Chestnut as a food allergen: identification of major allergens

Abstract

To evaluate the clinical significance of chestnut as a food allergen in Korea, skin prick test and ELISA were done in 1,738 patients with respiratory allergies. To identify the IgE binding components, IgE-immunoblotting, 2D IgE-immunoblotting and MALDITOF were performed. To observe the effects of digestive enzymes and a boiling treatment, simulated gastric fluid (SGF) and simulated intestinal fluids (SIF) were incubated with chestnut extracts, and IgE-immunoblotting were then repeated. Skin prick test revealed that 56 (3.2%) patients showed more than 2+ of allergen to histamine ratio to chestnut. Among the 21 IgE binding components, 9 bands were found in more than 50% of the sera tested and the 24 kDa protein had the highest binding intensity. The amino acid sequence of the 24 kDa protein (pI 6.3) had homology with legume protein of oak tree. SGF, SIF and boiling treatment were able to suppress the IgE binding components. In conclusion, chestnut ingestion was shown to induce IgE mediated responses with a 3.2% sensitization rate. Twenty one IgE binding components and one new allergen (the 24 kDa protein) were identified. Digestive enzymes and boiling treatment were able to decrease the allergenic potency.

Fig. 1

Specific IgE bindings to chestnut by ELISA according to Allergen/Histamine (A/H) ratio of chestnut on skin prick test. Horizontal bars indicate the mean values.

Fig. 2

Chestnut-ELISA inhibition tests with the additions of chestnut (●), chestnut pollen (△) and D. pteronyssinus (■) extracts.

Fig. 3

IgE-immunoblot analysis of chestnut extracts in the sera from the sensitized patients. M: standard molecular marker. Lane 1-9: subjects of the sensitized patients. Lane 10, 11: control. Lane 12: buffer. Arrow indicates the major IgE binding component (24 kDa).

Fig. 4

Result of IgE-immunoblot analysis of the chestnut extracts using the sensitized sera. Shadow bar indicate 10 major auergeric components.

Fig. 5

Two dimensional SDS-PAGE (A) and the IgE binding component (B) of the fresh chestnut extracts using the pooled sera of two patients with high levels of specific IgE. The iso-electric point was 6.3.

Fig. 6

Effect of simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) treatment on chestnut allergens as analyzed by 12% SDS-PAGE (A) and IgE-immunoblotting (B). 1, Standard; 2, SGF-30 sec; 3, SGF-2 min; 4, SGF-10 min; 5, SGF-30 min; 6, SIF-15 min; 7, SIF-2 hr; 8, SIF-16 hr.

Fig. 7

Effect of simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) treatment on the boiled chestnut allergen as analyzed by SDS-PAGE (A) and IgE-immunoblotting (B). 1, Standard; 2, SGF-2 min; 3, SGF-15 min; 4, SGF-30 min; 5, SGF-1hr; 6, SIF-15 min; 7, SIF-2 hr; 8, SIF-16 hr.