1. ImmunoCAP ISAC Customer days
CRD and technical validation of ImmunoCAP ISAC
Phn. Biol. E. Cavalier
Phn. R. Gadisseur
19/11/2009

2. Timeline…allergy diagnosis
Standardization of allergen extracts
Characterization & designation of IgE
Recombinant allergen panels recreating allergen extracts
ImmunoCAP ISAC (Phadia/ VBC Genomics)
First allergens cloned
First allergen microarray
Provocation tests (SPT)
RAST
1880
1967
2000
2008
Simplified overview of the historical development of allergy diagnosis and treatment.
IN-VIVO testing
IN-VITRO testing
Component – Resolved -Diagnostics

3. Components : Production

4. Components - Resolved Diagnosis (CRD)
Component-Resolved Diagnosis uses defined recombinant allergens to dissect the individual patient’s IgE reactivity profile with the aim of identifying the disease-eliciting molecules.
Valenta R et al, Clin Exp Allergy 1999
Molecules, genuine markers tell you the truth !
Mari A., VIP Customers day, Brussels, 17th November 2009.
The presence of IgE to cross-reactive allergen components can be determined and used to predict clinically relevant sensitization to allergen sources which contain immunologically related allergens.

5. Biochemical property of the allergens
Allergens, data bases
Peanut allergens
Biochemical property of the allergens

6. « specific » IgE for extracts
From a laboratory point of view…
What does one value of sIgE means for allergen extracts ?
How to manage this sIgE rate ?
Concerning the care of the patient ?
Concerning the risks ?
Impact on overall patient management and health outcomes (severity assessment, risk, therapy choices, improve patient’s quality of live).
Is there an interest of Component-resolved diagnosis in the laboratory routine practice?
How can we realize CRD in the laboratory?
Our experience with peanut sensitization…
+ sIgE for f13, anamnesis, components
Our question is :”Should we recommend the strict avoidance of peanut ?”

7. Clinical Cases (I) 1. Julie, F : f13 (peanut) = 27.4 kUA/L.
SPT + : peanut, walnut, hazelnut, sesame seed, weeds, birch, pine nut.
SPT - : soybean, egg white et walnut.
Asthma, gastrointestinal intolerance and erythema after ingestion of nuts.

8. Clinical cases (II) 2. Laurent, H : Maud, F :
f13 (peanut) = 1.22 kUA/L.
sIgE + : birch, apple, pear, peach, apricot, hazelnut, walnut and recently to celery and to fennel.
Atopy, polysensitised, oral allergy syndrome (OAS) and a recent evolution to breathing difficulties.
Maud, F :
f13 (peanut) = 1.23 kUA/L
sIgE + : pollens of weeds, cow milk, carrot, orange, tomato, wheat, potato, garlic, onion, celery, banana, peach and peanut.
Sensitization to a wide variety of foods from plants.
Gastro-intestinal disorders.

9. Clinical cases (III) Eddy, M : f13 (peanut) = 0,44 kUA/L
SPT+ : birch, apple, peach, banana, garlic, peanut
Swollen lips
Sensitization to birch…What can we conclude?

10. Clinical cases (IV) 5. Nadia, F : In those 5 clinical cases…
f13 (peanut) = 1,73 kUA/L
f17 (hazelnut) = 4,23 kUA/L
sIgE + : apple, peach, hazelnut, peanut
SPT +++ : peanut, hazelnut, walnut.
Atopy, polyallergy, rhinoconjonctivitis, OAS, urticaria et angiœdema
In those 5 clinical cases…
sIgE rates for peanut extracts are they proportional to the gravity of the allergy ?
What are the risks for those patients?
Eviction of diet or not ?
How to manage the patient?
What will be the future of the patient ? ?

