Component resolved diagnosis in food allergy

Sadudee Boonmee, MD
Division of Allergy and Immunology
Department of Pediatrics
King Chulalongkorn Memorial Hospital

Topic outline
• Introduction
• Important protein families
• Allergen from animal origin (Egg, CM)
• Allergen from plant origin (Soy, wheat
and peanut)

Currently available tests detect only sensitization,
not clinical allergy, cannot predict prognosis and severity

Nature
Allergenic
source

contain a mixture of allergenic and
non-allergenic molecules

Introduction
• Specific IgE determined using allergen extracts as test
allergens have to 2 type of problem
1. the difficulty of starndardizing the allergens
used as substrates ( differ in allergenicity
variability of allergen source)

2. can not differentiated between primary
sensitization and immunological cross-reactivity

Borres et.al,Pediatric Allergy and Immunol 2011;22:454-461.

Introduction
• Both skin prick testing and specific IgE blood
testing employ heterogeneous protein
preparations  not always the best indicators
of clinical reactivity
• Component testing  designed to pinpoint
the allergen source when cross-reactive
allergen components are present.

Borres et.al,Pediatric Allergy and Immunol 2011;22:454-461.

Introduction
• Identifying whether the sensitization is
genuine in primary (species-specific) or if it is
due to cross-reactivity to similar protein
structure may help to evaluate the risk of
reaction on exposure to difference allergen
source

Clinical and Experimental Allergy;2010 ,40: 896-904

Basic of allergen component

1. Almost anything containing proteins can be
an allergen source
- each allergen source contains many different
allergenic protein (allergen component)
- each allergen component commonly has several
different epitopes
- epitope is the actual three-dimensional binding
site for an antibody



2. Protein stability
- allergen that stable to heat and digestion
are more likely to cause severe reaction

- allergen that heat and digestion labile are
more likely to be tolerateed or only cause
mild/local symptom



3. Allergen component name
- the first 3 letter of the genus, follow by the first
letter of the species and the number indicating
the order of allergen identification

- Latin designation of peanut is
Arachis hypogaea thus Ara h 1 designated
allergen number 1 from peanut



• Production technique of allergen component
can be
- biotechnological in “recombinant” from
- “purified” from (original source)

Important allergen families

• Most allergen component belong to a limited
number of protein families

• IgE antibodies in the same protein family
often cross-reactive, and classifying allergen
component into protein families can
answering question on cross-reactivity

Plant allergen

LTP (non-specific Lipid Transfer Protein, nsLTP)
- A protein stable to heat and digestion causing reactions also to
cooked foods.
- Often associated with systemic and more severe reactions in
addition to OAS.
- Often associated with allergic reactions to fruit and vegetables in
southern Europe.

Storage proteins
- Proteins found in seeds serving as source material during the growth
of a new plant.
- Often stable and heat-resistant proteins causing reactions also to
cooked foods.

PR-10 protein,Bet V1 homologue
- A heat labile protein, cooked foods are often tolerated.
- Often associated with local symptoms such as oral allergy syndrome
(OAS).
- Often associated with allergic reactions to fruit and vegetables in
northern Europe.

Profilin
-An actin-binding protein showing great homology and cross-reactivity even
between distant related species.
- Recognized as a minor allergen in plants and plant related foods.
- Seldom associated with clinical symptoms but may cause demonstrable or even
severe reactions in a small minority of patients.

CCD
- Cross-reactive carbohydrate determinants are seldom associated with clinical
symptoms but may cause demonstrable or even severe reactions in a small
minority of patients.

Animal allergen

Lipocalin
- Stable proteins and important allergens in animals
- Allergen components displaying limited cross-reactivity between
species.

Parvalbumin
- A major allergen in fish.
- A marker for cross-reactivity among different species of fish and
amphibians.
- A protein stable to heat and digestion causing reactions also to
cooked foods.


Tropomyosin
- An actin-binding protein in muscle fibres.
- A marker for cross-reactivity between crustaceans, mites and
cockroach.

