Since the 1990s, a substantial increase has occurred in the incidence of food allergy, particularly in early childhood.1 Although some vaccines contain potential allergens,2,3 social media and Internet Web sites have promulgated the concern that vaccines contain food proteins, which then induce sensitization. Parental unease regarding the immunologic impact of vaccines appears to be quite common as 72.5% of pediatricians report that parents are concerned that the number of vaccines recommended stress their child's immune system and may be the cause of various diseases.

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 mu g/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 mu g/g Ara h 1, 2,522 mu g/g Ara h 2, and 2,036 mu g/g Ara h 6. In contrast, peanut flours contained 787 to 14,631 mu g/g Ara h 2 and exceeded Ara h 1 levels by 2- to 20-fold. Flour extracts used for OIT contained 394 to 505 mu g/mL Ara h 1, 1,187 to 5,270 mu g/mL Ara h 2, and 1,104 to 8,092 mu g/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.

RATIONALE: Methods currently used for the detection of total as well as allergen-specific IgE are labor intensive, time consuming and require large sample volumes. Our aim was to develop a fluorescent multiplex array to allow simultaneous detection of total and allergen-specific IgE in serum in a single quantitative test. METHODS: Purified allergens or mAb were covalently coupled to fluorescent microspheres to develop a suspension array for detection of total IgE and IgE specific to Der p 1, Der p 2, Fel d 1, Can f 1, Bet v 1 and Phl p 5. The array was validated by comparing total and allergen-specific IgE levels in sera from allergic patients using ELISA and ImmunoCAP. RESULTS: Comparison of total IgE levels in 63 sera between fluorescent array and ImmunoCAP showed an excellent correlation (r = 0.96, range: <20 - >5000 IU/ml, p<0.001). Allergen-specific IgE levels also correlated very well with ELISA results (r = 0.81 - 0.95, p<0.001). Assay sensitivity was <0.5 IU/ml and inter-assay CVs ranged between 6 - 20%. The required sample volume for a 7-plex assay was less than 20μl per sample. CONCLUSIONS: The multiplex array is a high throughput assay platform that allows simultaneous quantification of allergen-specific and total IgE, while using only a fraction of the serum required in other methods. This highly reduced sample volume requirement will be particularly valuable for pediatric studies. Our results suggest that fluorescent multiplex technology will facilitate clinical and epidemiological studies of allergic sensitization.

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.

