April 23, 2013

ISAC and Component-resolved Diagnosis

Douwe de Boer, Chris Nieuwhof, Judith Bons en Paul Menheere

CRD and ISAC | De Boer, Nieuwhof, Bons and Menheere | April 23, 2013 2

Allergy: clinical relevance “in vitro” testing

Ebo et al., University of Antwerp

A

B

C

allergen = mixture of allergen components

Component-resolved Diagnosis (CRD)

intra-allergenic heterogeneity

ED

CRD and ISAC | De Boer, Nieuwhof, Bons and Menheere | April 23, 2013 3

Multiplex assay: multiple analysis of specific

IgE directed against allergen components in one

single run

RAST

Single-analyte assay: single analysis of specific IgE directed against

single allergens, single allergen components or mixtures of allergens

classical ImmunoCAP assay

ISAC (Immuno Solid-phase Allergy Chip )

ImmunoCAP ISAC (16 spots per mm2)

ISAC112

40 µLRAST1

30 µL

CRD and ISAC | De Boer, Nieuwhof, Bons and Menheere | April 23, 2013 4

intra-allergenic heterogeneity

non-specific Lipid Transfer Protein

Pathogenesis Related-10 protein

Profilin

Storage protein

Storage protein

Storage protein

Storage protein

Storage protein

Storage protein

Component-resolved Diagnosis (CRD)

CRD and ISAC | De Boer, Nieuwhof, Bons and Menheere | April 23, 2013 5

Complications Detecting specific IgEs - 1

Ara h 9

Ara h 8

Ara h 5

non-specific Lipid Transfer Proteins

Pathogenesis Related-10 proteins

Profilins

inter-allergenic homogeneity

Pan-allergens

intra-allergenic heterogeneity

CRD and ISAC | De Boer, Nieuwhof, Bons and Menheere | April 23, 2013 6

Complications Detecting specific IgEs - 2

intra-allergenic heterogeneity

inter-allergenic homogeneity

Component-resolved Diagnosis (CRD)

CRD and ISAC | De Boer, Nieuwhof, Bons and Menheere | April 23, 2013 7

Pan-allergens – risks and severity

Cross-reactive CarbohydrateDeterminants

specific glycan epitope

initially CCD was considered to be important to explain cross-reactivity

mainly “plant” derived pan-allergens

CRD and ISAC | De Boer, Nieuwhof, Bons and Menheere | April 23, 2013 8

Profilins

rMal d 4

rHev b 8

rPhl p 12rBet v 2rMer a 1 rAra h 5

rPru p 4

Cytoskeleton proteins

Highly conserved proteinsTrue pan-allergen

(high degree of cross-reactivity)

CRD and ISAC | De Boer, Nieuwhof, Bons and Menheere | April 23, 2013 9

Pathogenesis Related-10 Protein (PR-10)

rMal d 1

rDau c 1

rPru p 1

rAra h 8rCor a 1.01 rCor a 1.04

rApi g 1

rGly m 4rBet v 1

rPru ar 1rPyr c 1

rPru av 1

Unknown function

Large family and conserved proteinsHeat and acid labile proteins

CRD and ISAC | De Boer, Nieuwhof, Bons and Menheere | April 23, 2013 10

non-specific Lipid Transfer Protein (LTP)

rCor a 8rPru p 3

rAra h 9 rMal d 3

rZea m 4

rPru av 3

Relatively conserved proteinsHeat and acid stable protein

Shuttle protein of

lipids

CRD and ISAC | De Boer, Nieuwhof, Bons and Menheere | April 23, 2013 11

Storage Protein

rBer e 1

rAra h 1rAra h 2rAra h 3

rAra h 6

rCor a 9

rSes i 3

nGly m 5nGly m 6

Proteins for storage of energy mainly in seedsRelatively less conserved proteins

pseudo-Pan-allergen (low degree of cross-reactivity)

“Voorraad of opslag eiwitten”

CRD and ISAC | De Boer, Nieuwhof, Bons and Menheere | April 23, 2013 12

Pan-allergen sensitization in Europe

CRD and ISAC | De Boer, Nieuwhof, Bons and Menheere | April 23, 2013 13

ISAC and Component-resolved Diagnosis

Besides separate allergen components groups of pan- allergens can be distinguished

Conclusions and remarks

Components and pan-allergens are being associated with risks

Component-resolved Diagnosis can give insights in sensibilisations for separate allergen components

ISAC is a multiplex assay (an extensive number of components)

RAST is a single analyte assay (limited number of components)

inter-allergenic homogeneity

intra-allergenic heterogeneity