CRD and technical validation of ImmunoCAPISAC

(1)

CRD and technical validation of

ImmunoCAP ISAC

Customer days

Phn. Biol. E. Cavalier

Phn. R. Gadisseur

19/11/2009

(2)

Timeline…allergy diagnosis

1880

1967

1988-91

1995-99

2000

2008

Provocation

tests (SPT)

First

allergens

cloned

RAST

Characterization

& designation of

IgE

Recombinant allergen

panels recreating

allergen extracts

First allergen

microarray

ImmunoCAP ISAC

(Phadia/ VBC Genomics)

IN-VITRO testing

IN-VIVO testing

Component – Resolved -Diagnostics

Standardization of

(3)

(4)

Components - Resolved Diagnosis

(CRD)

« 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

(5)

Allergens, data bases

Peanut allergens

_{Biochemical property}

(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

(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 :



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.

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.



Sensitization to a wide variety of foods from plants.

(9)

Clinical cases (III)

4. Eddy, M :



f13 (peanut) = 0,44 kUA/L



SPT+ : birch, apple, peach, banana, garlic, peanut



Swollen lips

(10)

Clinical cases (IV)

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

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

rBer e 1

Ara h 1

Ara h 2

Ara h 3

Cor a 9

rSes i 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.

Mal d 1

Dau c 1

Pru p 1

Ara h 8

Cor a 1.01

Cor a 1.04

Api g 1

Gly m 4

Bet v 1

Pru ar 1

Pyr c 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).

