Egg allergy: new allergens and molecular diagnosis Presented by Suparat Sirivimonpan, MD. September7, 2012

1. Egg Allergy
7/9/2012
Suparat Sirivimonpan,MD.

2. Egg allergy
Introduction
Prevalence
Egg allergen : new allergen
Diagnosis : molecular diagnosis

3. 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

4. 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

5. 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• Unpublished data 2012, Chulalongkorn university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

6. Egg allergen

7. Egg allergens
• 5 major allergenic proteins : Gal d 1 to Gal d 5 (Gallus
domesticus)
Curr Allergy Asthma Rep 2006;6(2):145–52

8. Egg White allergen

9. Egg white allergens
Allergy 2010; 65: 283–289

10. 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

11. 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

12. 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.

13. Newly identified egg white allergen
Allergology International. 2010;59:175-183

14. • Diluted egg white proteins were separated by 2-dimensional
(2-D) gel electrophoresis
63 spots
Allergology International. 2010;59:175-183

15. • 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
Allergology International. 2010;59:175-183

16. Allergology International. 2010;59:175-183

17. 15/25
• Spots of egg white proteins that bound to the patients’ IgE
were identified by mass spectrometry-based proteomics
10/25
Allergology International. 2010;59:175-183

18. 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
Allergology International. 2010;59:175-183

19. L-PGDS
Cystatin
• Reaction to LPGDS and cystatin was confirmed using each purified
protein
Allergology International. 2010;59:175-183

20. 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
Allergology International. 2010;59:175-183

21. 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
Allergology International. 2010;59:175-183

22. 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
Allergology International. 2010;59:175-183

23. Egg yolk allergen

24. 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
Pediatr Clin N Am 2011;58:427–443

25. 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

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

27. • 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

28. • 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

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

30. • 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

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

32. 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
Pediatr Clin N Am 2011;58:427–443

33. Diagnosis

34. 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

35. 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
Pediatr Clin N Am 2011;58:427–443

36. 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

37. 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 ; ( ): –

38. 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

39. 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

40. 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

41. 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

42. • 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

43. • FCT resulted positive in 22/46 (48%) patients with suspected
HE allergy
Clinical & Experimental Allergy, 2010 (40) 1561–1570

44. 95% predicted probability (CDP)of a positive FCT Clinical & Experimental Allergy, 2010 (40) 1561–1570

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

46. ↓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
Clinical & Experimental Allergy, 2010 (40) 1561–1570

47. • 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

48. • 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
Clinical & Experimental Allergy 2012 :42;441-451

49. No significant differences
significant differences,
P<0.01
significant differences
Clinical & Experimental Allergy 2012 :42;441-451

50. ImmunoCAP
Clinical & Experimental Allergy 2012 :42;441-451

51. 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
Clinical & Experimental Allergy 2012 :42;441-451

52. Clinical & Experimental Allergy 2012 :42;441-451

53. 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
Clinical & Experimental Allergy 2012 :42;441-451

54. 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;
Clinical & Experimental Allergy 2012 :42;441-451

55. 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
Clinical & Experimental Allergy 2012 :42;441-451

56. 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
Clinical & Experimental Allergy 2012 :42;441-451

57. • 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
Clinical & Experimental Allergy 2012 :42;441-451

58. • 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

59. • 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
J Allergy Clin Immunol 2012:129(6);168132

60. • 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
J Allergy Clin Immunol 2012:129(6);168132

61. • 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
J Allergy Clin Immunol 2012:129(6);168132

62. • 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)
J Allergy Clin Immunol 2012:129(6);168132

63. 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
J Allergy Clin Immunol 2012:129(6);168132

64. 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
J Allergy Clin Immunol 2012:129(6);168132

65. • 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
J Allergy Clin Immunol 2012:129(6);168132

66. 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

67. Thank you