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.
BACKGROUND
Peanut is a most common and potent food allergen. Many peanut allergens have been characterized using, in particular, IgE-binding studies.


OBJECTIVE
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 beta-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).


CONCLUSIONS
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.
Blanc
F
F
INRA, UR496, Unité d'Immuno-Allergie Alimentaire, Gif sur Yvette, Cedex, France.
Adel-Patient
K
K
Drumare
M-F
MF
Paty
E
E
Wal
J-M
JM
Bernard
H
H
D055516:2S Albumins, Plant / Q000097:blood / Q000276:immunology*; D000293:Adolescent; D000485:Allergens / Q000097:blood / Q000276:immunology*; D000818:Animals; D052179:Antigens, Plant / Q000097:blood / Q000276:immunology*; D015549:Basophil Degranulation Test; D015153:Blotting, Western; D015550:Cell Degranulation; D002478:Cells, Cultured; D002648:Child; D002675:Child, Preschool; D005260:Female; D005434:Flow Cytometry; D006023:Glycoproteins / Q000097:blood / Q000276:immunology*; D006801:Humans; D007073:Immunoglobulin E / Q000097:blood; D008297:Male; D021183:Peanut Hypersensitivity / Q000097:blood / Q000276:immunology*; D051381:Rats; D017455:Receptors, IgE / Q000096:biosynthesis
D055516:2S Albumins, Plant; D000485:Allergens; D052179:Antigens, Plant; C500890:Ara h 2 allergen, Arachis hypogaea; C492737:Ara h 6 allergen, Arachis hypogaea; D006023:Glycoproteins; D017455:Receptors, IgE; D007073:Immunoglobulin E