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.1Nelson H.S. Allergen immunotherapy: where is it now?.J Allergy Clin Immunol. 2007; 119: 769-779Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar 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.2Platts-Mills T.A. Vervloet D. Thomas W.R. Aalberse R.C. Chapman M.D. Indoor allergens and asthma: report of the Third International Workshop.J Allergy Clin Immunol. 1997; 100: S2-24Abstract Full Text Full Text PDF PubMed Scopus (668) Google Scholar 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.3van R.R. Chapman M.D. Ferreira F. Vieths S. Bryan D. Cromwell O. et al.The CREATE project: development of certified reference materials for allergenic products and validation of methods for their quantification.Allergy. 2008; 63: 310-326Crossref PubMed Scopus (175) Google Scholar, 4Chapman M.D. Ferreira F. Villalba M. Cromwell O. Bryan D. Becker W.M. et al.The European Union CREATE project: a model for international standardization of allergy diagnostics and vaccines.J Allergy Clin Immunol. 2008; 122: 882-889Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar 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.5Earle C.D. King E.M. Tsay A. Pittman K. Saric B. Vailes L. et al.High-throughput fluorescent multiplex array for indoor allergen exposure assessment.J Allergy Clin Immunol. 2007; 119: 428-433Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar 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.6Filep S. Tsay A. Vailes L.D. Gadermaier G. Ferreira F. Matsui E.C. et al.A multi-allergen standard for immunoassays: CREATE principles applied to eight purified allergens.Allergy. 2011 Nov 18; ([Epub ahead of print])https://doi.org/10.1111/j.1398-9995.2011.02750.xCrossref PubMed Scopus (44) Google Scholar 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.7Ford A.W. Rawle F.C. Lind P. Spieksma F.T. Lowenstein H. Platts-Mills T.A. Standardization of Dermatophagoides pteronyssinus: assessment of potency and allergen content in ten coded extracts.Int Arch Allergy Appl Immunol. 1985; 76: 58-67Crossref PubMed Google Scholar 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.8Yasueda H. Saito A. Akiyama K. Maeda Y. Shida T. Sakaguchi M. et al.Estimation of Der p and Der f I quantities in the reference preparations of Dermatophagoides mite extracts.Clin Exp Allergy. 1994; 24: 1030-1035Crossref PubMed Scopus (38) Google Scholar, 9Meyer C.H. Bond J.F. Chen M.S. Kasaian M.T. Comparison of the levels of the major allergens Der p I and Der p II in standardized extracts of the house dust mite, Dermatophagoides pteronyssinus.Clin Exp Allergy. 1994; 24: 1041-1048Crossref PubMed Scopus (70) Google Scholar 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 IAllergen concentration of reference standards measured by ELISA using the UASUnitsAllergenConcentrationWHO-IUIS standards∗Mean of duplicate determinations. Dermatophagoides pteronyssinus 82/518100,000 IU/ampouleDer p 17.2 μg/ampoule Dog hair 84/685100,000 IU/ampouleCan f 120.4 μg/ampoule†Based on these data, 1 IU Can f 1 = ∼5 ng Can f 1.FDA standards‡Mean data from measurements by 2 operators on 3 separate occasions. The values for Fel d 1 units in the cat hair and pelt standards were determined by radial immunodiffusion by the FDA (data kindly provided by Cherry Valerio, FDA Center for Biologics Evaluation and Research). D pteronyssinus E810,000 AU/mLDer p 120.3 μg/mL D pteronyssinus E1010,000 AU/mLDer p 127.6 μg/mL D pteronyssinus E810,000 AU/mLDer p 223.4 μg/mL D pteronyssinus E1010,000 AU per mLDer p 223.5 μg/mL D farinae E910,000 AU/mLDer f 119.9 μg/mL D farinae E1010,000 AU/mLDer f 129.2 μg/mL D farinae E910,000 AU/mLDer f 2§Determined using a separate in-house natural Der f 2 standard with a concentration of 1.25 mg/mL by amino acid analysis.19.2 μg/mL D farinae E1010,000 AU/mLDer f 2§Determined using a separate in-house natural Der f 2 standard with a concentration of 1.25 mg/mL by amino acid analysis.15.0 μg/mL Cat hair E410,000 BAU/mL or 7.0 Fel d 1 units/mLFel d 15.4 μg/mL‖1 FDA unit Fel d 1 = ∼1 μg Fel d 1. Cat pelt E410,000 BAU/mL or 14.8 Fel d 1 units/mLFel d 115.6 μg/mL‖1 FDA unit Fel d 1 = ∼1 μg Fel d 1. Cat hair E510,000 BAU/mL or 22.0 Fel d 1 units per mLFel d 124.8 μg/mL‖1 FDA unit Fel d 1 = ∼1 μg Fel d 1.BAU, Bioequivalent allergy unit.∗ Mean of duplicate determinations.† Based on these data, 1 IU Can f 1 = ∼5 ng Can f 1.‡ Mean data from measurements by 2 operators on 3 separate occasions. The values for Fel d 1 units in the cat hair and pelt standards were determined by radial immunodiffusion by the FDA (data kindly provided by Cherry Valerio, FDA Center for Biologics Evaluation and Research).§ Determined using a separate in-house natural Der f 2 standard with a concentration of 1.25 mg/mL by amino acid analysis.‖ 1 FDA unit Fel d 1 = ∼1 μg Fel d 1. Open table in a new tab BAU, Bioequivalent allergy unit. 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.10Turkeltaub P.C. Biological standardization.Arb Paul Ehrlich Inst Bundesamt Sera Impfstoffe Frankf A M. 1997; 91: 145-156PubMed Google Scholar 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.8Yasueda H. Saito A. Akiyama K. Maeda Y. Shida T. Sakaguchi M. et al.Estimation of Der p and Der f I quantities in the reference preparations of Dermatophagoides mite extracts.Clin Exp Allergy. 1994; 24: 1030-1035Crossref PubMed Scopus (38) Google Scholar, 9Meyer C.H. Bond J.F. Chen M.S. Kasaian M.T. Comparison of the levels of the major allergens Der p I and Der p II in standardized extracts of the house dust mite, Dermatophagoides pteronyssinus.Clin Exp Allergy. 1994; 24: 1041-1048Crossref PubMed Scopus (70) Google Scholar 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.6Filep S. Tsay A. Vailes L.D. Gadermaier G. Ferreira F. Matsui E.C. et al.A multi-allergen standard for immunoassays: CREATE principles applied to eight purified allergens.Allergy. 2011 Nov 18; ([Epub ahead of print])https://doi.org/10.1111/j.1398-9995.2011.02750.xCrossref PubMed Scopus (44) Google Scholar 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.1Nelson H.S. Allergen immunotherapy: where is it now?.J Allergy Clin Immunol. 2007; 119: 769-779Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar 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.4Chapman M.D. Ferreira F. Villalba M. Cromwell O. Bryan D. Becker W.M. et al.The European Union CREATE project: a model for international standardization of allergy diagnostics and vaccines.J Allergy Clin Immunol. 2008; 122: 882-889Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar 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. We are grateful to the National Institute for Biological Standards and Control (Potters Bar, United Kingdom) and to the US Food and Drug Administration, Center for Biologics Evaluation and Research (Rockville, Md), for providing allergen reference materials.

