Gebruik van componenten in “in vitro”diagnostiek van allergie (CRD).

Major and minor components

Purified and recombinant allergen components

Allergy-elicitingbiological material

Standard AllergenImmunoCAP

Extraction,Immobilizationon solid phase

mRNAextraction

poly-(A)

poly-(A)

poly-(A)

poly-(A)

poly-(A)poly-(A)

cDNA synthesis, cloning andselection of cDNA clones encoding

individual allergenic proteins

Recombinant AllergenImmunoCAP

Production of recombinant allergens,Immobilization on solid phase

Standardization andresearch reagents

Nomenclature of components

• t3: Betula verucosa

• g6: Phleum pratense

• d1: Dermato. pteronys.

• e1: Felis domesticus

• f13 : Arachis hypogaea

Bet v 1, 2, 4, 6, 7

Phl p 1,..5,…7…12

Der p 1, 2, 3,….10,..

Fel d 1, 2, 3…

Ara h 1, 2 , 3, …..8,

Preceded by:

n : natural purified protein nDer p1

r : recombinant protein rBet v 1

Component - Resolved Diagnostics (CRD)

o More sensitive tests (Spiked extracts)

o Tailor-made immunotherapy (Specific components)

o Predict cross-reactivity (Cross reactive components)

o Predict severity of reactions (Link with symptoms)

Why?

Birch t3 Timothy g6 Wall pellitory w21 Peach f95 Peanut f13 Braz nut f18 Soy f14 Celery f85 Latex k82 Asp. Fum. m3 Hazelnut f17

PR 10 Bet v1 Pru p1 Ara h8 Gly m4 Api g1,01 Cor a1

Profilin Bet v2 Phl p12 Pru p4 Ara h5 Hev b8 Cor a2

Calcium binding protein Bet v4 Phl p7

Lipid Transfer Protein LTP (Par j2) Pru p3 Ara h9 Hev b12 Cor a8

Vicilin 7S globulin Ara h1 Cor a11

Conglutin 2S Albumin Ara h2 Ber e1 X

Glycinin 11S globulin Ara h3 X Cor a9

Enolase Hev b9 Asp f22

Mn-SOD Hev b10 Asp f6

Shrimp f24 Fish f3 Cat e1 Dog e5 Cow e4

Tropomyosin Pen a1

Parvalbumin Cyp c1

Serum albumin Fel d2 Can f3 Bos d6

Lipocalin Fel d4 Can f1, Can f2 Bos d2

ImmunoCAP™ Panallergens

Panallergens and crossreactions

Panallergens – important proteins in food allergy:

Proteins with similar functions in different species may have more or less similar molecular structures. Some of the most well-studied allergen components belong to protein families listed in this table. They are widely distributed and IgE antibodies to these proteins often show cross-reactivity.

The term “panallergens” is often used for these widely distributed proteins.

PR-10 protein, Bet v 1 homologue• A heat labile protein, cooked foods are often tolerated

• Often associated with local symptoms such as oral allergy syndrome (OAS)

• Often associated with allergic reactions to fruit and vegetables in northern Europe

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

• Often associated with allergic reactions to fruit and vegetables in southern Europe

Profilin•Seldom associated with clinical symptoms but may cause demonstrable or even

severe reactions in a small minority of patients or specific allergens

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

CCD• A marker for sensitization to cross-reactive carbohydrate determinants

• Seldom associated with clinical symptoms but may cause demonstrable or even

severe reactions in a small minority of patients

Tropomyosin• An actin-binding protein in muscle fibers

• A marker for cross-reactivity between crustaceans, mites and cockroach

Parvalbumin

• One of the major allergens in fish

• A marker for cross-reactivity among different species of fish and amphibians

• A protein stable to heat and digestion causing reactions also to cooked foods

Serum albumin

• A common protein present in different biological fluids and solids e.g cows milk

and beef, eggs and chicken

• Cross-reactions between albumins from different animal species are well known,

for example between cat and dog and cat and pork

d1: Dermatophagoïdes pteronyssinus

Prevalentie: 25% van de bevolking (35.2%)

Kruisreakties: Andere mijten (Dermatophagoïdes farinae)

Andere ongewervelden (slak, garnaal) (Verantwoordelijke eiwit: Tropomyosin)

1) Inhalatie allergenen (binnenshuis)

“Allergen cross-reactivity between house-dust mites and other invertebrates”

K. E. Sidenius, Allergy 2001: 56: 723-733nDer p1, rDer p2, rDer p10

Mite extract

A possible decision tree for Der p-immunotherapy.Serological testing with mite allergen extract confirmsIgE-mediated allergy. Componentresolved diagnosis (CRD) with major (Der p 1, Der p2) and cross-reactive (Der p 10) allergens may beperformed to further determine suitability for Der p-specificimmunotherapy.

