Egg allergy: new allergens and molecular diagnosis

Egg Allergy
7/9/2012

Suparat Sirivimonpan,MD.

Egg allergy

Introduction
Prevalence
Egg allergen : new allergen
Diagnosis : molecular diagnosis

Introduction

• Egg allergy may be defined as an adverse reaction of
immunologic nature induced by egg proteins
– IgE ,non-IgE , mixed IgE-mediated disorders

• hen’s egg allergy is the second most common food
allergy in infants and young children

• closely associated with atopic dermatitis
• Increase risks of sensitization to aeroallergens and
asthma in children with egg allergy

Pediatr Clin N Am 2011;58:427–443

Prevalence

• estimated prevalence : varies depending on method
of data collection or definition

• A meta-analysis of the prevalence of egg allergy in
children
– Self-reported prevalence : up to 7%
– challenge-confirmed egg allergy : up to 1.7%

Allergy 2010; 65: 283–289

Prevalence

In Thailand
• Santadusit S, et al.
– Egg allergy : 1.52%

J Med Assoc Thai 2005;88:S27-32

• Unpublished data 2012, Chulalongkorn university
– egg allergy : 1.1% (parent reported and physician diagnosis) in young
children

Egg allergens

• 5 major allergenic proteins : Gal d 1 to Gal d 5 (Gallus
domesticus)

Curr Allergy Asthma Rep 2006;6(2):145–52

Egg white allergens

Allergy 2010; 65: 283–289

Ovomucoid

• dominant allergen in egg
• unique characteristics
– relative stability against heat and digestion with proteinases
– presence of strong disulfide bonds that stabilize protein
• children with persistent egg allergy had significantly higher
specific IgE levels to OVM than children who outgrew their egg
allergy
• A favorable prognosis was associated with the absence or a
decline in OVM-specific IgE titers

Allergy 2007; 62:758–765
Curr Opin Allergy Clin Immunol 2011, 11:210–215

Ovalbumin

• heat-labile and less allergenic

• IgE-binding epitopes on OVA might be destroyed
after heating : children who have specific IgE
primarily to OVA are likely to tolerate heated egg

Curr Opin Allergy Clin Immunol 2011, 11:210–215

Ovalbumin

• The Maillard reaction occurs between reducing sugars and
proteins during thermal processing of foods

• It produces chemically glycated proteins termed advanced
glycation end products (AGEs)  T-cell immunogenicity of
chemically glycated ovalbumin

• The glycation structures of AGEs are suggested to function as
pathogenesis-related immune epitopes in food allergy

J Allergy Clin Immunol 2010;125:175-83.

Newly identified egg white allergen

Allergology International. 2010;59:175-183

• Diluted egg white proteins were separated by 2-dimensional
(2-D) gel electrophoresis

63 spots


• Immunolabeling was performed on individual patient sera
• 19 child patients with egg white allergy
• 11 negative control subjects

19 patients 11 negative control subjects
-Aged 2.2 ± 1.7 years -Aged 4.0 ± 3.7 years
-Total IgE 434.1 ± 424.7 Iuml -no clinical history of any allergic
-Diagnosis was based on clinical history symptoms
and CAP-RAST results to hen’s egg
white
-Open food challenges : 9/19 patients


15/25
• Spots of egg white proteins that bound to the patients’ IgE
were identified by mass spectrometry-based proteomics

10/25


Identification of IgE-reactive
spots
• spots were excised
• digested in-gel with trypsin
• The resulting peptide mixtures were analyzed by
26% pt – LC-MS/MS (Liquid chromatographytandem mass
16% pt
spectrometry)
L-PGDS
– MALDI-TOF/MS (matrix assisted laser desorption
(1 anaphylaxis)

ionization time of flight mass spectrometry)

53% pt


L-PGDS

Cystatin

• Reaction to LPGDS and cystatin was confirmed using each purified
protein

Ch21 protein and L-PGDS

• Ch21 protein and L-PGDS belong to the lipocalin Family
• has been reported as allergenic
• Ch21 protein and L-PGDS were minor allergens

