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Background Most studies of indoor allergens have focused on the home environment. However, schools may be an important site of allergen exposure for children with asthma. We compared school allergen exposure to home exposure in a cohort of children with asthma. Correlations between settled dust and airborne allergen levels in classrooms were examined.

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.

Allergy diagnostics is being transformed by the advent of in vitro IgE testing using purified allergen molecules, combined with multiplex technology and biosensors, to deliver discriminating, sensitive, and high-throughput molecular diagnostics at the point of care. Essential elements of IgE molecular diagnostics are purified natural or recombinant allergens with defined purity and IgE reactivity, planar or bead-based multiplex systems to enable IgE to multiple allergens to be measured simultaneously, and, most recently, nanotechnology-based biosensors that facilitate rapid reaction rates and delivery of test results via mobile devices. Molecular diagnostics relies on measurement of IgE to purified allergens, the "active ingredients" of allergenic extracts. Typically, this involves measuring IgE to multiple allergens which is facilitated by multiplex technology and biosensors. The technology differentiates between clinically significant cross-reactive allergens (which could not be deduced by conventional IgE assays using allergenic extracts) and provides better diagnostic outcomes. Purified allergens are manufactured under good laboratory practice and validated using protein chemistry, mass spectrometry, and IgE antibody binding. Recently, multiple allergens (from dog) were expressed as a single molecule with high diagnostic efficacy. Challenges faced by molecular allergy diagnostic companies include generation of large panels of purified allergens with known diagnostic efficacy, access to flexible and robust array or sensor technology, and, importantly, access to well-defined serum panels form allergic patients for product development and validation. Innovations in IgE molecular diagnostics are rapidly being brought to market and will strengthen allergy testing at the point of care.

Allergy diagnostics is being transformed by the advent of in vitro IgE testing using purified allergen molecules, combined with multiplex technology and biosensors, to deliver discriminating, sensitive, and high-throughput molecular diagnostics at the point of care. Essential elements of IgE molecular diagnostics are purified natural or recombinant allergens with defined purity and IgE reactivity, planar or bead-based multiplex systems to enable IgE to multiple allergens to be measured simultaneously, and, most recently, nanotechnology-based biosensors that facilitate rapid reaction rates and delivery of test results via mobile devices. Molecular diagnostics relies on measurement of IgE to purified allergens, the “active ingredients” of allergenic extracts. Typically, this involves measuring IgE to multiple allergens which is facilitated by multiplex technology and biosensors. The technology differentiates between clinically significant cross-reactive allergens (which could not be deduced by conventional IgE assays using allergenic extracts) and provides better diagnostic outcomes. Purified allergens are manufactured under good laboratory practice and validated using protein chemistry, mass spectrometry, and IgE antibody binding. Recently, multiple allergens (from dog) were expressed as a single molecule with high diagnostic efficacy. Challenges faced by molecular allergy diagnostic companies include generation of large panels of purified allergens with known diagnostic efficacy, access to flexible and robust array or sensor technology, and, importantly, access to well-defined serum panels form allergic patients for product development and validation. Innovations in IgE molecular diagnostics are rapidly being brought to market and will strengthen allergy testing at the point of care.

