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.

Serum tryptase level and inflammatory markers in exhaled breath condensate of children with exercise-induced symptoms.

BACKGROUND: The pathogenesis of exercise-induced bronchoconstriction (EIB) is poorly understood. OBJECTIVE: To evaluate the biomarkers concentration in exhaled breath condensate (EBC) in schoolchildren with postexercise symptoms. We also evaluated changes in fractional exhaled nitric oxide (FeNO) value and the serum tryptase level after exercise. METHODS: One hundred children with postexercise symptoms were included. Methacholine challenge testing (MCT) was performed at visit 2, and exercise challenge testing (ECT) was performed at visit 3. Before and after ECT serum tryptase levels and FeNO values were measured. EBC was collected after ECT from 10 randomly selected children from each group. The children were assigned to the following groups: ECT(+) MCT(+), ECT(+) MCT(-), ECT(-) MCT(+), ECT(-) MC(-). We measured the following molecules: eotaxin, interleukin (IL) 8, IL-1ra, IL-1 beta, IL-6, IL-1 alpha, IL-12(p40), IL-5, granulocyte-macrophage colony-stimulating factor, IL-7, IL-15, IL-4, IL-2, IL-10, tumor necrosis factor alpha, interferon gamma, IL-13, tumor necrosis factor beta, monocyte chemoattractant protein-1, IL-17A, macrophage inflammatory proteins-1 alpha, macrophage inflammatory proteins-1 beta, IL-12(p70), and regulated on activation, normal T-cell expressed and secreted by using a multiplex immunoassay. Prostaglandin E2 (PGE2), leukotriene B4, and cysteinyl leukotriene were analyzed by using separate enzyme-linked immunosorbent assay kits. RESULTS: In the MCT(+) group, a detectable level of IL4 in EBC and detectible levels of eicosanoids were seen in the ECT(+) group. We observed the opposite direction of ECT-induced changes in FeNO and serum tryptase concentrations in patients with detectable compared with patients without detectable levels of cytokines in EBC. We showed ECT-induced reduction in the tryptase level in patients with a nondetectable PGE2 level in EBC and an increase in tryptase levels in patients who had detectable levels of PGE2 in EBC. CONCLUSIONS: EBC was a useful method to estimate inflammation but only in children with symptoms and with EIB shown by a positive ECT. Children with a positive ECT had detectable levels of eicosanoids in EBC; the opposite direction of ECT-induced changes in FeNO and serum tryptase concentrations was observed. The results of above study confirm the role of mast cells and eicosanoids in the pathogenesis of EIB in children.