Simultaneous quantification of specific food allergen proteins using a fluorescent multiplex array.

The aim was to develop a fluorescent multiplex array for simultaneously measuring regulated food allergens using specific allergen protein molecules from peanut, tree nut, cow's milk, egg, soy, fish, shellfish, sesame, mustard and celery. Microspheres coupled to specific monoclonal antibodies were used for allergen detection, with purified allergens as reference standards.Standard curves for 17 allergens covered a 5-log dynamic range. Intra- and inter-assay acceptance criteria were within 70-130% recovery and a CV of ≤15%. Food reference materials contained high levels of their respective major allergens (2000-175,000 µg/g), Similar high allergen levels were found in 10 selected foods analysed using a 9-plex array. Egg, milk, peanut, hazelnut and walnut allergens were detectable in chocolate bars with incurred allergens at 3, 10, 30, and 100 ppm. The multiplex array is an efficient tool for measuring specific food allergens, with applications for risk assessment and standardization of therapeutic products for food allergy.

Doses of Specific Allergens in Early Introduction Foods for Prevention of Food Allergy.

BACKGROUND: Consumption of common allergenic foods, such as peanut, in early life can reduce the risk of food allergy among high-risk children and is recommended in revised clinical guidelines. Commercial early allergen introduction foods (EIF) containing single or multiple allergenic foods for feeding infants are promoted to consumers and health care providers as aids to prevent food allergy. OBJECTIVE: To determine the concentration and doses of major food allergens in EIF. METHODS: Extracts from 32 EIF and 4 control foods were analyzed for 17 allergens: Ara h 1, Ara h 3, Ara h 6, Bos d 5, Bos d 11, Gal d 1, Gal d 2, Ana o 3, Cor a 9, Jug r 1, Gly m 5, Ses i 1, Api g 1, Sin a 1, Cyp c 1, shrimp tropomyosin, and Tri a 19 using a validated fluorescent multiplex array. Ara h 2 was measured by enzyme-linked immunosorbent assay. RESULTS: The EIF comprised 1-8 samples of 32 foods (n = 86). Combined peanut allergen levels of up to 26,000 µg/g were measured in peanut puffs (doses of 65-182 mg per 7 g serving). Peanut allergens were not detected in mixed food blend puffs. Major allergen levels of >10,000 µg/g were found in several milk, egg, and peanut powders, or combinations thereof, with cumulative allergen doses of 159-2946 mg in the EIF. Mixed food blend powders, puffs crackers, and fruit sauces contained much lower allergen levels, often <10 µg/g, and some had undetectable allergens. The allergen concentration in these EIF varied over a >3 log range and provided lower cumulative doses of allergen. CONCLUSIONS: Significant variability in allergen composition, concentration, and dose per serving were observed in EIF containing the same foods. The doses of allergens consumed by potentially at-risk infants in early life were EIF dependent. Guidelines should be established to enable consumers and health care providers to make informed decisions about EIF and to improve the formulation and standardization of EIF for prevention of food allergy.

Detection of Food Allergens in School and Home Environments of Elementary Students.

BACKGROUND: Little is known about environmental food allergen exposure on school surfaces. OBJECTIVE: To compare the distribution of major food allergens in floor dust and table wipe samples from elementary schools and dust samples from students' homes. METHODS: In this substudy of the School Inner-City Asthma Study-II, 103 table wipe samples and 98 floor dust samples from cafeterias and classrooms in 18 elementary schools were analyzed for milk, peanut, cashew, hazelnut, and egg using a multiplex array. Home kitchen floor and bed dust samples from 90 students were also analyzed. RESULTS: Food allergens were detectable in schools, but at significantly lower levels than in homes (P < .001). In schools, milk and peanut were detected in all table wipe samples; milk and egg were detected in all floor dust samples. Cafeteria table wipe samples contained significantly higher levels of milk, peanut, hazelnut, and egg, compared with classrooms. Cafeteria floor dust samples contained higher levels milk than classrooms. Peanut-restrictive policies did not consistently reduce environmental peanut exposure in schools. Peanut allergen was lower in dust from homes of students with peanut allergy (n = 5) compared with those without peanut allergy (n = 85) (P < .001). Reassuringly, peanut allergen in the schools of peanut-allergic students was not significantly different than in their homes. CONCLUSION: Food allergens were readily detectable on tables and floors in elementary schools, but at levels lower than in students' homes. For peanut-allergic students, the levels of detectable peanut in their schools were not higher than their homes. The low levels of detectable food allergens in school environments are unlikely to result in severe allergic reactions.