Adapted from: G. Pittner et al.,Clinical and Experimental Allergy, 2004; 34:597–603

e1: Kat, epitheel Felis domesticus

Prevalentie: 9.5% van de bevolking (16%)

Kruisreakties: Hond, Paard, Varkensvlees (Verantwoordelijke eiwit: Serum Albumine)

rFel d1, nFel d2e220: Kat serum albumin (nFel d 2)

Cat avoidance

• Rate of decline of Fel d1

after cat removal.

• From: Wood RA & al.

« The effect of cat removal on allergencontent in househod dust samples »

J allergy Clin Immunol 1989; 83: 730-35

Source material: Serumalbumin

Common names: Cat, Domesticcat

Family: Felidae

Felis domesticus

e220 Cat serum albumin

= nFeld d2

Source material: Serum albumin

Family: Bovidae

Bos spp.

e204 BSA (bovine serumalbumin)

= nBos d 6

Source material: Serum

Common names: Chicken, Hen, Cock,

Family: Phasianidae

Gallus domesticus

e219 Chicken, serum proteins

Contains

nGal d 5

Source material: Serum albumbin

Common names: Dog, DomesticDog,

Family: Canidae

Canis familiaris

e221 Dog serum albumin

= nCan f 3

f233 Ovomucoid (ovotransferrin) = nGal d 1f232 Ovalbumin = nGal d 2f323 Conalbumin = nGal d3k208 Lysozyme = nGal d4

f76 a-lactalbumin = nBos d4f77 b-lactoglobulin = nBos d5f78 Casein = nBos d8f334 Bovine lactoferrin = nBos d Lactoferrin

Native Components

Common names: Swine, Pig, Hog, Boar

Family: Suidae

Sus scrofa

e222 Swine, serum albumin

=nSus s PSA

e5: Hond, haar en huidschilfersPrevalentie: 3-14% van de bevolking (9.9%)

Kruisreakties: Kat (Verantwoordelijke eiwit: Serum Albumine)

rCan f1, rCan f2, nCan f3

m3:Aspergillus fumigatus

Prevalentie: (mx1) + 5% van de bevolking (4.3%)

Kruisreakties: Andere Aspergillus soorten

rAsp f1, rAsp f2, rAsp f3,rAsp f4, rAsp f6

i6: Kakkerlak (Blatella germanica)

Prevalentie: 4.7% van de bevolking

Kruisreakties: Andere insecten (en geleedpotigen)

(Verantwoordelijke eiwit: Tropomyosine Bla g7)

(rDer p10, rPen a1)

Latex

allergen

Molecular

mass (Kda)

Protein name Significance

allergen

Significance

Cross-reaction

IgE Binding

Prevalence

Hev b 1 14,6 Rubber

elongation factor

High (SB) Not observed HCW:52%

SB:81%

Hev b 2 34-36 Beta-1,3-

glucanase

Medium Medium HCW:65%

SB:54%

Hev b 3 24-27 Small rubber

particle protein

High (SB) Not observed HCW:13-20%

SB:76-78%

Hev b 4 50-57 Beta-

glucosidase

Not known Not observed Not Clear

Hev b 5 16-24 Acidic structural

protein

High in all risk

groups

Not observed

(Homology Kiwi)

HCW:68-92%

SB:33-56%

Hev b 6.01 20 Prohevein High in all risk

groups

High (Banana-kiwi-

avocado)

LAP:75-83%

Hev b 6.02 4,7 Hevein High in all risk

groups

High (Banana-kiwi-

avocado)

LAP:56%

HCW:75%

SB:27%

Hev b 6.03 14 C-domaine of

prohevein

High High (Homlogy

plant stress

proteins)

LAP:15-21%

Hev b 7.01 42 Patatin-like

protein

Low-Medium Unclear

(Homology potato-

tomato)

LAP:11-49%

Hev b 7.02 44 Patatin-like

protein

Medium only in

SB

Unclear

(Homology potato-

tomato)

