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Seminario: Identification of major rice allergen and their clinical significance in children
1. Identification of major rice allergen and
their clinical
significance in children
You Hoon Jeon, MD, Se Jo Oh, PhD, Hyeon Jong
Yang, MD, Soo Young Lee, MD, Bok Yang Pyun, MD
Manuela Colorado
Carolina Giraldo Benítez
III Semestre Medicina
UPB
2. INTRODUCTION
Abnormal, inappropriate and exaggerated
Allergy reaction of immune system to substances that
typically are not well tolerated. Is mediated by
IgE
Is produce by allergens
• Food allergy is different from food intolerance
• Allergen: is a substance that can induce a hypersensitivity (allergic)
reaction in susceptible people, who have previously been in contact
with the allergen.
3. INTRODUCTION
• Rice is known to be a relatively low allergenic food, but have become a problem.
• A study shows that not eat rice has helped at 76% of 25 patients with atopic
dermatitis with IgE-rice positive.
• Is a few reports on the correlation of rice allergen sensitization and their clinical
symptoms.
• The immunologic characteristics of rice allergen are unclear.
4. INTRODUCTION
• Some allergens have been mentioned as possible major allergens of rice:
-Globulin in the embryonic bud of rice
-Albumin with protein bands of 14-16 kDa
• Protein bands of 26, 33, 56 kDa are the major IgE-binding components in rice.
• Some studies reported that rice is less allergenic if it is heating first.
• Heat- and acid-stable protein, lipid transfer protein (LTP) has emerged recently as
a relevant factor in symptomatic rice allergies.
5. GENERAL OBJECTIVE
• Determine the major allergens in rice and their
clinical significance.
Objetivo
http://freestyle.bligoo.com.co/objetivos
7. MATERIALES Y MÉTODOS
44 Pacientes
Grupo control
IgE-Arroz Negativa
2 Grupos
n=18 n=26
Sujetos sensibilizados, pero Sujetos no sensibilizados
no al arroz
8. MATERIALES Y MÉTODOS
2. Métodos
1. Análisis de sujetos y grupo control
• Historial médico y pruebas de laboratorio Edad, sexo,
síntomas de la alergia, IgE en suero, ECP, IgE específica.
Erupción cutánea, eritema, urticaria, angioedema, el síndrome
de alergia oral, rino-conjuntivitis, sibilancias, anafilaxia tras la
ingesta de arroz con leche, arroz hervido, galletas de arroz,
etc.
* Agravación de la dermatitis atópica se excluyó.
11. MATERIALES Y MÉTODOS
1. Sodio dodesil sulfato policrilamida gel electroforesis. (SDS PAGE)
• Muestra + Buffer Se calentó
• Electroforesis durante 90 minutos SDS (amortiguación con el marcador)
• Visualización por tinción con azul de coomassie brillante
• Cuantificación de bandas Densitometría
12. MATERIALES Y MÉTODOS
4. Análisis de las bandas de proteínas de arroz y arroz
cocido
• Arroz crudo
• Arroz cocido
Electroforesis:
• Arroz integral cocido
• Arroz semicocido
Comparación y análisis de distribución de bandas
SDS PAGE Electroforesis
http://www.invitrogen.com/site/us/en/home/Products-and-Services/
Applications/Protein-Expression-and-Analysis/Protein-Gel-
Electrophoresis/Protein-Gels/Novex-NuPAGE-SDS-PAGE-Gels.html
13. MATERIALES Y MÉTODOS
5. SDS PAGE de arroz tratado con calor, fluido gástrico simulado (SGF) y fluido
intestinal simulado (SIF)
•Electroforesis en serie
•Proteína de arroz se hirvió a 100°C 10, 30 y 60 minutos Electroforesis
Se trató con enzima digestiva, SGF y SIF 0, 10, 30 y 60 minutos
5 μL de mezcla en
12 μL de buffer tubos de ensayo
y 1 μL de 0,2 M
NaCO3
Electroforesis
Comparación de bandas
de proteínas
14. MATERIALES Y MÉTODOS
6. Inmunoblot de IgE del arroz
En 18 de los 24 pacientes 4 Sueros para el control
Bandas de proteínas Electrotransferidas desde Bloqueada con 3% de albúmina
el gel a una membrana de suero bovino-fosfato-buffer-
nitrocelulosa salino por una hora
>16 Horas a 4°C Adición de suero Lavado 3 veces con PBS-T
alérgico y suero control durante 10 minutos
a la membrana
Lavado con PBS-T
15. MATERIALES Y MÉTODOS
Conjugación con 2 horas a Conjugación con
anticuerpo biotinilado temperatura avidinfosfatasa por
anti IgE humana ambiente 30 minutos
* Las bandas de proteínas del arroz calentado
se electrotransfirieron y se les realizó
inmunoblot de forma similar.