11. Predict cross reactions
Storage protein
Protein 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
rSes i 3
rBer e 1
Cor a 9
Ara h 1 Ara h 2 Ara h 3
nGly mβ-conglycinin
nGly m glycinin

12. Predict cross reactions
PR-10 protein, Bet v 1 homologue
A heat labile protein, cooked foods are often tolerated.
Breiteneder H, Biotechnol Adv, 2005
Often associated with local symptoms such as oral allergy syndrome (OAS).
Often associated with allergic reactions to fruits, vegetables and spices in northern Europe.
Cor a Cor a 1.04
Gly m 4
Api g 1
Ara h 8
Dau c 1
Bet v 1
Pru p 1
Mal d 1
Pyr c 1
Pru ar 1
Pru av 1

13. Oral Allergy Syndrome (OAS)
Oral Allergy Syndrome : allergic reactions that occur rapidly, within minutes of eating a trigger food.
An itching or burning sensation in the lips, mouth, and/or pharynx.
Associated to a sensitization in one or more proteins like PR-10 (Bet v 1-like) (birch).
Hazelnut, 46%
Apple, 39%
Peach, 24%
Cherry, 22%
Nut, 21%
Pear, 20%
Almond, 19%
Plume, 17%
Brazil nut, 16%
Potato, 15%
Carrot, 13%
Peanut, 10%
Strawberry, 10%
Orange, 9%
Apricot, 7%
PR-10
allergic reactions that occur rapidly, within minutes of eating a trigger food. The most common reaction is an itching or burning sensation in the lips, mouth, and/or pharynx.
Allergies to a certain pollen are associated with OAS reactions to certain foods. For Alder pollen: almonds, apples, celery, cherries, hazel nuts, peaches, pears, parsley
Birch pollen: almonds, apples, apricots, avocados, carrots, celery, cherries, chicory,[4] coriander, fennel, fig,[5] kiwifruit, nectarines, parsley, parsnips, peaches, pears, peppers, plums, potatoes, prunes, soy, wheat; Potential: hazel nuts, and walnuts
Grass pollen: fig,[5] melons, tomatoes, oranges
Mugwort pollen : carrots, celery, coriander, fennel, parsley, peppers, sunflower
Ragweed pollen : banana, cantaloupe, cucumber, honey dew, watermelon, zucchini; Potential: Dandelions or chamomile tea
Possible cross-reactions (to any of the above): berries (strawberries, blueberries, raspberries, etc), citrus (oranges, lemons, etc), grapes, mango, figs, peanut, pineapple, pomegranates, watermelon
rBet v 1
rPru p 1
rGly m 4
rAra h 8
rApi g 1.01
rAln g 1
rCor a 1
nAct d 8
rMal d 1
rDau c 1

14. Predict cross reactions
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
LTPs often cause food allergy to fruit in the absence of pollen allergy.
Often associated with allergic reactions to fruit and vegetables in southern Europe.
LTP
Zea m 14
nPru p 3
rCor a 8
rPar j 2
nArt v 3
Ara h 9
Mal d 3
Zea m 4
Jug r 3
Cor a 8
Pru p 3
Pru av 3
Mal d 3
Pru ar 3
Ara h 9

15. Predict cross reactions
Profilin
Seldom associated with clinical symptoms but may cause demonstrable or even severe reactions in a small minority of patients
Profilins are present in a broad range of pollens and foods including trees, grasses and weeds.
There is a wide range of homology between profilins from different fruits and vegetables.
Ara h 5
PFL
rBet v 2
rPhl p 12
rMer a 1
rPhl p 12
rBet v 2
nOle e 2
rMer a 1
rHev b 8
rMal d 4
rPru p 4
nAct d 9
nAna c 1
nAra h 5
profilin in cross-reactivity between Latex profilin and other plant profilins.
rPhl p 12
rBet v 2
nOle e 2
rMer a 1
rMal d 4
rPru p 4
nAct d 9
nAna c 1
Fra a 4
nAra h 5
Cor a 2
rApi g 4
rDau c 2
rHev b 8
rMal d 4
Pru p 4
rOle e 2
rCor a 2
rPyr c 4

16. Characteristic symptoms Sensitization profiles
To resume…
Characteristic symptoms
Sensitization profiles
PR-10 proteins
ns-LTP
Local (OAS)
Systemic
Profilins

17. In-vitro cross-reactions
CCD (MUXF3) :
Marker of sensitization to Carbohydrate determinants (cross-reacting carbohydrate determinants). Glyco-epitopes.
Present in most plants,
sIgE directed towards glycans appear to show the widest pattern of cross-reactivity among allergenic extracts and are often responsible for observed in-vitro cross-reactions.
« in-vivo » effects ?
Seldom associated to clinical symptoms …
But, publications ?
To compare to the clinical informations and to SPT…
Test if…
Sensitization to fruits and vegetables, seeds.
Sensitization to latex in a pollinic patient without occupational risk exposure.
Sensitization to Hymenoptera venom with/without sensitization to pollens.

