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
Article first published online: 15 JUN 2009
© 2009 Blackwell Publishing Ltd
Clinical & Experimental Allergy
Volume 39, Issue 8, pages 1277–1285, August 2009
How to Cite
Blanc, F., Adel-Patient, K., Drumare, M.-F., Paty, E., Wal, J.-M. and Bernard, H. (2009), 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. Clinical & Experimental Allergy, 39: 1277–1285. doi: 10.1111/j.1365-2222.2009.03294.x
- Issue published online: 9 JUL 2009
- Article first published online: 15 JUN 2009
- Submitted 10 September 2008; revised 9 April 2009; accepted 13 April 2009
- mast cell activation;
- peanut allergens
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 pm, respectively, while they were much higher for Ara h 3 and Ara h 1 (65 and 150 pm, 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.