To cite this article: Permaul P, Hoffman E, Fu C, Sheehan W, Baxi S, Gaffin J, Lane J, Bailey A, King E, Chapman M, Gold D, Phipatanakul W. Allergens in urban schools and homes of children with asthma. Pediatr Allergy Immunol 2012: 23: 543–549.AbstractBackground: 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.Methods: Settled dust and airborne samples from 12 inner‐city schools were analyzed for indoor allergens using multiplex array technology (MARIA). School samples were linked to students with asthma enrolled in the School Inner‐City Asthma Study (SICAS). Settled dust samples from students’ bedrooms were analyzed similarly.Results: From schools, 229 settled dust and 197 airborne samples were obtained. From homes, 118 settled dust samples were obtained. Linear mixed regression models of log‐transformed variables showed significantly higher settled dust levels of mouse, cat and dog allergens in schools than homes (545% higher for Mus m 1, estimated absolute difference 0.55 μg/g, p < 0.0001; 198% higher for Fel d 1, estimated absolute difference 0.13 μg/g, p = 0.0033; and 144% higher for Can f 1, estimated absolute difference 0.05 μg/g, p = 0.0008). Airborne and settled dust Mus m 1 levels in classrooms were moderately correlated (r = 0.48; p < 0.0001). There were undetectable to very low levels of cockroach and dust mite allergens in both homes and schools.Conclusion: Mouse allergen levels in schools were substantial. In general, cat and dog allergen levels were low, but detectable, and were higher in schools. Aerosolization of mouse allergen in classrooms may be a significant exposure for students. Further studies are needed to evaluate the effect of indoor allergen exposure in schools on asthma morbidity in students with asthma.

BACKGROUND:Current enzyme immunoassay methods for detection of common indoor allergens in environmental dust samples are labor-intensive and time consuming.OBJECTIVE:To develop and validate a fluorescent multiplex array to measure 6 (Der p 1, Der f 1, Der p 2, Der f 2, Fel d 1, and Can f 1) indoor allergen levels simultaneously.METHODS:A multiplex array for 6 allergens, using mAbs covalently coupled to fluorescent microspheres, was developed using a single universal standard composed of purified natural allergens. The multiplex array was validated by comparing the measured dust mite, cat, and dog allergen levels in household dust samples to those obtained by standard ELISA methods.RESULTS:Linear regression analysis showed a highly significant quantitative correlation between the multiplex array and ELISA for dust mite, cat, and dog allergens: R(2) values ranging from 0.90 to 0.99 (P < .001). In addition, the sensitivity, limit of detection (<0.1 ng/mL), reproducibility, intra-assay coefficient of variance (<5%), and interassay coefficient of variance (<25%) of the fluorescent multiplex array were shown to be equal to or better than the ELISA method.CONCLUSION:A multiplex array has been developed to measure simultaneously 6 indoor allergens from a single sample. The array will facilitate epidemiologic studies and indoor air quality assessments and can, in principle, be expanded to include other allergens and biologics.CLINICAL IMPLICATIONS:The multiplex array lends itself to clinical studies, population-based environmental surveys, and allergen avoidance studies comparing allergen exposure in large populations over several time points.