18. Arachis Hypogea Allergenicity :
Ara h 9 : nsLTP
Stable to heat and digestion.
Severe reactions >> systemic reactions !
Ara h 8 : Profilin
Cross-reactions between many fruits and vegetables.
CCD (MUXF3)
Cross-reactive Carbohydrate Determinants
Seldom associated to symptoms.
Present in most plants.
Ara h 1 : Storage protein (vicillin)
Major allergen
Ara h 2 : Storage protein (conglutin)
+ frequent, sensitizing potential ++, stability ++ to digestion.
Ara h 3 : Storage protein (glycinin)
Minor allergen, rare sensitizations.
Ara h 8 : PR-10
Major allergen of peanut in birch pollen allergic patients.
Sensitive to heat and digestion.
Allergenicity :
Ara h 2 is 100 times stronger than Ara h 1
Koppelman SJ et al, Clin Exp Allergy 2004,34:583
Ara h 2 possesses high proteolytical stability
Lehmann K et al, Biochem J, 2006:
After roasting peanuts, allergenicity of Ara h 2 is multiplicated by 3
Maleki SJ et al, J Allergy Clin Immunol, 2003

19. Laboratory practice : How to diagnose peanut allergy ?
Peanut (f13) + Ara h 2 (f423)
Peanut (f13) : Neg
Ara h 2 : Neg
Peanut (f13) : Pos
Ara h 2 : Neg
Peanut (f13) : Pos
Ara h 2 : Pos
Low risks of severe reactions
Risk of severe reaction?
Very high risk of severe reactions
How to explore? Recommendations
How to explore ?
Recommendations
Test : Risk
Ara h 1 (f422)
Ara h 3 (f424)
Ara h 9 (f427)
Ara h 8 (f352)
CCD (Ro214)
Birch (north Europe) : test Ara h 8 (f352)

20. Clinical Cases (1) 1. Julie, F : f13 (peanut) = 27.4 kUA/L.
SPT + : peanut, walnut, hazelnut, sesame seed, birch, weeds, pine nut.
SPT - : soybean, egg white et walnut.
Asthma, gastrointestinal intolerance and erythema after ingestion of nuts.
What are the risks ? Birch?

21. Very high risk of severe reactions How to explore? Recommendations
Clinical cases (1)
Peanut (f13) = 27,4 kUA/L
Ara h 2 (f423) = 14,2 kUA/L
Peanut (f13) : Pos
Ara h 2 : Pos
Very high risk of severe reactions
Sensitization to Ara h 8 due to birch allergy (PR-10).
Sensitization to Ara h 2 : Storage protein (conglutin), Stable ++ to digestion, Diet eviction of peanut because risks of severe reactions +++.
Sensitization to 3 major allergens of peanut.
How to explore? Recommendations
Cross-reaction : storage proteins ??
Tests : Risks
Ara h 1 = 3,46 kUA/L
Ara h 3 = 0,6 UA/L
Ara h 9 <0,1 kUA/L
Ara h 8 = 2,27 kUA/L
CCD < 0,1 kUA/L
Major allergens of peanut
Storage proteins
peanut PR-10 protein !

22. Clinical cases (2) 2. Laurent, H : f13 (peanut) = 1.22 kUA/L.
sIgE + : birch, apple, pear, peach, apricot, hazelnut, walnut and recently to celery and to fennel.
Atopy, polysensitised, oral allergy syndrome(OAS) and a recent evolution to breathing difficulties.
What are the risks?
What about the Birch?
Recent evolution of the allergy?

