WAC 2011 Debates 4, 6 December 2011: 10:45 - 11:45
Skin test or in-vitro Test for Food Allergy?
“In vitro”
Motohiro Ebisawa, MD, PhD
IgE
Disclosures of Motohiro Ebisawa, MD, PhD
1) Employee of Sagamihara National Hospital
2) Academic activities
WAO: Board member at large,
AAAAI: International Assembly vice-chair
Japanese Society of Allergology: Board member
Japanese Society of Pediatric Allergy: Board member
3) Grant support from Japanese government, Ministry of Labor,
Health, and Welfare for 12 years as PI
4) No Conflicts of Interests
RAST: First Generation
Allergen bound
to paper disc
All antibody isotypes
bind: Ig of A,M,G,E
class
Bound IgE detected
with polyclonal I125
Anti-IgE
Results reported as log-related
classes or arbitrary units by
interpolation of heterologous IgE
anti-birch pollen curve
RAST 1st on the market in 1974, considerable variability & questionable
quantification-no longer in use and term is no longer appropriate
Hamilton R, Adkinson F. J Allergy Clin Immunol 2004;114:213-25.
3
Historical Manual Chemistries







RAST = disc allergosorbent 1o (transitioned) 1968
Hycor Hy-Tec (paper disc based)
FAST = Allergenics/Biowhittaker, fluorescent allergosorbent test
MAST = Hitachi: thread pipette
EAST = Sanofi Dignostics Pasteur
Magic Lite = ALK/Corning/Bayer
Matrix = Abbott
Historical Semi-automated Chemistries


Alastat, Diagnostic Products Corp. (biotinylated-allergen)
AutoCAP, Pharmacia (Allergen insolubilized on sponge)
“The Pearls and Pitfalls of Diagnostic Allergy Testing” developed
by the ACAAI/AAAAI Specific IgE Test Task Force (SETTaF)

ImmunoCAP (250, 1000):
Phadia (changed from Pharmacia,
Jan 06)

HyTec-288: Hycor BiomedicalAgilent (June 07)

Immulite 2000/2500:
Siemens Medical Solutions
Diagnostics (Jan 07)
• The antibody binds to the allergen on the solid phase
• Enzyme anti-IgE detects bound IgE
• All assays report in similar units (kUa/L) with comparable
analytical sensitivities of 0.1 kUa/L
• All assays primarily use allergens from extracts
ImmunoCAP perceived as gold standard for
in vitro IgE testing
”The predicitive values associated with clinical evidence for ImmunoCAP
cannot be applied to Turbo-MP and Immulite”NIH/NIAID
Food allergy guideline. J Allergy Clin Immunol 2010; 126:S1-S58
”The Pharmacia CAP system is in world wide use and is a de facto
standard to which other methods are compared”
Dolen WK. Allergy 2003; 58: 717-723
In-vitro
IgE Antibody
In-vivo
SPT
High sensitivity*
High specificity*
High reproducibility
Quantitative results in kIU/L^
WHO Standard calibrated
Quality assurance test program
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
No
No
Can be used independently
of pharmaceutical treatment
Yes
No
Can be used independently
of patient skin status
Yes
No
Time factor
Cost factor
Usefulness in motivating patients
1-7 days
more expensive
obscure
15-30 minutes
inexpensive
dramatic
*Results may vary between specific bioassays
^Although all are expressed with same units, cannot compare results between different bioassays
“The Pearls and Pitfalls of Diagnostic Allergy Testing” developed
by the ACAAI/AAAAI Specific IgE Test Task Force (SETTaF)
IgE testing in vitro
Standardization of reagents!

No common standardization exists for SPT extracts

Huge variability between extracts from different producers,
and also from the same producer

