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. Qualification of a candidate ELISA system was performed with regard to range, robustness and cross-reactivity in preliminary studies. The assay specifically detected Phl p 5 with a quantification range from 3.9 ng/mL to 62.5 ng/mL. Suitability to quantify recombinant and natural Phl p 5 was further assessed in a collaborative study including 14 laboratories in Europe and the USA. Precision and accuracy of the assay was satisfactory with 93% of calculated Phl p 5 concentrations and 100% of total recoveries being within the ± 30% acceptance range. Similar results were obtained for spike recoveries, with exclusion of the lowest concentration spike, showing spike recoveries exceeding the acceptance range for six laboratories. Inter-assay (repeatability) and inter-laboratory (reproducibility) variability were satisfactory, in the format used in the present study. Robustness towards different statistical methods for data analysis was demonstrated. In conclusion, the assay can easily be established in routine testing and results of the preliminary testing and collaborative study support the proposal of the assessed Phl p 5-specific ELISA as a European Pharmacopoeia general method.

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.

BASALIT trial: double-blind placebo-controlled allergen immunotherapy with rBet v 1-FV in birch-related soya allergy.

BACKGROUND: Conflicting results exist on the effect of allergen immunotherapy (AIT) on pollen-related food allergy. We aimed to investigate the efficacy of one-year AIT with the folding variant (FV) of recombinant (r) Bet v 1 on birch-related soya allergy. METHODS: Of 138 subjects with Bet v 1 sensitization, 82 were positive at double-blind placebo-controlled food challenge (DBPCFC) with soya. A total of 56 of 82 were randomized in the ratio of 2:1 (active: placebo). Per-protocol population (PPP) had received ≥150 µg of allergen or placebo preparation. OUTCOME MEASURES: lowest observed adverse effect levels (LOAEL), postinterventional occurrence of objective signs (objS) at any dose level, sIgE/IgG4 against Bet v 1 and Gly m 4. Between-group changes were investigated (ancova, Mann-Whitney U-test, Fisher exact test). RESULTS: Baseline characteristics including LOAELs were comparable in both groups with objS and subjS occurring in 82% and 95% of active (n = 38) vs 78% and 83% of placebo group (n = 18). After AIT, objS occurred in 24% and 47%, respectively. LOAEL group differences showed a beneficial tendency (P = 0.081) for LOAELobjective in PPP (30 active, 15 placebo). sIgG4 raised only in active group (Bet v 1: P = 0.054, Gly m 4: P = 0.037), and no relevant changes occurred for sIgE. Only 56% of the intended sample size was recruited. CONCLUSION: For the first time, we present data on the effect of rBet v 1-FV on birch-related soya allergy. rBet v 1-FV AIT induced significant immunogenic effects. Clinical assessment showed a tendency in favour of the active group but did not reach statistical significance.

Standardization of double blind placebo controlled food challenge with soy within a multicentre trial.

BACKGROUND: Multicentre trials investigating food allergies by double blind placebo controlled food challenges (DBPCFC) need standardized procedures, challenge meals and evaluation criteria. We aimed at developing a standardized approach for identifying patients with birch related soy allergy by means of DBPCFC to soy, including determination of threshold levels, in a multicentre setting. METHODS: Microbiologically stable soy challenge meals were composed of protein isolate with consistent Gly m 4 levels. Patients sensitized to main birch allergen Bet v 1 and concomitant sensitization to its soy homologue Gly m 4 underwent DBPCFC. Outcome was defined according to presence and/or absence of ten objective signs and intensity of eight subjective symptoms as measured by visual analogue scale (VAS). RESULTS: 138 adult subjects (63.8% female, mean age 38 years) underwent DBPCFC. Challenge meals and defined evaluation criteria showed good applicability in all centres involved. 45.7% presented with objective signs and 65.2% with subjective symptoms at soy challenge. Placebo challenge meals elicited non-cardiovascular objective signs in 11.6%. In 82 (59.4%) subjects DBPCFC was judged as positive. 70.7% of DPBCFC+ showed objective signs and 85.4% subjective symptoms at soy challenge. Subjective symptoms to soy challenge meal in DBPCFC+ subjects started at significantly lower dose levels than objective signs (p < 0.001). Median cumulative eliciting doses for first objective signs in DBPCFC+ subjects were 4.7 g [0.7-24.7] and 0.7 g [0.2-4.7] total soy protein for first subjective symptoms (p = 0.01). CONCLUSIONS: We present the hitherto largest group of adults with Bet v 1 and Gly m 4 sensitization being investigated by DBPCFC. In this type of food allergy evaluation of DBPCFC outcome should not only include monitoring of objective signs but also scoring of subjective symptoms. Our data may contribute to standardize DBPCFC in pollen-related food allergy in multicentre settings. TRIAL REGISTRATION: EudraCT: 2009-011737-27.