• However, the sera from patient (MT,HS), which reacted to spot no.
28 did not seem to react to the major spots, such as nos. 3, 4, 5, 48, or 49
 not only the hidden allergen components, but there
are some patients who are preferentially reacting to those
minor egg allergens
• minor allergen does not always mean that it is allergenically
poor


Ch21 protein and L-PGDS

• PGD2 synthesized by L-PGDS is related to allergic
inflammation in vivo and that L-PGDS from chicken shows
PGDS activity

• These reports suggest that L-PGDS induces allergic reactions
in addition to binding to IgE, and that PGD2 may exist in eggs


Cystatin
• cysteine protease inhibitor
• allergen in cats and dogs
• considered to be a major allergen because it showed high
reactivity to IgE from patients with egg allergy

• 2 possible reasons for the lack of literature on cystatin as an
allergen
– similarity in molecular weight of cystatin and lysozyme, a
major allergen, makes it difficult identify cystatin without
using 2-D gel electrophoresis
– presence of only a small amount of cystatin in eggs makes
it difficult to identify without development of LC-MSMS


Egg yolk allergens

• alpha-livetin or chicken serum albumin (Gal d 5) is
the major allergen and is involved in the bird-egg
syndrome
• Several other allergens have been identified in egg yolk
– vitellenin (apovitellenin I) and
– apoprotein B (apovitellenin VI)
– roles in food allergy remain unclear


Bird-egg syndrome

• primary sensitization is to airborne bird allergens (feathers,
droppings, serum) and there is secondary sensitization or cross-
reactivity with albumin in egg yolk (Gal d 5)
• respiratory symptoms such as rhinitis and/or asthma with bird
exposure
• allergic symptoms (respiratory and gastrointestinal symptoms) when
egg is ingested
• In children with allergy to birds and egg, egg allergy is usually more
persistent
• Sensitization to other aeroallergens is also greater in individuals with
allergy to birds and egg

Allergy 2001: 56: 754-762
Allergol et Immunopathol 2003;31(3):161-5

J. Agric. Food Chem. 2010, 58, 7453–7457

• Sera from 27 patients (ages 2-74 yrs) with egg allergy
(clinical history and both positive-specific IgE and SPTs to egg)
• sera from 2 non-allergic subjects as negative controls

• The study was performed by
– sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and
– IgE-immunoblotting and
– IgEimmunoblotting- inhibition assays
• egg yolk extract was fractioned by reverse-phase high performance
liquid chromatography (RP-HPLC)
J. Agric. Food Chem. 2010, 58, 7453–7457

• A new allergen with an apparent molecular weight around 35 kDa was identified
from the yolk extract and enriched by RPHPLC (Figure 1A)
• 5/27 patients studied (18%of patients) showed specific IgE binding to the allergen
(Figure 1B)

J. Agric. Food Chem. 2010, 58, 7453–7457

• new allergen detected was characterized by N-terminal amino acid analysis

• revealed that allergen was the yolk glycoprotein 42 (YGP42) protein,
a fragment of the vitellogenin-1 (VTG-1) precursor (positions 1628-1912)

• The allergen has been designated Gal d 6.0101 by the World Health
Organization (WHO)/International Union of Immunological Societies
(IUIS) Allergen Nomenclature Subcommittee

J. Agric. Food Chem. 2010, 58, 7453–7457

• heat-resistant but digestible by pepsin
J. Agric. Food Chem. 2010, 58, 7453–7457

J Allergy Clin Immunol 2004;113:805-19

• Conformational epitopes can be destroyed with heating or partial hydrolysis

• Egg-specific IgE molecules that identify sequential or
conformational epitopes of OVM and OVA can distinguish
different clinical phenotypes of egg allergy
– patient with IgE antibodies reacting against sequential epitopes
tend to have persistent allergy
– IgE antibodies primarily to conformational epitopes tend to
have transient allergy

Diagnosis

• Double-blind, placebo-controlled food
challenge : gold standard

• open food challenges (OFCs)
– Less resource-intensive
– are generally considered sufficient in clinical
practice

