Premium
Capacity of purified peanut allergens to induce degranulation in a functional in vitro assay: Ara h 2 and Ara h 6 are the most efficient elicitors
Author(s) -
Blanc F.,
AdelPatient K.,
Drumare M.F.,
Paty E.,
Wal J.M.,
Bernard H.
Publication year - 2009
Publication title -
clinical and experimental allergy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.462
H-Index - 154
eISSN - 1365-2222
pISSN - 0954-7894
DOI - 10.1111/j.1365-2222.2009.03294.x
Subject(s) - degranulation , allergen , immunoglobulin e , peanut allergy , immunology , chemistry , in vitro , sensitization , ec50 , allergy , pharmacology , biology , biochemistry , antibody , receptor
Summary Background Peanut is a most common and potent food allergen. Many peanut allergens have been characterized using, in particular, IgE‐binding studies. Objectives We optimized an in vitro functional assay to assess the capacity of peanut allergens to degranulate humanized rat basophilic leukaemia cells, RBL SX‐38 cells, after sensitization by serum IgE from peanut‐allergic patients. We thus compared the activity of the main peanut allergens, i.e. Ara h 1, Ara h 2, Ara h 3 and Ara h 6, purified from roasted peanut. Methods Sera of 12 peanut‐allergic patients were collected and total and peanut‐specific IgE were measured. They were used to sensitize RBL SX‐38 cells and the degranulation was induced by incubation with ranging concentrations of a whole peanut protein extract or of purified peanut allergens. The mediator release was quantified by the determination of β‐hexosaminidase activity in the supernatant. The intensity of the degranulation was expressed as maximum release and as EC50, corresponding to the dose of allergen that induced 50% of the maximum release. Results For each serum, only 10 IU/mL of human IgE was necessary to sensitize the cells and obtain an optimal degranulation. With all the allergens, the release was positively correlated with the concentration of allergen‐specific IgE in the serum used to sensitize the cells. The medians of EC50 obtained for Ara h 2 and Ara h 6 were 2.1 and 2.8 p m , respectively, while they were much higher for Ara h 3 and Ara h 1 (65 and 150 p m , respectively). Conclusion The RBL SX‐38 release assay proved to be sensitive, specific and reproducible. It allowed the comparison of the degranulation potential of different peanut allergens. For all the sera tested, Ara h 2 and Ara h 6 were more potent than Ara h 1 or Ara h 3.