The conformational IgE epitope profile of soya bean allergen Gly m 4.

BACKGROUND: Birch pollen-related soya allergy is mediated by Gly m 4. Conformational IgE epitopes of Gly m 4 are unknown. OBJECTIVE: To identify the IgE epitope profile of Gly m 4 in subjects with birch pollen-related soya allergy utilizing an epitope library presented by Gly m 4-type model proteins. METHODS: Sera from patients with (n = 26) and without (n = 19) allergy to soya as determined by oral provocation tests were studied. Specific IgE (Bet v 1/Gly m 4) was determined by ImmunoCAP. A library of 59 non-allergenic Gly m 4-type model proteins harbouring individual and multiple putative epitopes for IgE was tested in IgE binding assays. Primary, secondary and tertiary protein structures were assessed by mass spectrometry, circular dichroism and nuclear magnetic resonance spectroscopy. RESULTS: All subjects were sensitized to Gly m 4 and Bet v 1. Allergen-specific serum IgE levels ranged from 0.94 to > 100 kUA /L. The avidities of serum IgE were 5.06 ng (allergic) and 1.8 ng (tolerant) as determined by EC50 for IgE binding to Gly m 4. 96% (46/48) of the protein variants bound IgE. Model proteins had Gly m 4-type conformation and individual IgE binding clustered in six major surface areas. Gly m 4-specific IgE binding could be inhibited to up to 80% by model proteins harbouring individual IgE binding sites in an epitope-wise equimolar fashion. Receiver operating curve analysis revealed an area under fitted curve of up to 0.88 for model proteins and 0.66 for Gly m 4. CONCLUSION AND CLINICAL RELEVANCE: Serum levels and avidity of Gly m 4-specific IgE do not correlate with clinical reactivity to soya. Six IgE-binding areas, represented by 23 amino acids, account for more than 80% of total IgE binding capacity of Gly m 4. Model proteins may be used for epitope-resolved diagnosis to differentiate birch-soya allergy from clinical tolerance.

Standardization of allergen products: 3. Validation of candidate European Pharmacopoeia standard methods for quantification of major birch allergen Bet v 1.

BACKGROUND: The BSP090 project aims at establishing European Pharmacopoeia Reference Substances in combination with the corresponding ELISA methods for the quantification of major allergens in allergen products. Two sandwich ELISAs proved suitable for quantification of Bet v 1, the major birch pollen allergen, in preceding phases of BSP090. METHODS: Two Bet v 1-specific ELISA systems were compared with respect to accuracy and precision in a ring trial including 13 laboratories. Model samples containing recombinant rBet v 1.0101 as well as native birch pollen extracts were measured independently at least three times in each facility. The assessment was completed with a comparative quantification of Bet v 1 in 30 marketed birch allergen products in one laboratory, simulating the future use as reference method. RESULTS: In the collaborative study, both candidate ELISAs confirmed their suitability to quantify recombinant and native Bet v 1. ELISA-A showed higher precision and lower interlaboratory variability, yet ELISA-B exhibited slightly higher accuracy. Subsequent parallel measurement of Bet v 1 in a panel of 'real-life' birch allergen products indicated better repeatability of ELISA-B. Both systems detected substantial differences in Bet v 1 content between allergen products, but the effect was more pronounced using ELISA-B due to persistently higher values compared to ELISA-A. CONCLUSIONS: In the collaborative study, no deciding differences were observed between the two candidate ELISAs. Further comparison under conditions simulating the intended use combined with the criterion of long-term availability enabled the selection of one Bet v 1-specific ELISA for proposal as European Pharmacopoeia standard method.

EAACI Food Allergy and Anaphylaxis Guidelines. Protecting consumers with food allergies: understanding food consumption, meeting regulations and identifying unmet needs.