Curr Opin Allergy Clin Immunol2009;9:244–250

Diagnosis

• detailed history and physical examination
• in vitro and/or in vivo allergy tests
– used to support diagnosis
– food-specific IgE antibodies
– skin prick tests
– diagnostic elimination diet
– OFC


Skin prick tests

• used in screening for egg-specific IgE and should be
performed by trained personnel
• diagnostic accuracy is dependent on quality of the extract,
which should be standardized

• SPT shows a good sensitivity and NPV, but poor specificity and
PPV
– negative test essentially excludes an IgE-mediated egg
allergy
– positive test does not predict clinical reactivity accurately

Allergy 2010; 65: 283–289

Skin prick test for diagnosis
References Year Age group (yr) Method Numbers of PPV SPT Wheal
of Dx patients diameter (mm)

Sampson and 1997 Children and DBC 100 85 ≥3*
Ho1 adolescents
Sporik et al2 2000 <2 OC 39 100 ≥ 5 EW
>2 82 ≥ 7 EW
Boyano- 2001 <2 OC 81 93 ≥ 3 EW,EY
Martinez et al 3

Monti et al4 2002 <19 mo (mean 16 mo) OC 107 100 ≥ 5 EW, EY
Hill et al5 2004 <2 OC 30 100 ≥5*
Verstege et al6 2005 All(3 mo-14.5yr) DBC/OC 160 95 ≥ 13 fresh mixed EW&EY
<1 26 ≥ 11.2
>1 134 ≥ 13.3
1JAllergy Clin Immunol 1997;100(4):444–51 4Clin Exp Allergy 2002; : –
2Clin Exp Allergy 2000;30(11):1540–6 5Pediatr AllergyImmunol 2004;15(5):435–41
3Clin Exp Allergy 2001;31(9):1464–9 6Clin Exp Allergy ; ( ): –

Serum specific IgE
References Year Age group (yr) Method Numbers of PPV Egg-specific IgE
of Dx patients (kUA/L)
Sampson and 1997 Children and DBC 100 95 6*
Ho1 adolescents
Sampson2 2001 3 mo-14 yrs DBC 75 98 7*
(median 3.8yr)

Boyano- 2002 11-24 mo OC 58 95 2 EW
(mean 16 mo)
Martinez et al3
Osterballe and 2003 0.5-4.9 yr OC 56 95 1.5 EW
Bindslev-Jensen4 (median2.2yr)

Celik-Bilgili et 2005 0.1-16.1 yr OC All 277 95 12.6 *
al5 (median13mo) ≤ 1 yr 41 10.9
> 1 yr 186 13.2

1J Allergy Clin Immunol 1997;100(4):444–51 4 J Allergy Clin Immunol 2003; 112:196–201
2J Allergy Clin Immunol 2001;107:891-6 5Clin Exp Allergy 2005; 35:268–73
3 J Allergy Clin Immunol 2002;110(2):304–9

Serum specific IgE (cont.)
References Year Age group (yr) Method Numbers of PPV Egg-specific IgE
of Dx patients (kUA/L)
Komata et 2007 0.2-14.6 yr OC 764 95 ALL ≥ 25.5
al6 (median2.2yr) ≤ 1 : 13
1-2 : 23
≥ 2 : 30
Benhamou 2008 16 mo – 11.9yr OC/DBC 35 95 7*
et al7 (median 3.9 yr)

Ando et al 8 2008 14 mo – 13yr DBC 108 95 (raw EW EW 7.38
(median 34.5 mo) allergy) OVA 9.84
OVM 5.21

84-88 (heated EW 30.7
EW allergy)
OVA 29.3
OVM 10.8
6J Allergy Clin Immunol 2007; 119:1272–4
7Pediatr Allergy Immunol 2008; 19:173–9
8J Allergy Clin Immunol 2008; 122:583–8

MOLECULAR DIAGNOSIS IN FOOD ALLERGY

• specific responses at the level of individual allergenic proteins :
component-resolved diagnosis [CRD]
or
• IgE-binding epitopes of allergens : epitope mapping or profiling

