J Korean Med Sci 2005; 20: 573-8
Copyright ? The Korean Academy
of Medical Sciences
Chestnut as a Food Allergen: Identification of Major Allergens
To evaluate the clinical significance of chestnut as a food allergen in Korea, skin prick
Soo-Keol Lee, Sung-Ho Yoon*,
test and ELISA were done in 1,738 patients with respiratory allergies. To identify the
Seung-Hyun Kim*, Jeong-Hee Choi*,
IgE binding components, IgE-immunoblotting, 2D IgE-immunoblotting and MALDI-
TOF were performed. To observe the effects of digestive enzymes and a boiling
Department of Internal Medicine, College of Medicine,
treatment, simulated gastric fluid (SGF) and simulated intestinal fluids (SIF) were
Dong-A University, Busan; Department of Allergy and
incubated with chestnut extracts, and IgE-immunoblotting were then repeated. Skin
Rheumatology*, School of Medicine, Ajou University,
prick test revealed that 56 (3.2%) patients showed more than 2+ of allergen to his-
tamine ratio to chestnut. Among the 21 IgE binding components, 9 bands were found
Received : 22 February 2005
in more than 50% of the sera tested and the 24 kDa protein had the highest binding
Accepted : 9 March 2005
intensity. The amino acid sequence of the 24 kDa protein (pI 6.3) had homology with
Address for correspondence
legume protein of oak tree. SGF, SIF and boiling treatment were able to suppress
Hae-Sim Park, M.D.
the IgE binding components. In conclusion, chestnut ingestion was shown to induce
Department of Allergy and Rheumatology, Ajou
IgE mediated responses with a 3.2% sensitization rate. Twenty one IgE binding com-
University School of Medicine, San-5 Wonchun-dong,
Yeongtong-gu, Suwon 442-721, Korea
ponents and one new allergen (the 24 kDa protein) were identified. Digestive enzymes
Tel : +64.31-219-5196, Fax : +64.31-219-5154
and boiling treatment were able to decrease the allergenic potency.
E-mail : firstname.lastname@example.org
*This study was supported by ARPC Grant (204070-
Key Words : Chestnut; Food Hypersensitivity; Allergens; Digestive Enzyme
matrix-assisted laser desorption/ionization-time of flight
(MALDI-TOF) analysis were performed. The stability of chest-
Allergic reactions to tree nuts can be a serious and life threat-
nut to digestive enzymes and to a boiling treatment was also
ening condition. Fresh and boiled chestnuts are commonly
ingested food in Korea. A previous study demonstrated that
chestnuts were accounted for the third greatest prevalence
among the food allergens and it is the most prevalent tree
MATERIALS AND METHODS
nut allergen for both adult and pediatric allergy patients in
Korea (1). Chestnut food allergy has also been reported in
those patients presenting with latex-fruit or oral allergy syn-
drome (2). To date, there has been no published study to
Patients who complained of respiratory allergy symptoms
address the actual prevalence of IgE sensitization to chestnuts
were recruited during one year from the Allergy Clinic of
as a food allergen and its major allergic components in patients
Ajou University Hospital in Suwon, Korea. Among the 1,738
sensitized to this food (3).
patients, ELISA was performed on the sera from 56 patients
It has been proposed that food allergens that can provoke
showing ?2+ positive reactions to chestnuts on skin prick
systemic allergic reactions tend to be stable to digestion by
test for detecting serum specific IgE to homegrown chestnuts.
the gastrointestinal tract (4, 5). Furthermore, resistance to
Among them, the sera from 9 patients showing high specific
pepsin digestion has been recently incorporated to the Food
IgE levels on ELISA were used for the ELISA inhibitions test-
and Agriculture Organization/World Health Organization
ing and for IgE-immunoblotting.
decision-making approach to the safety assessment of foods
produced through agricultural biotechnology (6, 7).
Preparation of allergen extracts
In this study, we evaluated the clinical significance of chest-
nuts as a food allergen by using skin prick test and by perform-
Chestnut food allergen extracts were prepared according
ing serum specific IgE antibody determination via ELISA.
to the method described previously (8). Briefly, fresh chest-
To characterize the IgE-binding components, 10% SDS-PAGE,
nuts (the most commonly ingested one in this country, 1:5
IgE-immunoblot and 2D-IgE-immunoblot followed by
wt/vol) were homogenized in phosphate-buffered saline (PBS,
S.-K. Lee, S.-H. Yoon, S.-H. Kim, et al.
pH 7.5). The homogenate was kept at 4? overnight followed
that showed higher antibody titer by using a spline fit pro-
by centrifugation at 3,500 rpm for 40 min. The supernatant
gram (Microplate Manager, Bio-Rad), and they were expressed
was dialyzed (the cut-off molecular weight was 6,000 Da;
as arbitrary units (A.U.). The chestnut-specific IgE was con-
Spectrum Medical Industries, Houston, TX, U.S.A.) against
sidered positive if the absorbance value was higher than the
4 L of PBS at 4? for 48 hr. The supernatant was then passed
cutoff value, which was derived from the mean+3×S.D. of
through a Nalgen syringe filter (Rochester, New York, NY,
the absorbance values from the 15 control subjects.
