Methods and results
Subsequent allergy evaluation included skin prick test (SPT) to commercial extracts of common foods with positive results for almond, hazelnut and walnut. A pure cocoa prick-prick test (PPT) was also positive (4 mm) (Figure 1). SPT to common aeroallergens included positive results for Olea europaea pollen and cat dander.
Results of in vitro analysis included a total serum IgE 310 (kU/L), specific IgE (ImmunoCAP; kUA/L) to hazelnut: 20.30, walnut 0.14, and cocoa 0.12; and ImmunoCAP ISAC (ISU-E) being positive with following species-specific food components: nCor a 9 (11S globulin): 1.8, rJug r 1 (2S albumin): 28, nJug r 2 (7S globulin): 3.6, rAra h 1 (7S globulin): 0.5, nGly m 6 (Glycinin): 0.6, rOle e 1: 0.1 and Fel d 1: 1.2.
Protein extracts from edible cocoa powder (fermented and roasted cocoa) were prepared by homogenization in phosphate-buffered saline (10% V/V) (50 mM phosphate buffer, 100 mM NaCl, pH 7.5), dialyzation against distilled water and liophilization. In order to avoid contamination by tree nut proteins that sometimes appears in some edible manufactured cocoa products, we prepared a protein extract from a raw cocoa bean following the same method above described.
SDS-PAGE immunoblotting method according to Laemmli 3was used to estimate the molecular mass of IgE-reactive proteins of the following samples: cocoa bean powder, cocoa powder extract, raw cocoa bean, raw cocoa bean extract as well as walnut and almond extracts. IgE binding was detected in a band of approximately 17.5 kDa in three cocoa samples: cocoa powder, raw cocoa bean, raw cocoa bean extract, and a broad band between approximately 21-17.5 kDa in cocoa powder extract. IgE-reactive bands between approximately 45 – 35 kDa and 20-9 kDa were observed in walnut extract, and bands between approximately 58-13 kDa in almond extract (Figure 2.I).
With the aim to assess the presence of IgE cross-reactivity to proteins in cocoa, walnut, and almond, an SDS-PAGE Immunoblotting-inhibition assay was carried out using cocoa extract in solid phase and walnut and almond extracts as inhibitors. Both walnut and almond extracts produced a partial IgE-binding inhibition, although walnut extract showed a higher inhibition capacity, however, neither produced a total IgE-binding inhibition to cocoa extract (Figure 2.II).