Immunol Allergy Clin N Am 2012;32:97–109

Microarray-based component-resolved
diagnosis

• ImmunoCAP-ISAC or Immuno Solid phase Allergen Chip
(VBC Genomics-Vienna, Austria; Phadia, Uppsala, Sweden)
• It currently has 112 native/recombinant component allergens
from 51 allergenic sources

• two main advantages:
– assesses simultaneously specific IgE to different components and
requires small amounts of serum(relevant in children)
– cost-efficient approach, as it delivers results for over 100 components

Curr Opin Allergy Clin Immunol 2011;11:210–215
Immunol Allergy Clin N Am 2012;32:97–109

• infants and children ,suspected IgE-mediated food (CM or HE)
– Skin (42%), respiratory (6%), GI (16%), combination of skin, RS,GI (32%),
anaphylactic shock (4%)
– Only atopic eczema were excluded
• all patients SPT, sIgE (ImmunoCAP and microarray) and open
food challenge test were performed

• Total 104 patients (62 males and 42 females), median age of 4.9
yrs (range 0.7–15.1)
• hen’s egg allergy 46 patient

Clinical & Experimental Allergy, 2010 (40) 1561–1570

• FCT resulted positive in 22/46 (48%) patients with suspected
HE allergy


95% predicted probability (CDP)of a positive FCT Clinical & Experimental Allergy, 2010 (40) 1561–1570

↓FCT≈15% ↓FCT≈37%

Sequential use of the
two tests did not lead to
a further improvement in
clinical performance

False negative remains relatively high  FCT should still be performed

• 68 children (47 male, 69.1%),
• Age 1 - 11 years (median 4.1 years)
• suspected HE allergy based on
– reported history of reactions like asthma, rhinitis, conjunctivitis,
urticaria, worsening of eczema, vomiting, other gastro-intestinal tract
symptoms, anaphylaxis, after ingestion or contact with HE,
– and positive SPT or IgE to HE white extracts

• 42/68 (62%) reported atopic dermatitis as the main allergic
complain

Clinical & Experimental Allergy 2012 :42;441-451

• Skin prick test
– Commercial extracts (Allergopharma, Reinbek, Germany) and fresh eggs using
the prick-prick technique
– Fresh: white and yolk from both raw and processed boiled HE
– Positive : wheal greater than 7 mm2
• Total and specific IgE
– HE white and yolk : ImmunoCAP (Phadia AB, Uppsala, Sweden)
– Specific IgE detection for Gal d 1, Gal d 2, Gal d 3 and Gal d5 : ISAC 103
microarray test (PMD, Vienna, Austria)
• Double- blind, placebo-controlled food challenge (DBPCFC)
– First : boiled egg (totol HE protein 6 g)
– Patients tolerating boiled HE were then challenged with raw HE
– patients with positive response to boiled HE challenge were considered likely to
be allergic to both boiled and raw eggs

No significant differences

significant differences,
P<0.01

significant differences


ImmunoCAP


ISAC
20 of 21 Gal d 1 positive
patients (95%) reacted to
raw egg.

44/47 Gal d 1 negative
patients (94%) could
tolerate boiled egg


ISAC – Other sensitization

• 13 patients had no additional IgE detectable sensitizations on
ISAC (103 microarray system)
– 10 were in ST group
– 3 in PT group
– none in A group

• 54 subjects : positive for at least one non-HE allergen
– higher sensitization prevalence to other food and nonfood
allergens in group A


A
PT
ST

Allerrgen group sensitization
LTP: Cor a 8, Pru p 3
Parvalbumin: Cyp c 1, Gad c 1
PR-10: Act d 11, Api g 1, Ara h 8, Bet v 1, Cor a 1.0101, Cora1.0401, Dau c 1, Mald 1, Pru p 1, Gly m 4
Profilin: Bet v 2, Hev b 8, Mer a 1, Ole e 2, Phl p 12
Tropomyosin: Der p 10, Ani s 3, Pen a 1,Pen i 1, Pen m 1;


statistically significant differences

Anisakis: Ani s 1
Kiwi: Act d 1, Act d 2,Act d 5
Cow’s milk: Bos d 4, Bos d 5, Bos d 8
Seeds: Ana o 2, Ara h1, Ara h 2, Ara h 3, Ber e 1, Cor a 9, Gly m 5,Gly m 6, Ses i 1;
Wheat: Tri a 18, Tri a 19, Tri a Gliadin

