J ALLERGY CLIN IMMUNOL
VOLUME 125, NUMBER 2
864 Decrease in Allergenicity of Food Allegerns by
Polymerization with Glutaraldehyde
J. A. Patel1, A. R. Patel2; 1Colorado College, Colorado Springs, CO,
2
Academy Allergy, Asthma, and Sinus Center, Colorado Springs, CO.
RATIONALE: For the treatment of food allergies, many investigators are
pursuing oral and sublingual immunotherapy, which may induce long-term
tolerance. Side effects of the immunotherapy distress both patients and
physicians. How can we decrease the life-threatening side effects such as
anaphylaxis? Can polymerization of food allergens with glutaraldehyde
decrease the allergenicity?
METHODS: Conventional food allergens (CFA) such as milk, egg, pea-
nut, and Brazil nut were purchased form Greer. They were dissolved in
phosphate buffered saline (PBS) to achieve 5 mg of protein/mL concentra-
tion. Next, the CFA were cross-linked by incubating with 30 mM glutaral-
dehyde for 5 hours at room temperature. Non-cross-linked allergenic
proteins were eliminated by exhaustive diafiltration against water through
a 500kDa cut-off ultra filtration device (Pellicon XL BIOMAX 500 PE,
Millipore Co, USA). The final products were labeled as glutaraldehyde-
modified food allergens (GMFA). After the IRB approval, sixteen patients
with bona fide food allergies were recruited; informed consents were ob-
tained. Based upon patientsÕ history, appropriate prick skin tests were per-
formed simultaneously with CFA and GMFA, and after 15 minutes the
wheal sizes were measured in mm.
RESULTS: The paired t-test showed a statistically significant difference
(p-value<0.001) in wheal sizes with CFA and GMFA. The wheal sizes
with GMFA were significantly smaller than the wheal sizes with CFA.
CONCLUSIONS: Glutaraldehyde modification decreased the allergenic-
ity of the food allergens. Oral immunotherapy with glutaraldehyde-modi-
fied food allergens shows promise as a safer alternative because fewer side
effects may occur. Clinical research centers may investigate glutaralde-
hyde-modified allergens as safer option to induce tolerance to food
allergens.
865
S. W€
Purification of Native Shrimp Tropomyosin by Affinity
Chromatography with Cross-reactive Antibody
unschmann1, M. Uchida1, S. B. Lehrer2, L. K. Arruda3, M. D. Chap-
man1; 1INDOOR Biotechnologies Inc., Charlottesville, VA, 2Tulane Uni-
versity Health Sciences Center, New Orleans, LA, 3School of Medicine of
Ribeirão Preto, Division of Clinical Immunology, University of Sao Paulo,
Sao Paulo, BRAZIL.
RATIONALE: Shrimp and other shellfish species are a common cause of
food allergy in adults and can provoke serious allergic reactions.
Tropomyosin, the major allergen in shrimp shares a high degree of amino
acid sequence identity and immunological cross-reactivity with other in-
vertebrate tropomyosins. Our goal was to purify native shrimp tropomyo-
sin using a monoclonal antibody against mite tropomyosin that crossreacts
with tropomyosin from insects and crustacea.
METHODS: Native shrimp tropomyosin was purified from shrimp meat
extract by affinity chromatography using monoclonal antibody 1A6 cou-
pled to sepharose. Eluted proteins were analyzed by silver-stained SDS
PAGE and Western Blot using 1A6 mAb. IgE binding to affinity purified
native shrimp tropomyosin was analyzed using a chimeric ELISA.
RESULTS: SDS-PAGE of affinity purified shrimp tropomyosin identified
a major protein band at 36kD protein as well as minor protein bands below
the 36kD monomer. These reacted with 1A6 mAb in a Western Blot and
likely represent tropomyosin fragments. Specific IgE from 8/10 sera of
shrimp allergic patients reacted with native shrimp tropomyosin.
CONCLUSIONS: Utilization of a cross-reactive monoclonal antibody is
useful for affinity purification of native tropomyosin from shrimp.