Blood tests are standardized to WHO ref

World wide proficiency testing assure low CV% with ImmunoCAP IgE
Van Ree R. JACI 2007; 119: 270-277
Probability of Reacting to a Food at a Given IgE Value
1.0
Egg white
0.8
0.8
0.7
0.7
0.6
0.5
0.4
0.6
0.5
0.4
0.3
0.3
0.2
0.2
0.1
0.1
0.0
0.0
0.35
0.7
Cow's milk
0.9
Probability
Probability
0.9
1.0
3.5
17.5
50
100
0.35
IgE antibody concentration (kUA/L)
17.5
50
100
50
100
1.0
Fish
0.9
0.8
0.8
0.7
0.7
0.6
0.5
0.4
0.6
0.5
0.4
0.3
0.3
0.2
0.2
0.1
0.1
0.35
0.7
Peanut
0.9
Probability
Probability
3.5
IgE antibody concentration (kUA/L)
1.0
0.0
0.7
3.5
17.5
50
100
0.0
0.35
IgE antibody concentration (kUA/L)
0.7
3.5
17.5
IgE antibody concentration (kUA/L)
Retrospective study
Prospective study
Logit model using log(kU A /L)
Reference: Calculated from Sampson and Ho, J Allergy Clin Immunol 1997; 100: 444-51
1
1
0.8
0.8
0.6
0.6
Probability
Probability
Probability of failed challenge in relation to the
specific IgE antibody levels for egg and milk
respectively divided into three age groups
0.4
0.2
0.4
0.2
< 1 year
< 1 year
1 year
1 year
 2 year
0
0.3
1
3
10
IgE antibody concentration (kU
30
A /L)
Egg (n=764)
 2 year
100
0
0.3
1
3
10
IgE antibody concentration (kU
30
A
100
/L)
Milk (n=861)
T. Komata, L. Soderstorm, M.P. Borres, H. Tachimoto ,and M.Ebisawa
J Allergy Clin Immunol, 119(5); 1272-1274
Predicted threshold values for 90% and 95%
probabilities for failing oral 359 challenge, for children
below 1 years of age, 1 year of age and 2 years or older
n
< 1 year
1 year
2 years
or older
PPV
90%
95%
Egg
215
6.4
13.0
Milk
223
3.6
5.8
Egg
187
10.9
23.0
Milk
177
20.8
38.6
Egg
362
17.0
30.0
Milk
275
33.8
57.3
(UA/ml）
T. Komata, L. Soderstorm, M.P. Borres, H. Tachimoto ,and M.Ebisawa
J Allergy Clin Immunol, 119(5); 1272-1274
Positive and negative decision points using
specific IgE antibody measurements obtained
from challenge with raw or heated egg white
Raw egg white
Specific
Egg white
IgE
Positive
decision
point
7.38
Heated egg white
Ovomucoid
Egg white
Ovomucoid
5.21
30.7
10.8
(UA/ml）
H. Ando, A Urisu et al
J Allergy Clin Immunol, 122 ;583-588
Probability curves for wheat and soybean
Soybean (n= 272)
1
1
0.9
0.9
0.8
0.8
Probability for failed challenge
Probability for failed challenge
Wheat (n= 277)
0.7
0.6
0.5
0.4
0.3
0.2
0.6
0.5
0.4
0.3
0.2
0.1
0.1
0
0.1
0.7
0.3
1
3
10
30
Specific IgE antibody concentration
(kUA/L)
100
0
0.1
0.3
1
3
10
30
100
Specific IgE antibody concentration (kUA/L)
T. Komata, L. Soderstorm, M.P. Borres, H. Tachimoto ,and M.Ebisawa
Allergol Int. 2009
Probability curves of Wheat and its age effect
Wheat (n=277)
1
Probability for failed challenge
0.9
0.8
<1 y
0.7
0.6
0.5
0.4
>=1 y
0.3
0.2
0.1
0
0.1
0.3
1
3
10
30
Specific IgE antibody concentration (kUA/L)
100
T. Komata, L. Soderstorm, M.P. Borres, H. Tachimoto ,and M.Ebisawa
Allergol Int. 2009
Plant Food Allergens
Peanut
Pollen crossreactive
components*
LTP
Pollen non-cross-reactive
components**
Ara h 8
Ara h 9
Ara h 1; Ara h 2; Ara h 3
Arah 4; Ara h 6; Ara h 7
Cor a 8
Cor a 9
Cor a 11
Gly m 1
Gly m 5
Gly m 6
Tri a 14
Tri a 19 (ω-5 gliadin)
Tri a 21 - alfa gliadin
Tri a 26 - HMW glutenin
Tri a 28 - AAI dimer 0.19
Ara h 5
Hazelnut
Cor a 1
Cor a 2
Soybean
Gly m 4
Gly m 3
Wheat
Tri a 12
PRP-10
Profilin
16
Allergen components in wheat
albumins and
globulins
Tri a 15 - AAI monomer
Tri a 28 - AAI dimer
Tri a 29, 30 - AAI tetramer
Tri a 12 - profilin
Tri a 14 - LTP
Tri a 18 - hevein-like
Tri a 25 - thioredoxin
Tri a 33 - serpin
Homologs to components in
timothy
gluten
gliadins
Tri a 19 - omega-5 gliadin
Tri a 21 - alfa/beta gliadin
Tri a gamma gliadin
Tri a omega-2 gliadin
glutenins
Tri a 26 - HMW glutenin
Tri a 36 - LMW glutenin
17
Subjects and Methods
• 343 patients with suspected wheat allergy from
3 different hospitals in Japan.
• Age range: 6 months - 20.4 years.
Median age: 2.3 years old.
Oral wheat challenge were performed for 339