U.S.A.) and lyophilized until ELISA, ELISA inhibition, im-
munoblotting analysis, and digestibility tests were performed.
SDS-PAGE and IgE-immunoblot
For skin prick test, the 1:5 wt/vol extract was mixed with an
equal amount of glycerin (Sigma Co., St. Louis, MO, U.S.A.).
Electrophoresis in a 10% polyacrylamide separation gel
with 4% stacking gel was performed and the proteins were
Skin prick test
stained with colloidal blue (Novex, San Diego, CA, U.S.A.)
or the proteins were transferred onto a PVDF membrane
Routine skin prick tests were performed for 30 food aller-
(Millipore, Bedford, MA, U.S.A.), and this was then incu-
gens using 26-gage needles on the backs of the patients. The
bated with the pooled serum of patients with high serum
results were read 15 min later. The size of the wheal produced
specific IgE that had been diluted 1:5 vol/vol with PBST.
by each allergen and histamine was expressed as the mean
Bound specific IgE was detected by biotin-labeled goat anti-
diameter of maximum length and vertical length at the mid-
human IgE antibody (Sigma). This was followed by a treat-
portion of the maximal length. Skin reactivity was expressed
ment with 0.66% 4-nitroblue tetrazolium and 0.33% 5-
as the ratio of the wheal size of the allergen to the histamine
bromo-4-chloro-3-indolyl-phosphate (Sigma), which was
(A/H). If the A/H ratio was from 0.1 to 1, but the erythema
used as a substrate.
was >21 mm, it was read as 2+. If the A/H ratio was 1 to 2,
it was read as 3+. If the A/H ratio was from 2 to 3, it was read
Effect of simulated gastric fluid and intestinal fluid on the
as 4+. If the A/H ratio was greater than 3, it was read as 5+.
IgE binding components
A positive was defined as equal to or greater than 2+ on skin
prick test (9).
The fresh chestnut extracts were prepared by heating the
crude extracts at 100? for 5 min. The digestibility of the
ELISA for serum specific IgE to chestnut
crude extract per se and the digestibility of the preheated extract
that was incubated in simulated gastric fluid (SGF) and simu-
ELISA was performed to determine the presence of specific
lated intestinal fluid (SIF) were examined as previously des-
IgE to chestnut as previously described (10). Microtiter plates
cribed (4). Briefly, for the SGF digestibility test, a protein
(Costar, New York, NY, U.S.A.) were coated with chestnut
sample (680 g of naive crude protein or the preheated extract)
extract at 100 L/well (diluted in carbonated buffer, pH 9.6,
was dissolved in 200 L of prewarmed 100 mM/L HCl (pH
10 g/well) and the plates were left at 4? for 24 hr. Each
1.2), along with 30 mM/L NaCl containing a 0.32 wt/vol
well was washed three times with 0.05% PBS-Tween (PBST),
percentage of pepsin A (Sigma Chemical Co). Digestion was
and was blocked by incubation with 3% BSA-PBST at 200
carried out at 37? with continuous shaking, and aliquots
L/well for 1 hr at room temperature (RT). The wells were
of this digest solution (20 L) were withdrawn at 0, 0.5 and
then incubated for 2 hr at RT with either 50 L of the patient’s
60 min. These aliquots were quickly mixed with 26 L of a
sera or the control sera (all the sera was diluted 1 to 5). After
sample buffer (containing 2.5% 2-mercaptoethanol and 1%
washing three times with PBST, biotin-labeled goat anti-
SDS) together with 6.0 L of Na2CO3 solution (200 mM/L).
human IgE antibody (Vector Co, Burlingame, CA, U.S.A.)