23. Risk of severe reactions? How to explore? Recommendations
Clinical cases (2)
Peanut (f13) = 1,22 kUA/L
Ara h 2 (f423) < 0,10 kUA/L
Sensitization to peanut after birch allergy (PR-10 proteins).
Sensitization to other PR-10 :
- Apple (Mal d 1), pear (Pyr c1), peach (Pru p 1), apricot (Pru ar 1), hazelnut (Cor a 1) and recently to celery (Api g 1)
In this case, evolution of the OAS with respiratory distress explained by recent sensitization to Api g 1.
Peanut (f13) : Pos
Ara h 2 : Neg
Risk of severe reactions?
How to explore? Recommendations
Tests : Risk
Ara h 1 < 0,1 kUA/L
Ara h 3 < 0,1 kUA/L
Ara h 9 < 0,1 kUA/L
Ara h 8 = 8,4 kUA/L
CCD < 0,1 kUA/L
PR-10, Bet v 1-homologue

24. Clinical cases (3) Maud, F : f13 (peanut) = 1.23 kUA/L
sIgE + : pollens of weeds, cow milk, carrot, orange, tomato, wheat, potato, garlic, onion, celery, banana, peach and peanut.
Sensitisation to a wide variety of foods from plants.
Gastro-intestinal disorders.
What are the risks ?
Multiple sensitizations to fruits and vegetables.

25. Risk of severe reactions? How to explore? Recommendations
Clinical cases (3)
Peanut (f13) = 1,23 kUA/L
Ara h 2 (f423) < 0,1 kUA/L
Sensitization to CCD :
- Marker of sensitization to Carbohydrate Determinants.
- Multiple cross-reactions.
- Seldom associated to clinical symptoms.
Test CCD if sensitization to fruits and vegetables, seeds.
Peanut (f13) : Pos
Ara h 2 : Nég
Risk of severe reactions?
How to explore? Recommendations
Tests : Risks
Ara h 1 < 0,1 kUA/L
Ara h 3 < 0,1 kUA/L
Ara h 9 < 0,1 kUA/L
Ara h 8 < 0,1 kUA/L
CCD = 1,93 kUA/L
CCD

26. Clinical cases (4) 4. Eddy, M : f13 (peanut) = 0,44 kUA/L Swollen lips
SPT+ : birch, apple, peach, banana, garlic, peanut
Swollen lips
What are the risks ?
Sensitization to birch…

27. Risk of severe reactions? How to explore? Recommendations
Clinical Cases (4)
Peanut (f13) = 0,44 kUA/L
Ara h 2 (f423) < 0,1 kUA/L ?
Sensitization to profilins (Bet v 2)
Seldom associated with clinical symptoms except OAS and rarely severe reactions in a minority of patients.
Presence of profilins in many fruits and vegetables.
- Birch (Bet v 2), apple (Mal d 4), peach (Pru p 4), banana (Mus a 1), garlic (All c 4), peanut (Ara h 5).
Peanut (f13) : Pos
Ara h 2 : Neg
Risk of severe reactions?
How to explore? Recommendations
Bet v 2 = 4,6 kUA/L
Testés
Ara h 1 < 0,1 kUA/L
Ara h 3 < 0,1 kUA/L
Ara h 9 < 0,1 kUA/L
Ara h 8 < 0,1 kUA/L
CCD < 0,1 kUA/L

28. Clinical cases (5) 5. Nadia, F : f13 (peanut) = 1,73 kUA/L
f17 (Hazelnut) = 4,23 kUA/L
sIgE + : apple, peach, hazelnut, peanut
SPT +++ : peanut, hazelnut, walnut.
Atopy, polyallergy, rhinoconjonctivitis, OAS, urticaria et angiœdema
What are the risks ? …angioedema..;

29. Clinical cases (5) Peanut (f13) = 1,23 kUA/L
Ara h 2 (f423) <0,1 kUA/L
Hazelnut (f17) = 4,23 kUA/L
Cor a 8 (f425) = 3,87 kUA/L
Peanut (f13) : Pos
Ara h 2 : Neg
Hazelnut (f17) : Pos
Cor a 8 : Pos
nsLTP
Risk of severe reactions?
Very high risk of severe reactions
How to explore? Recommendations
Sensitization to nsLTP :
Marker of cross-reactions.
Associated to severe reactions.
Occurs mainly in South of Europe.
Sensitization to apple (Mal d 3), peach (Pru p 3), hazelnut (Cor a 8) and peanut (Ara h 9).
Test : Risks
Ara h <0,1kUA/L
Ara h 3 <0,1kUA/L
Ara h 9 = 8,2 kUA/L
Ara h 8 <0,1kUA/L
CCD <0,1kUA/L
nsLTP