Cat: Fel d 1,
Cypress: Cry j 1, Cup a 1;
Dog: Can f 1, Can f 3
Grasses: Phl p 1, Phl p 2, Phl p 4, Phl p 5, Phl p 6, Phl p 11
Mites: Der p 1, Der p 2, Der f 1, Der f 2, Eur m 2
Olive: Ole e 1

• IgE detection to Gal d 1 on the ISAC microarray system useful
tool to predict oral tolerance to boiled eggs

• DBPCFC in egg allergic children should be performed first with
boiled eggs to introduce at least well cooked eggs in HE
allergic children’s diet

• Outgrowing clinical allergy leads to negativity of tests later on,
and ISAC IgE detection reaches such condition before all other
tests
– majority of the subjects tested negative for HE allergens on ISAC were in the PT and ST
groups


• Patients without previous egg exposure were recruited
between January 2005 and October 2011
• examined to determine their sIgE titers (mostly because of
atopic dermatitis, other food allergies, or family history)

• n= 100, males/females = 69/31
• Median age of 17 months (range 12-23 months)

J Allergy Clin Immunol 2012:129(6);168132

• sIgE titers to EW (n = 100) and OVM (n = 80, males/females = 55/25)
: ImmunoCAP System (Phadia AB, Uppsala, Sweden)

• OVM was not determined in 20 patients because of inadequate
sample volume or cost of the testing

• 2 children were sIgE-negative to EW, but they had previously
tested positive to EW or had a positive skin prick test result


• open OFCs using boiled EW

• Each child ate EW, boiled for more than 20 minutes, in
increasing amounts (trace, 1, 2, 5, and 10 g) at 20-minute
intervals
• total amount : half a large egg, containing 2.0 g of EW protein

• OFC was not conducted in patients with extremely high sIgE
titers to EW and OVM (50 kUA/L or higher [n = 17]) to
decrease the risk of inducing reactions


• Positive challenges 33 patients
– dermal (n = 31), RS (n = 8), and GI (n = 4) symptoms
– sIgE to OVM was examined in 27 patients
• Multisystem reactions : 12 patients
• 3 required intramuscular epinephrine injection
• The median cumulative EW dose required to provoke allergic
symptoms was 10.6 g (range, 1.0-18.5 g), containing
approximately 1.7 g of EW protein


• The median concentration of sIgE against EW was
– 15.6 kUA/L (range, 1.58-51.3 kUA/L) in challenge-positive patients,
(significantly higher, P <0.01)
– 4.34 kUA/L (range, 0.35-51.5 kUA/L) in challenge-negative patients

• A similar difference was observed for OVM (P < 0.01)
– 8.12 kUA/L (range, 0.35-48.8 kUA/L)
– 1.00 kUA/L (range, 0.35-32.0 kUA/L; P < .01)


sIgE to EW
10 kUA/L: 30% PPV

30 kUA/L:65% PPV
The calculated sIgE
titer that indicated a
95% PPV was sIgE to OVM
61.8 kUA/L for EW 10 kUA/L: 54% PPV
26.6 kUA/L for OVM
30 kUA/L:97% PPV


Patients with undetectable
sIgE against OVM (<0.35 kUA/L; n = 24),
regardless of the sIgE titer to
EW (range, 0.35-27.5 kUA/L), showed
88% (21 of 24) likelihood of a
negative challenge

3 individuals demonstrating a
positive OFC showed only mild
cutaneous symptoms

indicate the advantage of using sIgE titers against OVM to identify
patients who can safely consume boiled egg products

• OVM IgE : new predictive marker
• sIgE titer against OVM has a better predictive value than does
a titer against EW

• But for safety, we recommend an office challenge to introduce
egg products to the sensitized children, even if sIgE to OVM is
negative


Treatment
• Standard therapy for egg allergy is strict
avoidance

Or a l i mmu n o t h e r a p y
(O I T ) o r
s p e c i f i c o r a l

Egg allergy: new allergens and molecular diagnosis

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