The mixture was then boiled for 5 min and stored at -20?
was added to the each well and incubated for 1 hr. After
until further analyses. For the evaluation of SIF and SGF, a
washing, the wells were incubated with 100 L of 1:1,000
sample (680 g of the naive crude protein or the preheated
vol/vol streptavidin-peroxidase (Sigma Co., St. Louis, MO,
extract) was dissolved in 260 L of prewarmed intestinal
U.S.A.) for 30 min before ano- ther washing step. The col-
control solution (0.05 M KH2SO4, pH 6.8) containing a 1.0
orimetric reaction was developed with B (3,3?,5,5?-tetram-
wt/vol percentage of pancreatin (Pancreatin USP, Sigma). This
ethylbenzidine) substrate solution for 10 min at RT. The
was then incubated at 37? with continuous shaking. Aliquots
reaction was stopped by the addition of 100 L of 2 N sul-
of this digest solution (26 L) were withdrawn at 1, 90 and
furic acid and the absorbance was read at 450 nm by an auto-
240 min and quickly boiled for 5 min after mixing it with
mated microplate reader (Benchmark, Bio-Rad, Hercules,
26 L of a sample buffer (containing 2.5% 2-mercaptoethanol
CA, U.S.A.). All assays were performed in duplicate. The
and 1% SDS), and then 12% SDS-PAGE and IgE immunoblot
amounts of specific antibodies in the samples were calculat-
analysis were repeated as described above.
ed from control curves that were created with the optical
densities from serial dilutions of the positive control samples
Identification of Chestnut Allergen
of serum specific IgE, as determined by ELISA, to the chest-
nut extracts were 34.1% in the 2+ reactors, 58.3% in the
Skin prick test and ELISA for detecting specific IgE to
3+ reactors and 0% in the 4+ reactors. The absorbance value
of specific IgE to chestnut, according to skin reactivity, is
shown in Fig. 1.
The results of the skin prick test revealed that 56 (3.2%)
of 1,738 patients showed more than 2+ A/H response to the
ELISA inhibition test
chestnut extracts, as is shown in Table 1. Among them, 15
(0.9%) showed more than 3+ A/H response. The prevalence
The ELISA inhibition test for wild chestnut using the pooled
sera from subjects with high serum specific IgE level showed
Table 1. Skin prick test and specific lgE bindings to chestnut
significant dose-dependent inhibitions by the chestnut and
extracts by ELISA
chestnut pollen extracts, and both extracts had similar poten-
Skin prick test
cies, while negligible inhibition was noted for D. pteronyssinus,
as is shown in Fig. 2.
Skin reactivity (%)
SDS-PAGE and IgE-immunoblot
Specific lgE (%)
14/41 (34.1) 7/12 (58.3) 0/3 (0)
Patients (positive rate on specific lgE bindings, %).
To observe the protein components of the fresh chestnut
9 10 11 12
Specific lgE to chestnut (A.U.)
Skin prick test (A/H)
Fig. 1. Specific IgE bindings to chestnut by ELISA according to
Allergen/Histamine (A/H) ratio of chestnut on skin prick test. Hori-
Fig. 3. IgE-immunoblot analysis of chestnut extracts in the sera from
zontal bars indicate the mean values.
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).
Percent inhibition (%)
Inhibitor added ( g/mL)
Fig. 4. Result of IgE-immunoblot analysis of the chestnut extracts
Fig. 2. Chestnut-ELISA inhibition tests with the additions of chest-
using the sensitized sera. Shadow bar indicate 10 major auergeric
nut ( ), chestnut pollen ( ) and D. pteronyssinus ( ) extracts.
S.-K. Lee, S.-H. Yoon, S.-H. Kim, et al.
extracts, they were analyzed by using 10% SDS-PAGE and
of two patients with high levels of specific IgE to chestnut
IgE-immunoblotting with the patients’ sera. As shown in
extract showed the 24 kDa IgE binding component and its
Fig. 3, 21 IgE-binding components were detected within
iso-electric point was 6.3, while a 29 kDa component was not
the chestnut extracts, while no bindings were noted with
detectable. Table 2 shows the result of amino acid sequencing
the control sera. The 90, 83, 76, 69, 56, 49, 43, 24 and 13
for this major allergen having a 24 kDa of molecular weight,
components were noted in more than 50% of the sensitized
which has homology with legume protein from oak trees (11).
patients (Fig. 4).
Effects of digestive enzymes on IgE-binding components
Two-dimensional SDS-PAGE, immunoblotting and N-ter-
minal amino acid sequencing
Immunoblotting using the pooled sera showed that most
of the IgE-binding components were substantially digested
Two dimensional SDS-PAGE (Fig. 5A) and immunoblotting
in 10 to 30 min by SGF in the preheated and heated condi-
(Fig. 5B) of the fresh chestnut extracts using the pooled sera
tions. After the treatment with SIF, although most of the pro-
teins within the chestnut extracts were not changed, IgE
Table 2. Amino acid sequence of the major allergen of chestnut
binding components were suppressed in the preheated and
heated conditions (Fig. 6, 7).