1 60 Birch MGVFNYETET TSVIPAARLF KAFILDGDNL FPKVAPQAIS SVENIEGNGG PGTIKKISFP Hazel MGVFNYETES TSVIPAARLF KAFILDGNNL IPKVAPQAVS SVENVEGNGG PGTIKKITFS Alder MGVFNYEAET PSVIPAARLF KAFILDGDKL LPKVAPEAVS SVENIEGNGG PGTIKKITFP Hornbeam MGVFNYEAET TSVIPAARLF KAFILDGNNL IPKVAPQAVS SVENVEGNGG PGTIKKITFS Beech MGVFTYESEN TSVIPPARLF KAFVLDADNL IPKVAPQSIK STETLEGDGG PGTIKKITFG Apple MGVFNYETET TSVIPAPRLF KAFILDGDNL IPKIAPQAIK STEIIEGDGG VGTIKKVTFG Cherry MGVFTYESEF TSEIPPPRLF KAFVLDADNL VPKIAPQAIK HSEILEGDGG PGTIKKITFG Apricot MGVFTYETEF TSVIPPEKLF KAFILDADNL IPKVAPTAVK GTEILEGDGG VGTIKKVTFG Pear MGLYTFENEF TSEIPPPRLF KAFVLDADNL IPKIAPQAIK HAEILEGNGG PGTIKKITFG Medicago MGVFNFEDET TSIVAPARLY KALVTDSDNL IPKVI.DAIQ SIEIVEGNGG AGTIKKLTFV Garden pea MGVFNVEDEI TSVVAPAILY KALVTDADNL TPKVI.DAIK SIEIVEGNGG AGTIKKLTFV Soybean MGVFTFEDEI NSPVAPATLY KALVTDADNV IPKAL.DSFK SVENVEGNGG PGTIKKITFL Tomato MGVNTYTYES TTTISPTRLF KALVLDFDNL VPKLLSQHVK NNETIEGDGG VGSIKQMNFV Celery MGVQKTVVEA PSTVSAEKMY QGFLLDMDTV FPKVLPQLIK SVEILEGDGG VGTVKLVHLG Carrot MGAQSHSLEI TSSVSAEKIF SGIVLDVDTV IPKAAPGAYK SVDV.KGDGG AGTVRIITLP Asparagus MSSSAVSHEI ESSVSAARLF KASMIEWHNL APKILPEIVS SASVVAVDGG VGSIRQINFT 120 Birch EGFPFKYVKD RVDEVDHTNF KYNYSVIEGG PIGDTLEKIS NEIKIVATPD GGSILKISNK Hazel EGSPFKYVKE RVEEVDHTNF KYSYTVIEGG PVGDKVEKIC NEIKIVAAPD GGSILKISNK Alder EGSPFKYVKE RVDEVDRVNF KYSFSVIEGG AVGDALEKVC NEIKIVAAPD GGSILKISNK Hornbeam EGSPVKYVKE RVEEVDHTNF KYSYTVIEGG PVGDKVEKIC NEIKIVAAPD GGSILKITSK Beech EGSQFKYVKH RIDEVDQANF SYGYSVIEGD VVSGIIEKIS YEIKIVASPD GGSLLKSTSK Apple EGSQYGYVKQ RVNGIDKDNF TYSYSMIEGD TLSDKLEKIT YETKLIASPD GGSIIKTTSH Cherry EGSQYGYVKH KIDSIDKENY SYSYTLIEGD ALGDTLEKIS YETKLVASPS GGSIIKSTSH Apricot EGSQYAYVKH RVDGIDKDNL SYSYTLIEGD ALSDVIENIA YDIKLVASPD GGSIVKTTSH Pear EGSQYGYVKH RVDSIDEASY SYAYTLIEGD ALTDTIEKIS YEAKLVASGS .GSTIKSISH Medicago EGGETKYDLH KVDLVDDVNF AYNYSIVGGG GLPDTVEKIS FESKLSAGPD GGSIAKLTVK Garden pea EDGETKHVLH KVELVDVANL AYNYSIVGGV GFPDTVEKIS FEAKLSAGPN GGSIAKLSVK Soybean EDGETKFVLH KIESIDEANL GYSYSVVGGA ALPDTAEKIT FDSKLVAGPN GGSAGKLTVK Tomato EGGPIKYLKH KIHVIDDKNL ETKYSLIEGD ILGEKLESIT YDIKFEANDN GGCVYKTTTE Celery EATEYTTMKQ KVDVIDKAGL AYTYTTIGGD ILVDVLESVV NEFVVVPT.D GGCIVKNTTI Carrot EGSPITSMTV RTDAVNKEAL TYDSTVIDGD ILLGFIESIE THLVVVPTAD GGSITKTTAI Asparagus SAMPFPYLKE RLDFVDEANF ECKSSLIEGG DLGTKLESAS SHFKLVPSSN GGCVVKLEGI 178 Birch YHTKGDHEVK AEQVKASKEM GETLLRAVES YLLAHSDAYN ~~~~~~~~~~ ~~~~~~~~ Hazel YHTKGDHEVD AEHIKGGKEK VEGLFRAVEA YLLAHSDAYN ~~~~~~~~~~ ~~~~~~~~ Alder FHTKGDHEIN AEQIKIEKEK AVGLLKAVES YLLAHSDAYN ~~~~~~~~~~ ~~~~~~~~ Hornbeam YHTKGDHEVP AEHIKGGKER VEGLLKPVEA YLLAHTAEYN N~~~~~~~~~ ~~~~~~~~ Beech YHIKGNHEIK EEEVKAGKEK AAGLFKAVEA YLLAHPDAYN ~~~~~~~~~~ ~~~~~~~~ Apple YHAKGDVEIK EEHVKAGKEK ASGLFKLLEA YLLAHSDAYN ~~~~~~~~~~ ~~~~~~~~ Cherry YHTKGNVEIK EEHVKAGKEK ASNLFKLIET YLKGHPDAYN ~~~~~~~~~~ ~~~~~~~~ Apricot YHTKGDVEIK EEQVKAGKEK AAGLFKLVEA YLLANPDAYN ~~~~~~~~~~ ~~~~~~~~ Pear YHTKGDIEIK EEHVKAGKEK AHGLFKLIES YLKDHPDAYN ~~~~~~~~~~ ~~~~~~~~ Medicago YFTKGDAAPS EEEIKGGKAR GDGLFKALEG YVLANPD.Y~ ~~~~~~~~~~ ~~~~~~~~ Garden pea YFTKGDAAPS EEQLKTDKAK GDGLFKALEG YCLAHPD.YN ~~~~~~~~~~ ~~~~~~~~ Soybean YETKGDAEPN QDELKTGKAK ADALFKAIEA YLLAHPD.YN ~~~~~~~~~~ ~~~~~~~~ Tomato YHTKGDHVVS EEEHNVGRRE NHEYFQGCRS IPSRESFCLR LNIDEKESGL HVRNYACT Celery YNTKGDAVLP EDKIKEATEK SALAFKAVEA YLLANLQFLA ~~~~~~~~~~ ~~~~~~~~ Carrot FHTKGDAVVP EENIKFADEQ NTALFKAIEA YLIAN~~~~~ ~~~~~~~~~~ ~~~~~~~~ Asparagus FKALPGVETT DEVAK.GKEM MTNAIKAAEA YLVANPTAYA ~~~~~~~~~~ ~~~~~~~~

o

Hazelnut, 46%

o

Apple, 39%

o

Peach, 24%

o

Cherry, 22%

o

Nut, 21%

o

Pear, 20%

o

Almond, 19%

o

Plume, 17%

o

Brazil nut, 16%

o

Potato, 15%

o

Carrot, 13%

o

Peanut, 10%

o

Strawberry, 10%

o

Orange, 9%

o

Apricot, 7%

PR-10

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

.