Individuals suffering from IgE-mediated food allergy usually have to practise life-long food allergen avoidance. This document aims to provide an overview of recent evidence-based recommendations for allergen risk assessment and management in the food industry and discusses unmet needs and expectations of the food allergic consumer in that context. There is a general duty of care on the food industry and obligations in European Union legislation to reduce and manage the presence of allergens alongside other food hazards. Current evidence enables quantification of allergen reference doses used to set-up reliable food safety management plans for some foods. However, further work is required to include a wider variety of foods and to understand the impact of the food matrix as well as additional factors which affect the progression and severity of symptoms as a function of dose. Major concerns have been raised by patients, carers and patient groups about the use of precautionary 'may contain' labelling to address the issue of unintended presence of allergens; these therefore need to be reconsidered. New and improved allergen detection methods should be evaluated for their application in food production. There is an urgent requirement for effective communication between healthcare professionals, patient organizations, food industry representatives and regulators to develop a better approach to protecting consumers with food allergies.

Component-resolved in vitro diagnosis of carrot allergy in three different regions of Europe.

BACKGROUND: Carrot is a frequent cause of food allergy in Europe. The objective of this study was to evaluate a panel of carrot allergens for diagnosis of carrot allergy in Spain, Switzerland and Denmark. METHODS: Forty-nine carrot allergic patients, 71 pollen allergic but carrot-tolerant patients and 63 nonatopic controls were included. Serum IgE to carrot extract, recombinant carrot allergens (rDau c 1.0104; rDau c 1.0201; rDau c 4; the isoflavone reductase-like proteins rDau c IFR 1, rDau c IFR 2; the carrot cyclophilin rDau c Cyc) were analyzed by ImmunoCAP. RESULTS: The sensitivity of the carrot extract-based test was 82%. Use of the recombinant allergens increased the sensitivity to 90%. The Dau c 1 isoforms were major allergens for Swiss and Danish carrot allergic patients, the profilin rDau c 4 for the Spanish patients. The rDau c IFR 1 and rDau c IFR 2 were recognized by 6% and 20% of the carrot allergics, but did not contribute to a further increase of sensitivity. Among pollen allergic controls, 34% had IgE to carrot extract, 18% to each of rDau c 1.0104, rDau c 1.0201 and rDau c 4, 8% to rDau c IFR 1 and 7% to rDau c IFR 2. Sensitization to rDau c Cyc occurred in one carrot allergic patient and one nonatopic control. CONCLUSION: Component-resolved in vitro analyses revealed a significant difference in IgE sensitization pattern between geographical regions and in the prevalence of sensitization to carrot components between carrot allergic and carrot-tolerant but pollen sensitized patients.

Thresholds for food allergens and their value to different stakeholders.

Thresholds constitute a critical piece of information in assessing the risk from allergenic foods at both the individual and population levels. Knowledge of the minimum dose that can elicit a reaction is of great interest to all food allergy stakeholders. For allergic individuals and health professionals, individual threshold data can inform allergy management. Population thresholds can help both the food industry and regulatory authorities assess the public health risk and design appropriate food safety objectives to guide risk management. Considerable experience has been gained with the double-blind placebo-controlled food challenge (DBPCFC), but only recently has the technique been adapted to provide data on thresholds. Available data thus vary greatly in quality, with relatively few studies providing the best quality individual data, using the low-dose DBPCFC. Such high quality individual data also form the foundation for population thresholds, but these also require, in addition to an adequate sample size, a good characterization of the tested population in relation to the whole allergic population. Determination of thresholds at both an individual level and at a population level is influenced by many factors. This review describes a low-dose challenge protocol developed as part of the European Community-funded Integrated Project Europrevall, and strongly recommends its wider use so that data are generated that can readily increase the power of existing studies.

Strategy for allergenicity assessment of 'natural novel foods': clinical and molecular investigation of exotic vegetables (water spinach, hyacinth bean and Ethiopian eggplant).

