 0.001) were significant. These results demonstrate the intense cross-reactivity between Cs, Ca and Ja. The allergenic potency of the Ja extract is superior to that of Cs and Ca extracts, both in vitro and in vivo. This superiority is correlated with a high concentration of the major allergen, Jun a 1. The non-standardized The now standardized extract of in vitro ashei pollen therefore represents an effective and documented solution for identification, and probably for treatment, of Cupressaceae pollen allergy.

Competitive immunoassay for antigenic latex protein measurement: rabbit antiserum-based assay compared to modified Lowry and human IgE-inhibition methods.

A new rabbit antiserum-based assay was designed and evaluated for the assessment of the allergen potency of latex medical gloves, since total protein measurement by modified Lowry method remains unsatisfactory and the human IgE-inhibition method is limited by the use of sera from type I allergic patients. Four rabbit sera against a nonammoniated latex extract were shown by immunoblotting to have similar binding patterns to those obtained by human IgE. One rabbit serum was used to develop a competitive immunoassay for antigenic latex proteins (CIALP). The same nonammoniated latex extract was used for coating and calibration. The lower detection limit of the method was 0.085 microgram/g of glove. Antigenic proteins measured by CIALP for 77 latex glove extracts (33 pairs of surgical gloves and 11 exam gloves) showed positive correlation with those of the modified Lowry method (r = 0.4; p < 0.001). For 36 extracts made from the right and the left hand of 18 out of the 33 surgical latex gloves tested, inhibition of human specific-IgE results did not correlate with the modified Lowry method but did with the CIALP results (r = 0.8, p < 0.0001). The CIALP, which is well correlated with the human IgE-inhibition test, enables the assessment of the allergenicity of latex medical gloves by measuring the antigenic proteins.

Latex allergy diagnosis: in vivo and in vitro standardization of a natural rubber latex extract.

For the diagnosis of IgE-mediated (immediate) hypersensitivity to natural rubber latex (NRL), skin prick testing with extracts of latex gloves has been widely used, but such extracts are difficult to standardize. The present study aimed to produce on an industrial scale an NRL extract from freshly collected NRL and to evaluate, calibrate, and standardize the extract by both in vivo and in vitro testing. The source material, latex of the rubber tree, Hevea brasiliensis (clone RRIM 600), was frozen immediately after collection in Malaysia and shipped in dry ice to Stallergènes SA, France. Protein and allergen profiles were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), immunoblotting, isoelectric focusing (IEF), crossed immunoelectrophoresis (CIE), and crossed radioimmunoelectrophoresis (CRIE). Allergen quantification was effected by RAST inhibition. The capacity of the preparation to elicit immediate hypersensitivity reactions in vivo was measured by skin prick testing in 46 latex-allergic patients and 76 nonallergic control subjects. SDS-PAGE and immunoblot profiles of the extract and an NRL standard (E8) provided by the US Food and Drug Administration were almost identical, disclosing several distinct IgE-binding proteins with apparent molecular weights of 14, 20, 27, 30, and 45 kDa, conforming to reported molecular weights of several significant NRL allergens. An arbitrary index of reactivity (IR) of 100 was assigned to the extract at 1:200 dilution (w/v), having a protein content of 22 micrograms/ml. Skin prick testing of latex-allergic patients and controls using the extract at 100 IR revealed 93% sensitivity, 100% specificity, 100% negative predictive value, and 96% positive predictive value. In conclusion, a skin prick test reagent for diagnosis of type I NRL allergy was successfully standardized. The reagent was demonstrated to contain most, if not all, of the currently known clinically significant NRL allergens, and it showed high sensitivity and specificity.