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).