16. RESULTADOS
Fig. 1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of (A) raw rice, (B) cooked rice, (C) cooked
unpolished rice, and (D) semi-cooked rice (Hatban). M, marker; kDa, kilodalton.
18. RESULTADOS
SDS-PAGE para alérgeno del arroz
Arroz cocido,
Arroz integral cocido
intensidad
Arroz semicocido
Bandas de proteínas 14-16,
31 - 35 kDa.
19. RESULTADOS
Fig. 2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of rice treated with (A) heat, (B) simulated
gastric fluid (SGF), and (C) simulated intestinal fluid (SIF). M, marker; kDa, kilodalton.
20. RESULTADOS
Cambios en las bandas de las proteínas del arroz después del tratamiento
con calor y enzimas digestivas
10’ bandas se mantienen.
30’ bandas se mantienen- desvanecimiento.
60’ bandas degradadas, menos 9,14, 31-35 kDa.
21. RESULTADOS
Cambios en las bandas de las proteínas del arroz después del tratamiento
con calor y enzimas digestivas
Bandas degradadas inmediatamente.
60’ bandas 9, 14, 28, 31, 35 kDa se mantuvieron.
22. RESULTADOS
Cambios en las bandas de las proteínas del arroz después del tratamiento
con calor y enzimas digestivas
Pocas bandas degradadas.
23. RESULTADOS
Cambios en las bandas de las proteínas del arroz después del tratamiento
con calor y enzimas digestivas
9, 14, 31 kDa son estables
después del procesamiento con calor
digerido con las enzimas en el estómago y el intestino
24. RESULTADOS
Fig. 3. Rice immunoglobulin E immunoblot of patient sera and control. M, marker; kDa, kilodalton.
25. RESULTADOS
Inmunoblot de IgE del arroz crudo
Gel de electro-transferencia
Inmunoblot de IgE suero 18 pacientes sensibilizados
grupo control
Proteínas alergénicas
Actividad de unión al IgE
29. RESULTADOS
Inmunoblot de IgE del arroz calentado
Gel de electro-transferencia
Inmunoblot de IgE suero 18 pacientes sensibilizados
control
bandas descoloridas
30. RESULTADOS
Inmunoblot de IgE del arroz calentado
Proteínas
14 kDa 38,9%
9 kDa 27.8%
31,33 kDa 38.9%
31. RESULTADOS
Inmunoblot de IgE del arroz calentado
Suero de 4 pacientes con
síntomas alérgicos (5,11,15,16)
Reaccionaron con bandas de proteína
31-33 kDa 75%
9 kDa 50%
14 kDa 50%
33. DISCUSSION
Name Approach True or False
Wu KG, et al. There are several studies that serum ECP levels in False: because in food allergy, the
Shin JE, et al. patients with atopic dermatitis is related with relationship between serum ECP level
disease severity. with clinical food allergy, sympton is
not evident.
Figueredo E, et al. An atopic woman, who developed anaphylaxis after True: because this suggests the
drinking beer with a corn made snack, was sensitized possibility of latent sensitization by a
to not only corn and barley, but also to wheat, rye, common epitope.
oat and rice.
34. DISCUSSION
Name Approach True or False
Yamada C, et al. Heat-processing or digestion can alter antigenicity True: because the allergen rice is
Untersmayr E, et of allergen. The rice allergen is weak after the weak after heating and the
al. heat-process. The antigen specific IgE of rice bands decreased in intensity and others
Kilshaw PJ, et al. decreases after heating at 100℃ for 60 minutes. disappear in cook rice.
Yum HY, et al. Studies have revealed cross reactivity of rice with False: Because the allergens
buckwheat, barley or wheat and one report that actually cause the allergic symptoms
compared the antigenicity of rice and low on the patients with sensitization to rice
allergenic rice. allergen are not obvious and an
interpretation of the sensitization to rice
is also unclear.
35. CONCLUSSIONS
• Although rice isn't has high allergenicity, in Asian countries like Japan and Korea,
the allergenicity has increased in the numbers of patients sensitized to rice
allergen, because it’s the main food in the meals.
• The allergenicity decrease when the rice is cooked, because the protein
bands decreased in intensity and others disappear in cook rice. Also the allergen
rice is weak after heating.
36. CONCLUSSIONS
• Total IgE, is an important factor in terms of
sensitivity to rice, because in these cases the IgE
is increased along with the ECP (eosinophil cationic
protein).
• This investigation is very important, because this area
is poorly studied, and also help to find the
inmunologic characteristics of rice allergen .