Variability in German Cockroach Extract Composition Greatly Impacts T Cell Potency in Cockroach-Allergic Donors.

German cockroach extract is used clinically to evaluate allergen-specific sensitization and for subcutaneous allergen-specific immunotherapy, though there are no guidelines for standardization in its manufacture. We performed an immunological evaluation of 12 different cockroach extracts prepared from different sources and their potency to induce allergen-specific T cell reactivity. PBMC from 13 cockroach allergic donors were expanded in vitro with 12 different German cockroach extracts. After culture expansion, cells were re-stimulated with the different extracts and T cell responses were assessed by FluoroSpot (IL-5, IFNγ and IL-10 production). In parallel to the extracts, single allergen peptide pools for allergens from groups 1, 2, 4, 5, and 11 were tested to determine allergen immunodominance. Furthermore, to assess allergy specificity, PBMC from 13 non-allergic donors were also tested with the most potent extract and T cell responses were compared to the allergic cohort. Dramatic variations in T cell reactivity were observed to the different cockroach extract batches. Response magnitudes varied over 3 logs within a single donor. IL-5 production in the allergic cohort was significantly higher compared to the non-allergic cohort (p=0.004). Allergen content determination by ELISA detected much lower concentrations of Bla g 5 compared to Bla g 1 and 2. Mass spectrometric analysis revealed that Bla g 5 was present in similar amounts to Bla g 1 and 2 in extracts made from whole body, whereas it was not detected in extracts made from fecal matter, suggesting that Bla g 5 is not excreted into feces. Different donors exhibit different response patterns to different extracts, potentially dependent on the donor-specific T cell allergen immunodominance pattern and the allergen content of the extract tested. These findings have dramatic implications for the selection of potent extracts used for diagnostic purposes or allergen-specific immunotherapy.

Allergen content in German cockroach extracts and sensitization profiles to a new expanded set of cockroach allergens determine in vitro extract potency for IgE reactivity.

BACKGROUND: Cockroach allergens are an important cause of IgE-mediated sensitization in inner-city asthmatic patients. However, cockroach extracts used for diagnosis and immunotherapy are not standardized. OBJECTIVE: We sought to determine the allergen content of nonstandardized German cockroach extracts and the levels of sensitization to an expanded set of cockroach allergens as determinants of in vitro extract potency for IgE reactivity. METHODS: Twelve German cockroach extracts were compared for allergen content and potency of IgE reactivity. Bla g 1, Bla g 2, and Bla g 5 were measured by using immunoassays. IgE antibody levels to 8 purified recombinant allergens from groups 1, 2, 4, 5, 6, 7, 9, and 11 were measured by using ImmunoCAP. IgE antibody binding inhibition assays were performed to assess extract in vitro potencies (concentration inhibiting 30% of the total IgE antibody-binding inhibition) relative to an arbitrarily selected reference extract in 5 patients with cockroach allergy. RESULTS: Allergen levels were highly variable. Three new major allergens (groups 6, 9, and 11), were identified among highly cockroach-sensitized subjects (CAP class ≥ 3). Sensitization profiles were unique per subject without immunodominant allergens. The sum of IgE to 8 allergen components showed a good correlation with cockroach-specific IgE levels (r = 0.88, P < .001). In vitro potencies varied among different extracts per subject and among subjects for each extract. CONCLUSIONS: The in vitro potency of German cockroach extracts for IgE reactivity depends on allergen content and allergen-specific IgE titers of patients with cockroach allergy. These factors are relevant for selection of potent extracts to be used for immunotherapy and for the design and interpretation of data from immunotherapy trials.