Serum albumin
- A common protein present in different biological fluids and solids e.g.
cow’s milk and beef, eggs and chicken.
- Cross-reactions between albumins from different animal species are
well known, for example between cat and dog and cat and pork.

Egg (Gallus domesticus)
Allergen component in egg white Gal d 1, 2, 3, 4

Gal d 5 alpha-livetin (serum chicken albumin)
Clinical& experimental allergy; 2010: 40: 1442-1460

Gal d 1(ovumucoid)
- 10% of total egg white protein
- stability against to heat and digestion by
protease (strong disulfide bonds)  capacity to
stimulate a specific immune response
- sIgE Ab to Gal d 1  helpful in prognosis
and diagnosis of egg allergy
- High concentration of ovomucoid-sIgE
associated with persistent egg allergy
(raw or cooked egg )
- Low concentration of ovomucoid-sIgE
associated with tolerance to heated egg

Gal d 2 (ovalbumin)
- heat-labile, IgE - binging epitopes on
OVA might be detroyed after heating 
less allergenic
- children who have sIgE primary to OVA
likely to tolerate heated egg


Gal d 3(ovotransferrin)
- nonheme iron-binding
- heat-labile allergen
- sIgE Ab to ovotransferrin in Dx of egg
allergy not been determined
Gal d 4 (lysozyme)
- lysozyme commomly used as a food
preservative(antibacterial)
- egg allergic pt. who sensitized to lysozyme
may react when exposured to such product

Allergenic component in egg yolk : Gal d 5
Gal d 5 (alpha-livetin) = chicken serum albumin
- bird-egg syndrome
Vitellenin(apovitellenin I) Role in egg allergy
apoprotein B (apovitellenin VI) remain unclear


• Objective: to evaluate the clinical usefulness and added
diagnostic value of measurements of IgE antibodies to egg
white, ovalbumin, and ovomucoid in children with egg allergy.

• Methods: 108 children age 14 mo – 13 y (median age, 34.5 mo)
with suspected egg allergy underwent double-blind, placebo
controlled food challenges with raw and heated egg.

• The outcomes of the challenges were related to the serum
concentration of specific IgE antibodies and total IgE by using
ImmunoCAP

JACI 2008;122:583-588

• Pt. devided into 3 group ( immediated reactions to OFC)
- Group A (n=38) : positive challenge for heated egg white
- Group B (n=29) : tolerated heated egg white, positive
challenge to raw egg white
- Group C (n=41) : tolerated both heated and raw egg white

• Serum of all pt. obtain before food challenge
- sIgE egg white, sIgE to ovalbumin, sIgE to ovomucoid, total
IgE  ImmunoCAP

JACI 2008;122:583-588

Results
Heated EW + Raw EW + Heated & raw EW -

Heated EW + Raw EW + Heated & raw EW -

JACI 2008;122:583-588

Heated Raw
Heated Raw Heated & raw EW -
EW + EW + Heated & raw EW -
EW + EW +

Heated Raw Heated Raw
EW + Heated & raw EW - EW + EW + Heated & raw EW -
EW +

JACI 2008;122:583-588

children allergic to raw egg white
(ie, groups A and B vs group C; A)

sIgE Ab to egg white well in diagnosis of reactions to raw egg
white
- positive decision point was 7.4 kUA/L
- negative decision point was 0.6 kUA/L

children allergic to heated egg white
(ie, group A vs groups B and C; B).

sIgE Ab to ovomucoid showed the best results in
patients who react to heated egg white
- positive decision point was 10.8 kUA/L
- negative decision point was 1.2 kUA/L

• Quantification of sIgE to OVM could be useful in guiding
physician in decision whether to perform a challenge or not
• OVM > 11 kUA/L  high risk of reaction to heated and raw
egg
• OVM < 1 kUA/L  low risk of reaction to heated egg (pt. may
react to raw egg)

Clinical implications: Measurement of specific IgE
antibodies to egg white and ovomucoid might be
helpful in guiding evaluations of children for allergy to
raw egg or heated egg (eg, in cakes/cookies).
JACI 2008;122:583-588

Cow’s milk (Bos domesticus)