30. Thanks to all the recombinants,
More complex…
Julien, M :
Complex clinical case !!
Atopy,
Atopic dermatitis,
Perannual rhinitis,
Giant eczema.
Food allergy (egg white, peanut, soybean, crab and garlic)
How to evaluate the symptoms ?
SPT Pneumallergens:
+ : D1 et D2, dog, weeds.
Suspiscious : birch.
SPT Trophallergens :
+ White egg, peanut, soybean, garlic.
IgEs for extracts
Peanut,f13
9.71
Soybean,f14
4.43
Crab,f23
1.98
Garlic,f47
4.11
Egg yellow,f75
0.61
Pityrosporum orbiculare,m70
3.93
Dermatophagoïdes pteronyssinus,d1
>10
Egg white,f1
5.37
Schrimp ,f24
2.64
Gx3:g1+g5+g6+g12+g13
93.6
Birch,t3
58.0
Thanks to all the recombinants,
We would be able to explain everything !!

31. In theory, this is possible…
Request form ImmunoCAP™Components
Patient: Nom : Mister Albert Gik
Adresse: Birch Street 1…
Birth date: 01/01/1950
Some other components less useful in Belgium could be available.
Don’t hesitate to ask to your Lab in case of specific clinical history.
June 2009
In theory, this is possible…
Practically, …
Docteur: Nom: Cavalier Etienne
Adresse : CHU of Liège
Date de demande: 19/11/2009
Signature:

32. ImmunoCAP© ISAC
VBC Genomics (expert in microarrays) and Phadia (expert in allergy in-vitro diagnostic tests) have combined “innovative biochip technology” with “cutting-edge research in molecular allergology” to develop ImmunoCAP®ISAC.
Harwanegg C et al, Expert Rev Mol Diagn 2004
Modern multiplex in vitro diagnostic tool for the allergy specialist, based exclusively on allergen components.
Miniaturized immunoassay platform for measurement of sIgE antibodies to over 40 common allergen sources in a single step.
103 purified natural or recombinant allergen components immobilized on a solid support.
Run CRD assays rapidly and cost-effectively.

33. Immuno Solid-phase AllergenChip (ISAC)
Allergen molecule
Allergen-triplet after
fluorescence-reaction
Fluorescence-labeled
secondary anti-human
IgE
IgE-antibody
(in the patient’s serum)
The allergen biochip is a solid substrate built from a glass plate that is chemically altered to retain allergenic protein molecules in a biologically active form. Picolitre quantities of each allergen preparation are deposited on spatially separate locations on the biochip. The resulting addressable microarray allows for many biological affinity reactions to be monitored in parallel (=multiplexing). In a single analytical step, the comprehensive allergen sensitisation profile of a patient can be measured using only a minute amount of serum or plasma; this is followed by automatic readout and report generation. The present chip used for in vitro diagnostics incorporates 103 allergens from about 50 different allergen sources.
4 reaction sites

34. ImmunoCAP® ISAC : Version 103.
Proteins, Cross-reactions :
11 PR-10 :
(Bet v 1-like proteins) :
Well represented, interest ++
4 nsLTP :
Some more ?
Which ones?
5 Profilins :
So many profilins exist…
Which ones are most representative in each botanic family ?
5 Storage proteins
2 Parvalbumins :
Important !
Which ones are most representative?
4 Tropomyosins :
5 serum albumins :
Honey bee and paper wasp venoms :
Lack…