Detection of tropomyosin sensitization is helpful for the diagnosis of shell-
fish allergy. Native shrimp tropomyosin may be a useful reagent for the im-
provement of diagnostic assays to determine sensitization and allergy to
shrimp and other shellfish species.
Abstracts AB221
866 Hidden Allergens in Breast Milk
M. Caminoa1, M. F. Martı́n-Muñoz1, F. Pineda de la Losa2,
G. Garcı́a-Parrado Garcı́a2, I. Bobolea1, J. Larco1, J. Diaz- Pena1,
S. Quirce1; 1La Paz Hospital, Madrid, SPAIN, 2Diater Laboratories,
R&D Department, Madrid, SPAIN.
RATIONALE: Food allergy is a rare affection among breastfed children.
Although cow’s milk is the most important causative allergen, any other
food allergen may be responsible for allergic reactions at that age. The
aim of our study was to identify breast milk allergens involved in allergic
reactions after breastfeeding.
METHODS: We report on 5 children who developed urticaria or gastroin-
testinal symptoms after breastfeeding. Prick tests with breast milk, cow’s
milk, white egg and the foods ingested by the mother before the reaction
were carried out. Total and specific IgE levels against the implicated
food were measured (CAP system). IgE immunoblot techniques were per-
formed with patientsÕ serum and different samples of breast milk obtained
before and after the intake of the specific foods implicated in the reactions.
Finally, the clinical evolution of the patients was evaluated after establish-
ing a maternal exclusion diet.
RESULTS: Skin prick test and specific IgE determinations were positive
with cow’s milk (n52), egg white (n55), hake (n51), or peanuts (n52).
The allergens responsible for the allergic reactions were identified by prick
testing with breast milk and the immunoblot studies. Immunoblot revealed
several IgE-binding bands in breast milk after the intake of peanut, white
egg or cow’s milk. These bands showed molecular masses that might cor-
respond with allergens from peanut, egg white or cow’s milk, which were
absent before the ingestion of these foodstuffs. After the mothers started
exclusion diets the infants remained asymptomatic.
CONCLUSIONS: Woman breast milk should be considered a source of
hidden allergens in infants with allergic reactions.
867 Decrease of Wheat Allergen by Treatment with Fermented
Soybean Extract
Y. Ko1, E. Kim1, J. Bahk1, S. Lee2, C. K. Oh3, C. Ryu1; 1Division of Ap-
plied Life Science(BK 21 Program), Gyeongsang National University, Jin-
Ju, Gyeongnam, REPUBLIC OF KOREA, 2Department of Pediatrics,
Sungkyunkwan University School of Medicine, Samsung Medical Center,
Seoul, REPUBLIC OF KOREA, 3Division of Allergy and Immunology,
Harbor-UCLA Medical Center, West Carson city, CA.
RATIONALE: Wheat is well known to induce food-associated allergic re-
actions in children and adults. In this study, we hypothesized that enzy-
matic solution from fermented soybean which have proteolytic activity
can reduce the allergenic protein of wheat such as v-gliadin which has
been reported as the major allergen in wheat-dependent exercise-induced
anaphylaxis(WDEIA).
METHODS: Fermented soybean was produced according to three-step
fermentation method. Fermented soybean extract treated to wheat was in-
cubated at 378 C for 30 hours. Serum levels of IgA and IgE specific to wheat
were measured by ELISA. Immunoblotting was performed using anti
v-gliadin peroxidase-labelled.
RESULTS: Wheat proteins were digested effectively on 50 U/ml concen-
tration of protease. The changes of allergenic proteins of wheat treated with
fermented soybean extract were confirmed by SDS-PAGE and immuno-
blotting. Digested wheat proteins showed reduced activity reacting with
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IgA and IgE antibodies from wheat allergic patients sera. v-gliadin and
gluten contents were significantly reduced in wheat extract treated with fer-
mented soybean extract. Treatment of wheat extract for 30 hours with the
fermented soybean extract effectively reduced the contents of some aller-
gens in wheat.
CONCLUSIONS: This study suggested that treatment of fermented soy-
bean extract to wheat should be useful tool in immunoprophylaxis as well
as functional wheat foods for the patients with wheat-associated allergic
disease.