Cor a 8

Pru p 3

Ara h 9

Mal d 3

Zea m 14

Pru av 3

LTP

nPru p 3

rCor a 8

rPar j 2

nArt v 3

Ara h 9

Mal d 3

Zea m 4

Jug r 3

Pru ar 3

(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.

rMal d 4

rHev b 8

rPhl p 12

rBet v 2

rMer a 1

Ara h 5

Pru p 4

rCor a 2

rOle e 2

rPyr c 4

PFL

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

(16)

To resume…

Local

(OAS)

Characteristic

symptoms

Systemic

Sensitization profiles

PR-10

proteins

ns-LTP

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

•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.

•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.

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, 2006:395-463

After roasting peanuts, allergenicity of Ara h 2 is multiplicated by 3

(19)

Laboratory practice :

How to diagnose peanut allergy ?

Peanut (f13) + Ara h 2 (f423)

Peanut (f13) : Neg

Ara h 2 : Neg

Low risks of severe

reactions

How to explore ?

Recommendations

Birch (north Europe) :

test Ara h 8 (f352)

Peanut (f13) : Pos

Ara h 2 : Neg

Risk of severe reaction?

How to explore?

Recommendations

Test : Risk

• Ara h 1 (f422)

•Ara h 3 (f424)

•Ara h 9 (f427)

•Ara h 8 (f352)

•CCD (Ro214)

Peanut (f13) : Pos

Ara h 2 : Pos

Very high risk of

(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.

(21)

Peanut (f13) = 27,4 kUA/L

Ara h 2 (f423) = 14,2 kUA/L

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

Peanut (f13) : Pos

Ara h 2 : Pos

Major allergens of peanut

Storage proteins

peanut PR-10 protein !

• 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.

Clinical cases (1)

Cross-reaction : storage proteins ??

Very high risk of

severe reactions

How to explore?

Recommendations

(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?

(23)

Peanut (f13) = 1,22 kUA/L

Ara h 2 (f423) < 0,10 kUA/L

Peanut (f13) : Pos

Ara h 2 : Neg

Risk of severe reactions?

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

Clinical cases (2)

PR-10, Bet v 1-homologue

• 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.

How to explore?

Recommendations

(24)

Clinical cases (3)

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 ?

(25)

Peanut (f13) = 1,23 kUA/L

Ara h 2 (f423) < 0,1 kUA/L

Peanut (f13) : Pos

Ara h 2 : Nég

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

Clinical cases (3)

• 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.

Risk of severe reactions?

How to explore?

Recommendations

(26)

Clinical cases (4)

4. Eddy, M :



f13 (peanut) = 0,44 kUA/L

–

SPT+ : birch, apple, peach, banana, garlic, peanut



Swollen lips



What are the risks ?

(27)

Peanut (f13) = 0,44 kUA/L

Ara h 2 (f423) < 0,1 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

?

Bet v 2 = 4,6 kUA/L

Clinical Cases (4)

• 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

(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

Peanut (f13) : Pos

Ara h 2 : Neg

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

Risk of severe reactions?

How to explore?

Recommendations

Hazelnut (f17) = 4,23 kUA/L

Cor a 8 (f425) = 3,87 kUA/L

Hazelnut (f17) : Pos

Cor a 8 : Pos

Very high risk of

severe reactions

nsLTP

•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

(30)

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.

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 IgEs for extracts

Thanks to all the recombinants,

(31)

Request form ImmunoCAP™Components

Patient: Nom : Mister Albert Gik

Adresse: Birch Street 1…

Birth date:

01/01/1950

Date de demande:

19/11/2009

Signature:

Docteur: Nom:

Cavalier Etienne

Adresse :

CHU of Liège

June 2009

Some other components less useful in Belgium could be available.

Don’t hesitate to ask to your Lab in case of specific clinical history.

In theory, this is possible…

Practically, …

(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.

(33)

Immuno Solid-phase AllergenChip (ISAC)

Allergen-triplet after

fluorescence-reaction

Allergen molecule

Fluorescence-labeled

secondary anti-human

IgE

IgE-antibody

(in the patient’s serum)

(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 :

 Important !

– 5 serum albumins :

 Which

ones

are

most

representative?

– Honey bee and paper wasp

venoms :

(35)

(36)

Validation of the method

• 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

Tot

IgE rate

(> 10.000 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 150.000kU/L of

Tot

IgE, 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 2

nd

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)

Pollens : Cyn d 1, Phl p 1,

Phl p 2, Phl p 4, Phl p 5,

Phl p 6 et Ole e 1

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

Profilins :

Bet v 2, Ole e 2, Hev b

8, Mer a 1, Phl p 12

Species markers :

Gal d 1, Bos d

lactoferrin, Fel d 1,

Fel d 4, Can f 1, Mus

m 1, Alt a 1

Tropomyosin : Der p 10

Der p 1, Der f 1, Der f

2, Der p 2, Eur m 2

Albert Gik

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.

(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: