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


Hervé Bernard, Unité d'Immuno-Allergie Alimentaire, CEA-SPI, iBiTec-S, Bat 136, F-91191 Gif sur Yvette cedex, France.


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.