SB:39.5%

Hev b 8 14 Profilin Low Medium

panallergen

LAP:11%

HCW:20-24%

SB:6-12%

Hev b 9 51 Enolase Low Medium (Molds) LAP:15%

Hev b 10 26 Manganese

superoxyde

dismutase

Low Medium (Molds) LAP:27%

HCW:0%

SB:10%

Hev b 11 32 Class I chitinase Low High (Fruits) LAP:19-29%

Hev b 12 9 Lipid transfer

protein

Low Medium (fruits)

panallergen

LAP:24%

Hev b 13 42 Esterase High Not determined HCW:63%

rHev b1, rHev b3, rHev b5, rHev b6.01,

rHev b6.02, rHev b8, (rHev b9, rHev b11)

2) Inhalatie allergenen (buitenshuis)

t4: Hazelaar (Corylus avellana)Kruisreakties: Berk, Hazelnoot (6.8%)

Bestuiving: Januari tot april

t3: Ruwe berk (Betula verrucosa)Kruisreakties:apple(rosaceae),hazelnoot, wortel, pinda (7.4%)

Bestuiving: Maart tot meirBet v1, rBet v2, rBet v4, rBet v6

Effect of seasonal exposure on IgE to Bet v 1

g6:Thimotheegras (Phleum pratense)Prévalentie: (gx3) 17% van de bevolking (22.8%)

Kruisreakties: Andere grassen, (Weegbree), (graangewassen)(tomaat)

Bestuiving: April tot septemberrPhl p1, rPhl p2, nPhl p4, rPhl p5, rPhl p6, rPhl p7, rPhl p11, rPhl p12

kU /lA

kU /lA

kU /lA kU /lA kU /lA

kU /lA

kU /lA

kU /lA

kU /lA

Immunotherapy?

w9: Smalle weegbree (Plantago lanceolata)Kruisreakties: Grassen (Phl p5?), Meloen

Bestuiving: Mei tot september

w6: Bijvoet (Artemisia vulgaris)Kruisreakties: Selderij, Wortel, Specerijen (Umbelliferae) (4.3%)

Bestuiving: Juli tot september

nArt v1, nArt v3

m2: Cladiosporum herbarum (mx1) Periode: Mei tot september (4.3%)

m6: Alternaria alternata (mx1) Periode: Juni tot september (4.3%)

rAlt a1

3) Voedingsallergenen bij kinderen: (In mengsel fx5 inbegrepen)

f1: EiwitPrevalentie: 35.7% van voedingsallergieën (50.9%)

Kruisreakties: Vogels (pluimen en vlees <5%)

nGal d1, nGal d2, nGal d3, nGal d4

f233 Ovomucoid (ovotransferrin) = nGal d 1f232 Ovalbumin = nGal d 2f323 Conalbumin = nGal d3k208 Lysozyme = nGal d4

f13: Pinda (Arachis hypogaea)

Prevalentie: 23.6% van voedingsallergieën (40.4%)

Kruisreakties: léguminosae (Erwt, Linze, Soja, Lupine)Berk

rAra h1, rAra h2, rAra h3, rAra h8, rAra h9

cicbaa 2007

http://www.cicbaa.com/

f2: KoemelkPrevalentie: 8.3% van voedingsallergieën (16.4%)

Kruisreakties: Geitemelk >50% Rundsvlees <5%

nBos d4, nBos d5, nBos d6, nBos d8f76 a-lactalbumin = nBos d4f77 b-lactoglobulin = nBos d5f78 Casein = nBos d8f334 Bovine lactoferrin = nBos d Lactoferrine204 BSA = nBos d6

f3: Kabeljauw (gadus morhua) Cluster Vissen Prevalentie: 4.3% van voedingsallergieën (6.9%)

Kruisreakties: Andere vissen (Verantwoordelijke eiwit: Parvalbumine)

rGad c1 (rCyp c1)

f4: Tarwe (Triticum aestivum) Cluster GranenPrevalentie: 2% van voedingsallergieën (3.4%)

Kruisreakties: Andere grassen (Rogge, Haver, Maïs, Rijst)

rTri a19

f14: Soja (Glycine max) Cluster Peuldragers

Prevalentie: 1% van voedingsallergieën (excl. Pinda (8.1%)

Kruisreakties: léguminosae (Erwt, Linze, Pinda) Berk

rGly m4

rCyp c 1: Parvalbumin from CarprGad c 1: Parvalbumin from CodParvalbumins are abundant and stable allergenswith a remarkable resistance to heat, denaturing chemicals, and proteolytic enzymes.sensitizing agents for > 95% of fish allergic patients

ω-5 gliadin has been reported as a major allergen in Wheat-Dependent Exercise-Induced Anaphylaxis (WDEIA)In addition,ω-5 gliadin has been shown to be a major allergen in children with immediate allergy to ingested wheat.