BACKGROUND: Foods not commonly consumed in the European Union must be proven safe before being brought to market, including an assessment of allergenicity. We present a three-stepwise strategy for allergenicity assessment of natural novel foods using three novel vegetables, namely, water spinach, hyacinth bean, Ethiopian eggplant. METHODS: First, vegetable extracts were analyzed for the presence of pan-allergens [Bet v 1 homologous proteins, profilins, nonspecific lipid transfer proteins (LTP)] by immunoblot analysis with specific animal antibodies. Secondly, the IgE-binding of the food extracts was investigated by EAST (Enzyme-allergosorbent test) and immunoblot analysis using sera with IgE-reactivity to known pan-allergens or to phylogenetically related foods from subjects (i) allergic to birch, grass and mugwort pollen, (ii) with food allergy to soy, peanut, tomato, multiple pollen-related foods and (iii) sensitized to LTP. Thirdly, the clinical relevance of IgE-binding was assessed in vivo by skin prick testing (SPT) and open oral food challenges (OFC). RESULTS: Profilin and LTP were detected by animal antibodies in all vegetables, a Bet v 1 homologue selectively in hyacinth bean. IgE-binding to LTP, profilin and a Bet v 1 homologue was proven by immunoblot analysis and EAST. Positive SPT and OFC results were observed for all vegetables in pollen-allergic patients. CONCLUSIONS: Our stepwise procedure confirmed the presence and IgE-binding capacity of novel vegetable proteins homologous to known allergens in endemic vegetable foods. In vivo testing proved the potential of the novel vegetables to elicit clinical allergy. Hence, our described algorithm seems to be applicable for allergenicity testing of natural novel foods.

Quantitative sandwich ELISA for determination of traces of hazelnut (Corylus avellana) protein in complex food matrixes.

A hazelnut-specific sandwich-type ELISA based on polyclonal antisera was developed for detection of hidden hazelnut protein residues in complex food matrixes. In the absence of a food matrix, extractable protein from different native and toasted hazelnuts was detected at rates of 94 +/- 13 and 96 +/- 7% applying standards prepared from native and toasted hazelnuts, respectively. From complex food matrixes, 0.001-10% of hazelnut was recovered between 67 and 132%, in average by 106 +/- 17%. Depending on the food matrix, hazelnut protein could be detected down to the ppb (ng/g) level. Intraassay precision was <6% for hazelnut >/= 0.001% and interassay precision was <15% for hazelnut >/= 0.01%. In 12 of 28 commercial food products without labeling or declaration of hazelnut components, between 2 and 421 ppm of hazelnut protein was detected, demonstrating a remarkable presence of potentially allergenic hazelnut protein "hidden" in commercial food products.

Indirect competitive ELISA for determination of traces of peanut (Arachis hypogaea L.) protein in complex food matrices.

An indirect competitive ELISA was developed allowing the detection of hidden peanut protein residues down to 2 ppm (micorgrams per gram) in various foods. The high-titer, peanut-specific polyclonal antiserum used recognized potentially allergenic proteins in both native and roasted peanuts. In the absence of a food matrix, extractable protein from roasted peanuts was detected at 104 +/- 13%. From various food items, peanut protein at > or =13 ppm was recovered between 84 and 126%, and at 2 ppm of peanut protein recovery was 143 +/- 6%. Intra- and interassay precision was <15%. In 5 of 17 commercial food products without declaration of peanut components, between 2 and 18 ppm of peanut protein was detected. This is the first assay based on commercially available reactants that allows the reliable determination of trace amounts of hidden peanut allergens in a variety of complex food matrices.

Factors influencing the quality of food extracts for in vitro and in vivo diagnosis.

Food extracts for diagnostic purposes often lack sufficient activity and consistency. Biologically standardized food extracts are not available on the market. Using extracts from plant-derived foods as examples, we investigated factors which may be important for the quality of such extracts. Divergent allergenic activities were found between strains of apples, but not within varieties of celery tuber (celeriac), hazelnut, and peanut, respectively. Heating of the food remarkably reduced the activity of apple, hazelnut, and celeriac, but had little effect on peanut. By contrast, heating of semipurified protein extracts from celery tuber and apple for 30 min at 100 degrees C did not deplete the immunoreactivity of the major allergens, indicating that this is an inappropriate test for identifying labile food allergens. Due to their high endogenous enzyme activities, apples and other fruits require special extraction procedures applying either low temperature or enzyme inhibitors. Variation of extraction conditions had little effect on the composition and activity of extracts from hazelnut. The storage stability of skin test solutions from plant foods can be improved by avoiding phenol as an additive and by including 50% of glycerol. For model studies considering neoallergens, IgE was raised in mice against native and heated celery tuber, respectively. When extracts from nonthermally and thermally processed celeriac were subjected to an RBL-cell mediator release assay with these sera, an inverse ranking was obtained with anti-heated celeriac IgE and anti-native celeriac IgE, respectively. These data indicated that new epitopes had been formed by the heating process. Since all parameters were tested in model experiments with either human or murine IgE, their relevance has to be proven in further clinical investigations.