Caseins

Major allergen in milk
Whey

Caseins= 80% of total protein
β lactoglobulin (β-LG) not present in human milk
Curr Opin Allergy Clin Immunol 11: 216-221

α-lactoglobulin (Bos d 4)
- Functional subunit of whey lactose syntase
- controversial role in milk sensitization
- 80% of CMPA react to Bos d 4
β-lactoglobulin (Bos d 5)
- most abundant whey protein
- Pt. have allergic reaction to β-lactoglobulin 13-76%
Bovine serum albumin (BSA) (Bos d 6)
- cow’s milk and beef allergy
- CMPA pt. have positive to BSA 0-88%
(only 20% have clinical reaction)


Bovine immunoglobulins (Bos d 7)
- in blood, tissue fluid, secretion
- seldom cause clinical symptom in CMPA
Caseins (Bos d 8)
- αs1-caseins, αs2-caseins, β-caseins, κ-caseins
- polysensitization to many caseins fraction
usually observed (closely epitope or cross-
sensitization )
- αs1-caseins major allergen inducing strong
immediated or delayed allergic reactions


• Objective
: to calculate the frequency of accidental
exposure reactions in children allergic to cow’s
milk during a 12-month period

: to analyze clinical characteristics and
circumstances surrounding the reactions

: to identify risk factors for severe reactions

(J Allergy Clin Immunol 2009;123:883-8.)

• Methods
: 88 children allergic to cow’s milk (44 boys; median age,
32.5 months)
: cross-sectional study at age >= 18 mo
: Systematized questionnaire about accidental
exposure (reactions were classified as mild,
moderate, and severe)
: sIgE to cow’s milk & casein titers were determined
periodically as clinical indicated in F/U visit


Severe reaction 15%

mo

• Result

Five of the 6 children had asthma


Conclusion : Reactions to accidental exposure are frequent in
children with cow’s milk allergy. The proportion of severe
reactions was 15%. The risk factors for severe reactions
included very high levels of specific IgE to cow’s milk and
casein and asthma.

P, Percentile.
Mann-Whitney U test: #/{P 5 .044; #/kP < .01; /P 5 .044; /**P < .01.
*P value: comparison between severe AARs versus mild or moderate AARs or no reaction.
Fisher test.
Mann-Whitney U test.
§x2 Test.


fir
- Retrospective studies of 52 patients (3 to 114
months) with proven CMA by DBPCFC
- All Pt were rechallenge at least once , some were
rechallenge 2 or 3 time
- 32 (61.5%) patients became tolerant in the analysed
time period.
- Serum was collected at all time point prior to OFC 
analysed for

B. Ahrens, et.al, Clinical & Experimental Allergy, 2012 ( ) –

• Result
sIgE CM

Children with initial sIgE CM
below 5.0 kU/L  4 time
likely to tolerant

18
33 mo
mo


• Conclusion
• Children became tolerant earlier if their specific IgE-antibody
levels against the α-lactalbumin, and β-lactoglobulin(Bos
d5.0102), αs1- casein and, κ-casein were low initial low
Children with
(<0.3ISU/L) sIgE to α lactalbumin,
• CM-specific IgE is a good prognostic marker for persistence of CM
β lactoglobulin(Bos d 5.0102),
κ casein and αs1- casein  7.2,
2.7, 3.8 and 2.7 time likely to
tolerant respectively
18
mo


Soy (Glycine max)
Allergen component in soy : Gly m 4, 5 and 6
Gly m 4
- PR-10 protein family (Bet v 1 homologue)
- pollen-associated soy allergy mainly discribed
in adult with mild symptoms
(primary sensitization to birch pollen)
- cross-reactive to Ara h 8
- In Europe approximately 2/3 of pt. allergic to
soy are allergic to peanut


Soy (Glycine max)
Gly m 5 and 6
- most important in primary sensitization to soy
protein (via GI tract) esp. in children
- sensitization rate to
Gly m 5 36%
In soy allergic pt.
Gly m 6 43%
Gly m 5 and Gly m 6 are diagnostic marker for
severe allergic reaction to soy


Paper soy2paper

• Aim : to identify relevant soybean allergens and correlate the
IgE-binding pattern to clinical characteristics in European
patients with confirmed soy allergy

• Methods : The IgE reactivity in 30 sera from subjects
(Switzerland, Denmark and Italy)— with a positive double-
blind, placebo-controlled soybean challenge (n = 25)
or a convincing history of anaphylaxis to soy (n = 5) was
analyzed by ELISA or CAP-FEIA

J Allergy Clin Immunol 2009;123:452-8.