4) Voedingsallergenen bij volwassenen

f49: Appel (Malus sylvestris) Cluster RosaceaePrevalentie: 7% van voedingsallergieën (27.3%)

Kruisreakties: Berkepollen, Andere rosaceae

(rPru p1, rPru p3, rPru p4)

f17: Hazelnoot(Corylus avellana) Cluster noten (rBer e1)Prevalentie: 5.9% van voedingsallergieën (16%)

Kruisreakties: Andere noten, Berkepollen, Hazelaar

Cor a1, Cor a8, (Cor a9)

f85: Selderij (Apium graveolens) Cluster SchermbloemigenPrevalentie: 5.5% van voedingsallergieën (17.5%)

Kruisreakties: Bijvoetpollen, Berkepollen;Wortel, Specerij

rApi g1

Multialignment of Bet v 1 and related proteins 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 ~~~~~~~~~~ ~~~~~~~~

Act d8

Pru p 1

Pru p 3

Pru p 4

Pru a 1

Pru a 3

Pru d 4

Mal d 1

Mal d 3

Mal d 4

Pru d 1

Pru d 3

Pru d 4

Pyr c 1

Pyr c 3

Pyr c 4

Pru av 1

Pru av 3

Pru av 4

Similarity of these components between different stone fruits

Rosaceae fruits

f96: Avocado (Persea americana) Cluster Latex Prevalentie: 5.1% van voedingsallergieën (23%)

Kruisreakties: Latex; Banana, Kiwi, Kastanje

(rHev b6.01, rHev b6.02, rHev b11)

f13: Pinda (Arachis hypogea)Prevalentie: 4% van voedingsallergieën (10.1%)

Kruisreakties: léguminosae (Erwt, Linze, Soja, Lupine)

rAra h1, rAra h2, rAra h3, rAra h8, rAra h9

f24: Garnaal (Pandalus borealis, Penaeus monodon Metapenaeopsis barbata, Metapenaus joyneri)

Prevalentie: 2.5% van voedingsallergieën (4.7%)

Kruisreakties: Schaaldieren, Mijten (tropomyosine)

rPen a1

(rAct d8)

Established immunological relations within invertebrates:

o Crustaceans: e.g. shrimp, crab, crawfish, lobster

o Mollusks: e.g. clams, mussels, oysters, snails, squid

o Arachnids: e.g. mites, spiders

o Insects: e.g. cockroaches

Reese G et al. Immunol 1999;119:247-58

Recombinant tropomyosin from Penaeus aztecus (rPen a 1) for measurement of

specific immunoglobulin E antibodies relevant in food allergy to crustaceans

and other invertebrates

Asa Marknell DeWitt, Lars Mattsson, Iris Lauer, Gerald Reese , Jonas Lidholm

Mol. Nutr. Food Res. 2004, 48, 370 – 379

Xylose and Fucose are parts of most common CCD. Characterization of IgE responses against N-glycans using purified glycoproteins turned out to be more problematic than we originally thought.”

CCD From the current literature:…..

R Van Ree et.al. J Biol Chem 2000;275:11451-11458

Red - carbohydrate epitope

Green - peptidic epitope

Inactive Active Active

From: Fötisch & Vieths (2001) Glycoconj J 18:373-90

Are CCD epitopes biologically active or not?

J. Lidholm/2004

”Our results demonstrate that IgE specific for CCD is common in celery allergic patients, and can represent the major proportion of IgE against this food……………...

Our data show that proteins carrying multiple glycan units can be biologigcally active in patients sensitized to CCD.”

Fötisch K et.al. Involvement of carbohydrate epitopes in the

IgE response of celery-allergic patients. Int Arch Allergy

Immunol 1999;120:30-42

Bromelin, Rk202Allergen CCD; MUXF3 from Bromelin, Ro214

Double positive Venoms

HB (i1) 2 kUA/L

YJ (i3) 5 kUA/L

CCD 0,2 kUA/L

HB (i1) 2 kUA/L

YJ (i3) 5 kUA/L

CCD 2 kUA/L

SIT

HB +YJ

or

YJ only

?