children.
Four children were included with a recent
convincing case history of anaphylaxis in relation
to wheat.
Ebisawa M, Söderström L, Ito K, Shibata R, Sato S, Tanaka A, Borres M and Morita E , EAACI ’09
Results
138 children had positive reactions
Summary of positive reactions by challenge
Skin
113 (82%)
Cough
Wheeze
Gastrointestinal
Anaphylaxis
50 (36%)
28 (20%)
9 (7%)
3 (2%)
Nausea
OAS
9 (7%)
10 (7%)
IgE to wheat and ω-5 gliadin in wheat allergics
and non-wheat allergics
WA= wheat allergics
137 challenge positives
36 convincing history
NoWA= no wheat allergics
78 challenge negative
60 convincing history
Ebisawa M et al,Int Arch Allergy Immunol, 2011(in press)
Probability curves for the outcome of wheat allergy
at a given IgE value for ω-5 gliadin
for all children and for children ≤ 1 year
and >1 years of age
1.0
0.9
Probability (%)
0.8
0.7
0.6
0.5
0.4
>1 year; n=291
0.3
1 year; n=20
All patients, n=311
0.2
0.1
0.0
0.1
1
10
100
-5 gliadin: sIgE antibody concentration (kU A/L)
Ebisawa M et al,Int Arch Allergy Immunol, 2011(in press)
Plant Food Allergens
Peanut
Pollen crossreactive
components*
LTP
Pollen non-cross-reactive
components**
Ara h 8
Ara h 9
Ara h 1; Ara h 2; Ara h 3
Arah 4; Ara h 6; Ara h 7
Cor a 8
Cor a 9
Cor a 11
Gly m 1
Gly m 5
Gly m 6
Tri a 14
Tri a 19 (ω-5 gliadin)
Tri a 21 - alfa gliadin
Tri a 26 - HMW glutenin
Tri a 28 - AAI dimer 0.19
Ara h 5
Hazelnut
Cor a 1
Cor a 2
Soybean
Gly m 4
Gly m 3
Wheat
Tri a 12
PRP-10
Profilin
22
Allergen components in soy bean
Cupin superfamily
Allergen
Prolamin superfamily
PR-10
Profilin
Gly m 1
Gly m 4
Gly m 3
Ara h 9
Ara h 8
Ara h 5
7S globulin
/Vicillin
11S globulin
/Legumin
2S albumin
LTP
Soy
Gly m 5
Gly m 6
Gly m 2S
Albumin
Corresponding
peanut allergen
Ara h 1
Ara h 3
Ara h 2
23
IgE to Gly m 5 and Gly m 6 (soy)
predict severe reactions
IgE to Gly m 5 and/or Gly m 6 were found in
86% of the cases with anaphylaxis against soy
Holzhauser et al, JACI 2009
24
Gly m 5 & Gly m 6 Are Associated with Systemic
Reactions in Soybean-allergic Japanese Children
Ito, Ebisawa et al, JACI 2011
25
Diagnostic value of measuring IgE to soybean 2S albumin in
clinical assessment of soybean allergic Japanese children
Specific IgE (kUA/l)
1000
p<0.01
100
10
1
0.1
0.01
Symptomatic
Non-Symptomatic
• Quantitative measurement of IgE to 2S albumin from soybean in sera
from 19 Japanese children allergic to soybean and 36 nonsymptomatic controls
• Comparison of IgE level between symptomatic and non-symptomatic
groups, the median levels are indicated.
Sigrid Sjölander et al, EAACI 2009
Plant Food Allergens
Peanut
Pollen crossreactive
components*
LTP
Pollen non-cross-reactive
components**
Ara h 8
Ara h 9
Ara h 1; Ara h 2; Ara h 3
Arah 4; Ara h 6; Ara h 7
Cor a 8
Cor a 9
Cor a 11
Gly m 1
Gly m 5
Gly m 6
Tri a 14
Tri a 19 (ω-5 gliadin)
Tri a 21 - alfa gliadin
Tri a 26 - HMW glutenin
Tri a 28 - AAI dimer 0.19
Ara h 5
Hazelnut
Cor a 1
Cor a 2
Soybean
Gly m 4
Gly m 3
Wheat
PRP-10
Profilin
Tri a 12
*Birch tree pollen, Timothy grass pollen for wheat
** Storage seed proteins, albumins and globulins
27
Peanut components
Ara h 11
Ara h 1
Oleosin
Vicilin
Ara h 10
Ara h 2
Oleosin
Conglutin
Ara h 9
nsLTP
Ara h 3
Peanut
Glycin
Ara h 8
Ara h 4
PR-10
Glycin
Ara h 5
Ara h 7
Conglutin
Ara h 6
Profilin
Conglutin
Annica Önell Nov 2010
With components you correctly identify
97.5% of the peanut allergics
29
Nicolaou et al. JACI. March 2011..
Allergy testing in the 21st century
For primary care
by eye or instrument
For specialists
Quantitative sIgE reduces the risk and need
for oral food challenge

Sampson et al reduced the need with 40%

Österballe et al reduced the need with 60%
Sampson and Ho. J Allergy Clin Immunol 1997; 100: 444-51
Österballe M. J Allergy Clin Immunol 2003;112:196-201
“Shifted to Immuno CAP from SPT,
more and more” by Dr. Hugh Sampson
APAPARI 2011, in Fukuoka (Japan)