Amino acid sequence
MW, Molecular weight; pI, Iso-electric point.
Castanea crenata var. dulcis, is a member of the beech family
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.
Identification of Chestnut Allergen
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.
(Fagaceae) that blooms with yellow flowers in spring and its
quently associated to actual clinical allergies not only to fruits,
nuts ripen in September and October, and the chestnut pollen
but also to other tree nuts. Also, allergy to chestnuts has been
has been found in the air of Korea. Chestnut is a commonly
widely reported in the latex-fruit syndrome (13-18), The per-
ingested food in this country in both the fresh and boiled
centage of individuals with latex allergy who have IgE anti-
form. A nationwide study on prevalence to commonly ingest-
bodies to a chestnut was estimated to be 34.6% (13). An
ed food allergens by skin prick test in Korea has demonstrat-
increasing number of plant sources has been associated with
ed chestnut to be the third most prevalent sensitizing food
this syndrome, but avocado, banana, chestnut and kiwi are
allergen in both adult and pediatric allergy patients (1). In
those most frequently reported foods (13, 19, 20). The rele-
this study, we demonstrated that the sensitization rate to
vant 32 kDa allergens of avocado and chestnut could be the
homemade chestnut extracts was 3.2% in the adult allergy
pan-allergens responsible for the latex-fruit syndrome. In this
patients, and the serum specific IgE antibody was detected
study, among the 9 subjects having high levels of specific IgE
by ELISA in the positive reactors on a skin prick test. The
antibody to chestnut, none had a history of latex allergy.
ELISA inhibition tests demonstrated that significant inhibi-
Typical food allergens are per oral sensitizers and, at the same
tions were noted with the addition of the chestnut extracts.
time, they are per oral inducers of allergic symptoms. Com-
These findings suggest that chestnut should be considered
plete food allergens are proteins having both the ability to
as one of the prevalent food allergens in this country.
sensitize and the ability to induce symptoms (21). They were
There have been a few studies to identify the IgE binding
also reported to be stable to digestion (19, 20), because they
components within chestnut allergen extracts (12). Previous
must be absorbed from the intestine and be recognized by
studies reported two allergens named Cas s 5 (chitinase Ib,
the immune system for a person to be sensitized. In contrast,
molecular weight was not identified) and Cas s 8 (lipid transfer
incomplete food allergens are those allergens having the capac-
protein, 9.7 kDa) from the chestnut. In this study, 21 IgE
ity to induce allergic symptoms per orally, even though they
binding components were identified and 9 of them could be
are digestible and cannot per orally sensitize people (21). Indi-
potential major allergens as they bound to ?the 50% of the
gestibility is not a prerequisite for a food protein to induce
subjects’ sera that we tested. Although the number of study
allergic symptoms. Digestible food allergens, as well as indi-
subjects was not large enough to fully evaluate all the aller-
gestible food allergens, have the ability to elicit allergic reac-
gen components, the 24 kDa component had the highest of
tions per orally (22, 23). In this study, we evaluated changes
the IgE binding intensities. The amino acid sequencing of
of the allergenic components after they were incubated with
the 24 kDa protein demonstrated a homology with the legume-
digestive enzymes and a boiling process in vitro. Gastric en-
like protein of oak tree. Further studies with a larger group
zyme treatment alone, and both the gastric enzyme treatment
of study subjects will be needed to investigate the IgE bind-
and the boiling treatment could decrease the IgE binding
ing components to differentiate between the symptomatic
components of most major allergens, while intestinal enzyme
and asymptomatic sensitizers, and this identification of the
treatment had minimal effects on the allergenicity. There
symptomatic sensitizers is an important issue for food aller-
has been a previous study suggesting that the possible lack
of detection of SGF-digested products from an allergen by
Rico et al. reported that one third of the chestnut-allergic
protein staining or IgE-immunoblotting after SDS-PAGE
patients experience severe anaphylactic episodes upon inges-
fractionation does not necessarily mean that it is inactivated
tion of chestnuts (3). Chestnut reactivity has also been fre-
in SGF (24). Further studies with a larger group of sensitized
S.-K. Lee, S.-H. Yoon, S.-H. Kim, et al.
patients will be needed to make any definite conclusion.
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