Jappe U et al. Allergy 2006 61: 1220-29

rApi m1, (rVes v5)

The development of recombinant hymenoptera allergens is the next step to be taken.

From the results of this and other studies it seems to be inevitable to develop and

include recombinant allergens in the diagnostic procedures to detect hymenoptera

venom allergy.

Polysensitisation/Crossreaction & Immunotherapy?

Immunotherapy?

Predict cross-reactivity

Common allergenic structures;

o due to close biological relation

o but also in seemingly unrelated species

Predict severity of reactions

Component - Resolved Diagnostics (CRD)

In vitro tests for true sensitization may give new applications :

o (More sensitive tests)

o Tailor-made immunotherapy

oT3: Bet v1, Bet v2, Bet v4

oG6: Phl p1, Phl p5, Phl p7, Phl p12

oW6: Art v1, Art v3

oD1: Der p1, Der p2

o Predict severity of reactions

oF13: Ara h1, Ara h2, Ara h3, Ara h8, Ara h9

oF17: Cor a1, Cor a8,

oF49: Pru p1, Pru p3,

oK82: Hev b1, Hev b3, Hev b5, Hev b6

o Predict cross-reactivity:

oProfilin: Pru p4, Hev b8, (Ara h5, Cor a2, etc....)

oCCD

Request form ImmunoCAP™Components

Grass pollens : � g6 Phleum pratense � rPhl p 1 Phleum pratense g205 � rPhl p 2 Phleum pratense g206 � nPhl p 4 Phleum pratense g208 � rPhl p 5b Phleum pratense g215 � rPhl p 6 Phleum pratense g209 � rPhl p 7 Calc. Bind. Prot. Phleum pratense g210 � rPhl p 11 Phleum pratense g211 � rPhl p 12 Profilin Phleum pratense g212 � Mix rPhl p 1, rPhl p 5b Phleum pratense g213 � Mix rPhl p 7, rPhl p 12 Phleum pratense g214

Weed pollens : � w6: Artemisia vulgaris � nArt v1 Artemisia vulgaris w231 � nArt v3 LTP Artemisia vulgaris w233 � w11: Salsola kali � nSal k1 Salsola kali w232

Tree pollens : � t3: Betula verrucosa � rBet v 1 PR10 Betula verrucosa t215 � rBet v 2 Profilin Betula verrucosa t216 � rBet v 4 Calc. Bind. Prot. Betula verrucosa t220 � rBet v 6 Betula verrucosa t225 � Mix rBet v 2, rBet v 4 Betula verrucosa t221 � t9: Olea europaea � nOle e 1 Olea europaea t224 � t222: Cupressus arizonica � nCup a1Cupressus arizonica t226

Mites �d1: Dermatophagoides pteronyssinus � nDer p1 Dermatophagoides pteronyssinus d202 � rDer p2 Dermatophagoides pteronyssinus d203 � rDer p10 Tropomyosin Dermatophagoides pteronyssinus d205

Microorganisms � m3 : Aspergillus fumigatus � rAsp f 1 Aspergillus fumigatus m218 � rAsp f 2 Aspergillus fumigatus m219 � rAsp f 3 Aspergillus fumigatus m220 � rAsp f 4 Aspergillus fumigatus m221 � rAsp f 6 Aspergillus fumigatus m222 � m6 : Alternaria alternata � rAlt a 1 Alternaria alternata m229

Epidermals & other proteins � e1: Cat Felis domesticus � rFel d 1 Felis domesticus e94 � nFel d 2 serum albumin Felis domesticus e220 � e5: Dog Canis familiaris � rCan f 1 Canis familiaris e101 � rCan f 2 Canis familiaris e102 � nCan f 3 Serum albumin Canis familiaris e221 � e4: Cow Bos spp � nBos d 6 BSA Serum albumin Bos spp. e204 � e83: Swine Sus scrofa � Swine serum albumin Sus scrofa e222

