Highly sensitive ELISA‐based assay for quantification of allergen‐specific IgE antibody levels

9. Bloomfield SF, Rook GA, Scott EA, Shanahan F, Stanwell-Smith R, Turner P. Time to abandon the hygiene hypothesis: new perspectives on allergic disease, the human microbiome, infectious disease prevention and the role of targeted hygiene. Perspect Public Health. 2016;136(4):213-224. 10. Herbst T, Sichelstiel A, Schar C, et al. Dysregulation of allergic airway inflammation in the absence of microbial colonization. Am J Respir Crit Care Med. 2011;184(2):198-205. SUPPORTING INFORMATION Additional supporting information may be found online in the Supporting Information section.

Highly sensitive ELISA-based assay for quantification of allergen-specific IgE antibody levels In order to establish a standard for ELISA-based quantification of allergen-specific human IgE, a human monoclonal chimeric IgE antibody (IgEmoAb) 7 consisting of the variable region of a mouse IgG 1 antibody specific for the major birch pollen allergen Bet v 1, which had been fused to the human epsilon heavy chain, was expressed and purified from hybridoma cells by affinity chromatography to >95% purity ( Figure S1A). Under non-reducing conditions,    Table S4) were compared, and a strong and significant correlation was noted (r = .988 P < .0001, 95% CI 0.967-0.996; Figure 2B).
The quantitative IgE ELISA assay should be useful for laboratories in less developed countries which cannot afford expensive IgE test systems for routine allergy diagnosis taking advantage of molecular allergy diagnosis. 9,10 Before extensive use, broader clinical studies correlating IgE concentrations to clinical signs may be warranted. In fact, the price for a quantitative IgE determination for one allergen should cost less than 10% than by ImmunoCAP; however, the time required for testing will be longer. Our assay will therefore be useful in places where work power cost is not a major issue and the results are desired in a higher-throughput format as well as for cases (eg, research) where regulations are of minor importance.
Furthermore, the assay should allow a flexible testing of different allergen molecules which can be kept on stock and used depending on the clinical questions and diagnostic needs required. To achieve these goals, the authors will make efforts to make available the key reagents for the standard curve (ie, IgEmoAb and rBet v 1) to the community. In summary, we provide a simple, inexpensive, sensitive and accurate method for measuring allergen-specific IgE concentrations in serum and body fluids to facilitate the diagnosis of IgEassociated allergy in the world.

S U PP O RTI N G I N FO R M ATI O N
Additional supporting information may be found online in the  1 The clustering analysis has already been used to identify phenotypes in allergic children, such as atopic dermatitis, asthma, and peanut allergy. 2,3 Such approach is of interest to better characterize the heterogeneity of allergic diseases and also to provide personalized patient management. Predicting who is the most at risk of severe anaphylaxis reactions would be of a great interest to reduce anaphylaxis morbidity and mortality but remains challenging.
We aimed to approach anaphylaxis heterogeneity by a nonsupervised statistical clustering analysis on a cohort of children (<18 years) admitted to emergency care unit for anaphylaxis.
This study was conducted in a cohort of 482 children (<18 years) admitted in emergency care units for anaphylaxis in three different French regions. The characteristics of these children have been reported before. 1,4,5 The anaphylaxis was defined according to Sampson's criteria. 6 The data have been recorded prospectively in one region 1 and retrospectively in the two others. 4,5 We performed a nonsupervised clustering analysis using 23 variables. The methods and statistical analysis are described (Supplementary Material S1).