35. Immuno Solid-phase Allergen Chip (ISAC)

36. Validation of the method
Selection of 19 sera of patients (6 males/13 females)
+ sIgE (>0.10 kUI/L) for recombinant allergens (ImmunoCAP ®250 Phadia).
positive and negative sIgE for different kind of allergenic sources (latex, peanut, birch, Timothy grass, hazelnut, peach, soybean, cat, dog…).
In all, we compared the results of 157 sIgE measured in CAP (recombinant allergens) with the ISAC®.
We tested 2 samples with a high TotIgE rate (> kU/L) on ISAC ®.
(rHev b 1; n=3, rHev b 3; n=3, rHev b 8; n=5), cross-reactive carbohydrate determinants (nAna c 2; n=2), soja (rGly m 4; n=4), peanut (rAra h 1; n=7, rAra h 2; n=7, rAra h3; n=5, rAra h 8; n=12), hazelnut (rCor a 8; n=3), Brazil nut (rBer e 1; n=1), peach (rPru p 1; n=11, rPru p 3; n=3), birch (rBet v 1; n=17, rBet v 2; n=11, rBet v 4; n=8), timothy grass (rPhl p 1; n=12, rPhl p2; n=2, rPhl p4; n=5; rPhl p 5; n=7, rPhl p 6; n=3, rPhl p11; n=1, rPhl p 12; n=5), Aspergillus fumigatus (rAsp f 6; n=2), Alternaria (rAlt a 1; n=3), cat (rFel d 1; n=7) or dog (rCan f 1; n=4, rCan f 2; n=2, rCan f 3; n=2).

37. Validation of the method
Results:
17 results out of the 122 measurements of positive sIgE in CAP® were found negative with ISAC ® (concordance of 86%).
2 results out of the 35 measurements of negative sIgE in CAP® were found positive with ISAC ® (concordance of 94%).
Until a rate of kU/L of TotIgE, we didn’t observe any unspecific binding.

38. Validation of the method
Discussion
Concordance of all positive results was 86%.
Can we explain the discordances?
Cut-off of the ISAC ® higher (>0,30 USI) than cut-off of CAP ® (>0,10 kUA/L)
Many measurements on ISAC ® were between the range of 0,10 and 0,30 USI.
Concordance of all negative results was 94%.
2 discordances concerned sera for which ISAC® found a positive Ara h1 and Ara h 3 (2 major allergens of peanut).
Clinical importance of those 2 allergens !
Look back the clinical anamnesis :
The first patient had positive sIgE in CAP for Ara h 1 and Ara h 2, his oral provocation test (TPO) to peanut was positive.
- Anamnesis well correlated with true positive Ara h 3 with ISAC ®
The 2nd patient had undergone one TPO for peanut which was suspiscious, sIgE Ara h 8 ++, LTP ++, CCD +, polysensitized patient and large food eviction (hazelnut, peanut, sea food…)
- Anamnesis correlated well with a possible true positive Ara h 1 with ISAC ®

39. Albert Gik Bet v 1 homologues (PR-10):
Bet v 1, Aln g 1, Cor a 1, Mal d 1, Pru p 1, Gly m 4, Ara h 8, Api g 1
Species markers :
Gal d 1, Bos d lactoferrin, Fel d 1, Fel d 4, Can f 1, Mus m 1, Alt a 1
Profilins :
Bet v 2, Ole e 2, Hev b 8, Mer a 1, Phl p 12
Der p 1, Der f 1, Der f 2, Der p 2, Eur m 2
Pollens : Cyn d 1, Phl p 1, Phl p 2, Phl p 4, Phl p 5, Phl p 6 et Ole e 1
Thanks to CRD, we can explain :
Many food allergies due to sensitization to profilins, PR-10 proteins (OAS), tropomyosin (sea food), cow milk…
Many weeds, mites, pets… which explain perannual rhinitis exacerbated during pollination.
Tropomyosin : Der p 10

40. Conclusion (I)
The way for the discover of sensitization profiles is open…
Allergen extracts are still useful :
In first intention,
Don’t permit to conclude every time (multiple positivity of the tests, lack of precision),
sIgE for extract not really significant.
CRD is in our hands.
Sensitizations can cause diagnosis errors (and therapeutic errors too).
Protein families have been described, similarities of structure (homology of amino-acids or structural homology)
Allergy to one protein can cause cross-reactions to other proteins of the same family.
Recombinant allergens permit to focus (one or more) allergen(s) of one source:
Available in CAP (and/or in ISAC) or to be developed…

41. Conclusion (II) Microarrays can help us, in the difficult cases :
103 recombinant or native purified allergens in one determination…
Analytical validation of the ImmunoCAP© ISAC performed:
Concordance : 86% (negative results) and 94% (positive results)
The ImmunoCAP© ISAC has a role to play in the diagnosis and the care of patients with complex sensitization profiles.
As this technique provides potentially unexpected results, the microarray determination has to be used in conjunction with the clinical findings.
Difficult interpretation of the results :
Should be limited to specialists !

42. Thank you
for your attention !!