Foods � f13: Peanut Arachis hypogaea � rAra h 1 Arachis hypogaea f422 � rAra h 2 Arachis hypogaea f423 � rAra h 3 Arachis hypogaea f424 � rAra h 8 PR10 Arachis hypogaea f352 � rAra h 9 LTP Arachis hypogaea f427 � f17: Hazelnut Corylus avellana � rCor a1 PR10 Corylus avellana f428 � rCor a8 LTP Corylus avellana f425 � f18: Brazil nut Bertholletia excelsa � rBer e 1 Bertholletia excelsa f354 � f4: Wheat Triticum spp � rTri a 19; Omega-5 Gliadin Triticum spp. f416 � f14: Soy Glycine max � rGly m 4 PR10 Glycine max f353 � f95: Peach Prunus persica � rPru p 1 PR10 Prunus persica f419 � rPru p 3 LTP Prunus persica f420 � rPru p 4 Profilin Prunus persica f421 � f85: Celery Apium graveolens � rApi g 1.01 PR10 Apium graveolens f417 � f84: Kiwi Actinidia chinensis � rAct d8 PR10 Actinidia chinensis f430

� f1 Egg white � nGal d 1 Ovomucoid Gallus spp. f233 � nGal d 2 Ovalbumin Gallus spp. f232 � nGal d 3 Conalbumin Gallus spp. f323 � nGal d 4 Lysozyme Gallus spp. k208 � f2: Milk Bos spp � nBos d 4 α-lactalbumin Bos spp. f76 � nBos d 5 β -lactoglobulin Bos spp. f77 � nBos d 8 Casein Bos spp. f78 � Bovine lactoferrin Bos spp. f334 � f24 Shrimp � rPen a 1 Tropomyosin Penaeus aztecus f351 � f3: Fish (cod) � rCyp c 1 Parvalbumin Cyprinus carpio f355 � rGad c1 Parvalbumin Gadus morhua f426

Occupational allergens � k82: Hevea brasiliensis � rHev b 1 Hevea brasiliensis k215 � rHev b 3 Hevea brasiliensis k217 � rHev b 5 Hevea brasiliensis k218 � rHev b 6.01 Hevea brasiliensis k219 � rHev b 6.02 Hevea brasiliensis k220 � rHev b 8 Profilin Hevea brasiliensis k221 � rHev b 9 Enolase Hevea brasiliensis k222 � rHev b 11 Hevea brasiliensis k224

Enzymes � nCar p 1 Papain Carica papaya k201 � α-amylase From Aspergillus oryzane k87 � Alkalase From Bacillus sp. k205 � Maxatase From Bacillus licheniformis k204 � Pepsin From Sus scrofa k213 � Savinase From Bacillus sp. k206

Venoms � i1Apis mellifera � rApi m 1 Phospholipase A2 Apis mellifera i208 Carbohydrate Determinants (CCD) � Bromelin k202 � CCD; MUXF3 (from bromelin) Ro214

r = Recombinant n = Natif

Patient: Name:………………………………………………

Address:…………………………………………….

Birth date:……/……/......…

Date of the request:…../……/……

Signature:Doctor: Name:……………………………………

Address:…………………………………

N°RIZIV/ INAMI:……………………….

December 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.

ImmunoCAP ISACImmunoCAP ISAC®®

IImmuno mmuno SSolidolid--Phase Phase AAllergen llergen CChiphip

What is the difference to ImmunoCAPWhat is the difference to ImmunoCAP®® ??

Size and matterSize and matter

Microscopic dimensions

~ 100 picogram / allergen spot> 100 allergens / cm²

ImmunoCAP ISAC®

~ 200 µm spotsize

Macroscopic dimensions

Microgram amount of allergen / CAP

103 - 104 times moreallergen per test required

Standard ImmunoCAP®

ImmunoCAP ISACImmunoCAP ISAC®®

Incubate sampleIncubate sample Stain with SAbStain with SAbPrepare serumPrepare serum

Scan biochipScan biochip Analyse imagesAnalyse images

Duration: 3 hoursDuration: 3 hours

Duration: 10 min.Duration: 10 min.

Create reportCreate report

Wash

Wash

•20 uL venous or finger stick serum sample

• Add serum to array, incubate and wash

• Add conjugate, incubate and wash

• Scan and analyze the arrays using MIA

Wash

Multiplex IgE testing with allergen component panelMultiplex IgE testing with allergen component panel

ImmunoCAP ISAC®

103 allergen components

47 allergen sources

Covers most relevant allergen components and

cross-reactive marker allergens

ImmunoCAP ISACImmunoCAP ISAC®®

ImmunoCAP ISACImmunoCAP ISAC®®

ImmunoCAP ISAC® Allergen Components