To the Editor: Allergen measurements require well-defined allergen standards. Allergists rely on these measurements for dosing patients on immunotherapy with the aim of achieving maintenance doses of 5 to 20 µg of specific allergen that have been associated with clinical efficacy.1 Allergists need to know that allergen measurements made by manufacturers are consistent and can reliably be used in clinical practice. Allergen measurements are widely used in the indoor air quality industry to assess exposure in homes, the workplace, schools, and commercial buildings. They are routinely used for assessing health risks associated with allergen exposure, for assessing the efficacy of allergen avoidance procedures, and for developing new allergen control products.2 Measurements of allergens by ELISA rely on standards of known allergen concentration, but few national or international allergen standards exist. The World Health Organization/International Union of Immunological Societies (WHO/IUIS) Allergen Standardization Committee initiated a program to develop purified allergen standards for calibration of in vitro measurements. This initiative was funded by the European Union to develop certified reference materials for allergenic products (“Development of Certified Reference Materials for Allergenic Products and Validation of Methods for their Quantification,” acronym CREATE). The aims of CREATE were to develop international reference materials with verifiable allergen content.3,4 Our goal was to apply the principles of allergen standardization developed in CREATE to other purified allergens. We developed a single “universal” allergen standard (UAS) for use in ELISA and in a fluorescent multiplex array for indoor allergens.5 Purified proteins are essential in multiplex systems to reduce nonspecific interactions. Eight purified allergens (Der p 1, Der f 1, Der p 2, Fel d 1, Can f 1, Rat n 1, Mus m 1, and Bla g 2) were combined in the UAS. The protein concentration of the purified allergens was determined by amino-acid analysis, in keeping with CREATE. A detailed validation of the UAS and comparison with previous ELISA standards will be published elsewhere.6 Here, we report the concentration of specific allergens in WHO/IUIS and US Food and Drug Administration (FDA) reference preparations using the single multiallergen standard. Specific allergen concentrations of WHO/IUIS and FDA reference preparations were determined by using ELISA (Table I). The WHO/IUIS Dermatophagoides pteronyssinus standard 82/518 has been widely used as a standard for measurements of allergen exposure with a reported concentration of 12.5 µg Der p 1 per ampoule.7 A value of 7.2 µg Der p 1 per ampoule was obtained by using the UAS, which is similar to estimates of 5 µg Der p 1 per ampoule reported previously.8,9 The WHO/IUIS dog hair standard has an assigned potency of 100,000 IUs per ampoule and contained 20.4 µg Can f 1 per ampoule as determined by using the UAS. TABLE I Allergen concentration of reference standards measured by ELISA using the UAS There are no published reports of the specific allergen concentrations of US FDA reference materials. The 10,000 allergy units/mL D pteronyssinus references had similar concentrations of Der p 1 and Der p 2 in both the E8 and E10 standards (range, 20.3–27.6 µg/mL) (Table I). The 10,000 allergy units/mL D farinae references (E9 and E10) contained comparable levels of Der f 1 to the levels of Der p 1 seen in the D pteronyssinus references. It was not possible to use the Der p 2 standard in the UAS for the calibration of Der f 2 in D farinae references. The Der p 2 standard in the UAS underestimated the amount of Der f 2 by ~8-fold (data not shown). The Der f 2 concentration of 10,000 allergy units/mL D farinae references was determined by using a purified in-house Der f 2 standard (Table I). The Der f 2 concentrations of the E9 and E10 D farinae references were very similar. It was reassuring that the allergen levels in E8 and E9 (previous mite standards) were broadly similar to those of the current E10 standard. Overall, Group 1 and Group 2 allergen levels were comparable, and in some cases almost identical, in the FDA references for both mite species. The FDA assigns potency to cat allergenic products based on the Fel d 1 content determined by radial immunodiffusion. Allergenic products containing 10 to 19.9 FDA units/mL of Fel d 1 have an assigned potency of 10,000 bioequivalent allergy units/mL.10 The Fel d 1 values in units of 2 of the FDA references (E4 and E5 hair) were outside of the recommended range. The Fel d 1 concentrations in micrograms per milliliter of the FDA cat references (E4 and E5) determined by ELISA using the UAS were consistent with the results obtained by radial immunodiffusion (Table I). Our data confirm that the Der p 1 level of 12.5 µg per ampoule assigned to the WHO/IUIS D pteronyssinus standard (National Institute for Biological Standards and Control 82/518) is an overestimate and that the actual value is in the range of 5 to 7 µg per ampoule.8,9 The data show that 1 unit Can f 1 in the WHO/IUIS dog hair extract (National Institute for Biological Standards and Control 84/685) corresponds to 5 ng Can f 1 and that 1 FDA unit Fel d 1 is ~1 µg Fel d 1. Values obtained by using previous standards have been applied to many studies involving thresholds or guidelines for exposure levels that result in allergic sensitization or symptom exacerbations.6 At this point, we do not believe that revision of such guidelines in the absence of further epidemiologic data would be worthwhile. Rather, future studies should incorporate purified allergen standards into the exposure assessment to generate a body of data based on standards that fulfill the CREATE principles. Dosing of immunotherapy based on measurements of specific allergens plays an increasing role in allergy practice.1 Although several US allergen manufacturers routinely measure specific allergens in their immunotherapy products, these measurements are not based on a national standard and may not be directly comparable. Our data provide the specific allergen concentrations in mite and cat allergen reference preparations used by the FDA for potency testing of all US standardized allergenic products. This provides a mechanism for standardizing allergen measurements in US products that allergists use for immunotherapy. The results suggest that the approach of using multiallergen standards could be extended to other sources, for example, pollens, molds, and foods, where purified allergens are available. It is critically important that regulatory agencies generate purified natural or recombinant allergen standards that researchers and companies can use as reference preparations. Two of the allergens used in CREATE, Bet v 1 and Phl p 5, are being formulated as biological reference preparations by the European Directorate for the Quality of Medicines.4 ELISA tests for each allergen are also being validated to produce certified ELISA products that can be used for analytical purposes. Once completed, the biological reference preparations will be incorporated into the European Pharmacopoeia and will become international standards for licensing of allergenic products. Our data demonstrate the feasibility of applying CREATE principles to other allergens and underline the need for validated purified allergens and assays to be developed and maintained by standardization agencies and regulatory authorities.