The 2 soybean major storage proteins
- Gly m 5 (β-conglycinin : 3 subunits)
: α (67kd)
: α ‘ (71 kd)
:β (50 kd))
- Gly m 6 (glycinin : 5 different subunits)
: G1 (A1aB1b, 53.6 kd)
: G2 (A2B1a, 52.4 kd)
: G3 (A1bB2, 52.2 kd)
: G4 (A5A4B3, 61.2 kd)
: G5 (A3B4, 55.4 kd)
• IgE reactivities major storage Protein (8 subunits) of the
proteins were correlated to the clinical characteristics of our patient group
to investigate the potential use as biomarkers for severe allergic reactions
to soybean.


Gly m 5 +ve = 13/30 (43%)
Gly m 6 +ve = 11/30 (37%) Clinical implications:
Gly m 5 ot Gly m 6 +ve =16/30(53%)
Component-resolved diagnosis
with purified Glym 5 and Glym 6
is potentially applicable for
identifying subjects at risk of
experiencing severe soy-related
allergic reactions.


Wheat (Triticium aestivum)
Tri aA_TI, Tri a 18, Tri a gliadin, Tri a 19 (Ѡ- 5 gliadin)
and high molecular weigh glutenin
Tri a 14
- Lipid transfer protein
- heat resistance and lack of cross-reactivity to
pollen
- major allergen in pt. living in southern Europe
and significant in baker’s asthma
Tri a 18
- Hev b 6 homologue (latex component)


Tri a 19 (Ѡ-5 gliadin)
- associated with risk of IgE-mediated reaction to
wheat

- risk of wheat-dependent exercise induce
anaphylaxis


Tri aA_TI (alpha-amylase/tyrpsin inhibitor)
- wheat allergen in raw and cooked food
- play role in wheat-depedent exercise induce
anaphylaxis

High molecular weigh glutenin
- associated with wheat-dependent exercise
induce anaphylaxis

Tri a gliadin
- primary wheat sensitization (low-risk of pollen cross-reactivity)


• Aim : to examine whether ω-5 gliadin is a clinically relevant allergen in
children with hypersensitivity reactions to ingested wheat.
• Method
: Sera were obtained from 40 children (mean age, 2.5 years; range,
0.7-8.2 years) with suspected wheat allergy (AD a/o GI a/o
respiratory symptoms
: Wheat allergy Dx with open or DBPC oral wheat challenge.
: Wheat ω-5 gliadin was purified by reversed-phase
chromatography,and serum IgE antibodies to ω-5 gliadin were
measured by ELISA
: In vivo reactivity was studied by SPT.
: Control sera were obtained from 22 children with no
evidence of food allergies

J Allergy Clin Immunol 2001;108:634-8

Immediated
- 16/19(84
%)
detected
• Result IgE to Ѡ-5
gliadin
- 6/7 SPT
+ve to Ѡ-5
gliadin

Delayed

- not detected
IgE to Ѡ-5 gliadin
- 2/2 SPT –ve to
Ѡ-5 gliadin


Conclusion: The results of this study
show that ω-5 gliadin is a significant
allergen in young children with
immediate allergic reactions to
ingested wheat. IgE testing with ω-5
gliadin could be used to reduce the
need for oral wheat challenges in
children.