Specific allergen profiles of peanut foods and diagnostic or therapeutic allergenic products.

BACKGROUND: Generic immunoassays for peanut cannot discriminate between allergen levels in peanut-derived food products or therapeutics. Clinical trials of oral immunotherapy (OIT) are strengthened by using standardized peanut preparations with defined doses of major allergens. OBJECTIVE: This article describes measurement of Ara h 1, Ara h 2, and Ara h 6 in peanut foods and in peanut flour extracts used for allergy diagnosis and OIT. METHODS: Monoclonal antibody-based enzyme immunoassays for Ara h 1, Ara h 2, and Ara h 6 were used to compare allergen levels in peanut (n = 16) and tree nut (n = 16) butter, peanut flour (n = 11), oils (n = 8), extracts used for diagnosis and OIT (n = 5), and the National Institute for Standards and Technology Peanut Butter Standard Reference Material 2387. RESULTS: Roasted peanut butters contained 991 to 21,406 µg/g Ara h 1 and exceeded Ara h 2 and Ara h 6 levels by 2- to 4-fold. Similarly, National Institute for Standards and Technology Peanut Butter Standard Reference Material 2387 contained 11,275 µg/g Ara h 1, 2,522 µg/g Ara h 2, and 2,036 µg/g Ara h 6. In contrast, peanut flours contained 787 to 14,631 µg/g Ara h 2 and exceeded Ara h 1 levels by 2- to 20-fold. Flour extracts used for OIT contained 394 to 505 µg/mL Ara h 1, 1,187 to 5,270 µg/mL Ara h 2, and 1,104 to 8,092 µg/mL Ara h 6. In most cases specific peanut allergens were not detected in tree nut butters or peanut oils. CONCLUSIONS: The results show marked differences in specific peanut allergen profiles in peanut butter and flour and peanut preparations for clinical use. Roasting can increase Ara h 1 levels in peanut butter. Variability in allergen levels could affect the outcome of clinical trials of peanut OIT, especially with respect to Ara h 1. Specific allergen measurements will improve standardization and provide accurate dosing of peanut preparations that are being used for OIT.

A multi-center ring trial of allergen analysis using fluorescent multiplex array technology.

BACKGROUND: Consistent performance of allergen assays is essential to ensure reproducibility of exposure assessments for investigations of asthma and occupational allergic disease. This study evaluated intra- and inter-laboratory reproducibility of a fluorescent multiplex array, which simultaneously measures eight indoor allergens in a single reaction well. METHODS: A multi-center study was performed in nine laboratories in the US and Europe to determine the inter-laboratory variability of an 8-plex array for dust mite, cat, dog, rat, mouse and cockroach allergens. Aliquots of 151 dust extract samples were sent to participating centers and analyzed by each laboratory on three separate occasions. Agreement within and between laboratories was calculated by the concordance correlation coefficient (CCC). RESULTS: Results were obtained for over 32,000 individual allergen measurements. Levels covered a wide range for all allergens from below the lower limit of detection (LLOD = 0.1-9.8 ng/ml) to higher than 6800 ng/ml for all allergens except Mus m 1, which was up to 1700 ng/ml. Results were reproducible within as well as between laboratories. Within laboratories, 94% of CCC were ≥ 0.90, and 80% of intra-laboratory results fell within a 10% coefficient of variance (CV%). Results between laboratories also showed highly significant positive correlations for all allergens (~0.95, p<0.001). Overall means of results were comparable, and inter-laboratory CV% for all allergens except Rat n 1 ranged between 17.6% and 26.6%. CONCLUSION: The data indicate that performance criteria for fluorescent multiplex array technology are reproducible within and between laboratories. Multiplex technology provides standardized and consistent allergen measurements that will streamline environmental exposure assessments in allergic disease.