Morita,et al. Allergology International.2009;58:493-498

Peanut (Arachis hypogaea)

- most common food associated with fatal
allergic reaction in western country, the
prevalence also increase in Asia
- 13 peanut allergen have been identified
- 5 peanut component are clinically
relevant and available

Ara h1, 2, 3, 6, 8, and 9


Ara h 1, 2, and 3
- major peanut allergen (peanut seed storage protein)
- associated with primary sensitization
- pt. with peanut allergy are sensitized to
Ara h 1 35-95%
Ara h 2 75-100%
Ara h 3 20-55%
- stable to heat and digestion  risk of reaction to
cooked or roast food


Ara h 1
- cross-reactive to nut and legume [lentil,pea,
and soy(Gly m 5)]
Ara h 2
- marker for primary sensitization to peanut and
risk marker for severe allergic reaction
- cross-reactive to lupine and tree nut
(eg. almond and brazil nut)
Ara h 3
- cross-reactive to soybean, pea, and tree nuts

Ara h 6
- high homology to Ara h 2
Ara h 8
- PR-10, Bet V 1 homologue
- heat-labile protein cooked food often tolerated
- primary sensitization through pollen : birch,cedar
- cross-reactive to lupine and soy (Gly m 4)
Ara h 9
- Lipid transfer protein (LTP)
- associated with severe OAS and systemic reaction
- primary sensitization probably due to primary
sensitization to peach or other LTP-containing fruits

• Aim
- To establish by oral food challenge the proportion of
children with clinical peanut allergy among those
considered peanut-sensitized by using skin prick tests
and/or IgE measurement
- To investigate whether component-resolved diagnostics
using microarray could differentiate peanut allergy from
tolerance.
• Method
- The Manchester Asthma and Allergy study 
unselected population based birth cohort
- participants recruited prenatally and F/U prospectively
at age 1, 3, 5 and 8 yr ( questionnaire, SPT, sIgE )

13 OFC

85 were sensitized to
peanut


- Peanut allergy fold change = 6.06  expression level 66.7 time higher than
negative control
- Peanut tolerant fold change = 0.28  expression level 1.2 time higher than
negative control

Clinical implications: Measurement of IgE response to
major peanut allergen Ara h 2 is more useful in predicting
clinical peanut allergy than currently used skin or blood
tests based on whole extract.
Green : peanut tolerant
Yellow : peanut allergy

Advantage of CRD
• Quantifying IgE antibodies to a single allergen
protein component rather than a
heterogeneous allergen preparation
• Providing a unique component-specific IgE
antibody profile of the patient for
personalized medical care
• Supporting a molecular-level understanding of
the patient’s individual constellation of
symptoms

Advantage of CRD
Advantage of CRD
• Better characterize the patient’s sensitization patterns
• Better understand the clinical risk for reactions in a
patient
• Determine the presence of cross-reactivity between
allergens
• Facilitate food challenge testing with increased
knowledge of what to expect
• Select appropriate patients for specific immunotherapy
and customize treatment

Allergen Components as severity markers

Peanut Ara h 1, 2 and 3

Soy Gly m 5 and 6

Wheat Omega-5-gliadin

Milk Bos d 8 (casein)

Egg Gal d 1 (ovomucoid)

Important allergen
Ara h 1, 2, 3, 8 and 9 (peanut)
• IgE abs to Ara h 1, 2, 3 and 9 (LTP) are associated with
peanut reactions, which in many cases can be severe.
• IgE abs to Ara h 8 (PR-10) are usually associated with
milder, local symptoms such as OAS, originting from birch
sensitzation.

Ovumucoid (egg white)
• IgE abs to ovomucoid are associated with persistent egg
allergy and usually neither raw or cooked is tolerated.

Important allergen
Omega-5 gliadin (wheat)
• IgE abs to omega-5 gliadin (Tri a 19) in adults are associated with a
risk of exercise- or NSAIDs-induced reactions in connection with
wheat ingestion.
• IgE abs to omega-5 gliadin in children are associated with a risk of
immediate reactions to wheat.

Gly m 4,5 and 6 (soy)
• Gly m 5 and 6 are associated with clinical reactions to soy.
Gly m 5 - Ara h 1 and Gly m 6 - Ara h 3 share homologoues
structures.
• IgE abs to Gly m 4 (PR-10) are usually associated with milder, local
symptoms such as OAS, originating from birch sensitzation.

Component resolved diagnosis in food allergy

Component resolved diagnosis in food allergy

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