New technologies in allergologyWorkshop

Ph.Biol. Romy GADISSEUR

16/10/2010

Timeline…allergy diagnosis

1880 1995-991988-911967 2000 2008

Provocation tests (SPT)

First allergens

clonedRAST

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 allergen extracts

Natural allergen extracts

• Problems: – Diagnostic accuracy problems :

Difficulty of standardising the allergens used as substrates, – Similar biological potency BUT differ in terms of their allergenic content,

» natural variability of the sources, » manufacturing process involved, » instability of the allergenic proteins.

– The use of raw extracts :» False-positive results : Presence of high-cross reactivity components or

contamination with allergens from other sources,» False-negative results : Absence or degradation of certain allergenic proteins

in the extract – fundamentally those of plant origin. – These considerations have led to major efforts to improve allergenic extract

quality and standardisation. Incapacity to differentiate among clinical cross-reactivity, true co-

sensitisation to different allergenic sources, and immunological cross-reactivity lacking clinical relevance,

… in the increasingly numerous population of polysensitized patients.

Difficulty in identifying the clinically relevant allergens

• Sensitizations :– monosensitisation, oligosensitisation or polysensitisation.– IgE targeted to a concrete allergenic source

true sensitisation immunological cross-reactivity (not always of a clinical nature), as a result of the

sharing of homologous proteins with the source in question. – Certain groups of proteins may be found in several not always taxonomically

related sources Panallergens : profilins or polcalcins.

– Immunological cross-reactions : cross-reactive carbohydrate determinants (CCDs).

– The determination of specific IgE against the allergenic source, but not against its individual allergenic components, does not allow differentiation between true sensitisation and immunological cross-reactivity.

Development of recombinant allergens and purification of native allergens

Development of recombinant allergens

• The developments in molecular allergy (last three decades)– Characterise the main allergens,– Purify them from their native sources,– Produce them as recombinant proteins from recombinant DNA, thanks

to advances in molecular engineering techniques.• Growing number of allergenic sequences and panels of

purified native and recombinant allergens :– Complexity of the epitopes of the natural sources – offering the

possibility of determining the reactivity pattern or profile of each individual patient by identifying the allergenic molecules that trigger disease.

Allergens described, sequences, studies of interest regarding the known allergenic components The Allergome database,

Freely accessible on the Internet !!

Component-resolved diagnosis

• Allergenic components have allowed diagnostic resolution at molecular level, in the form of component-resolved diagnosis (CRD),

– A new era in allergy. • Main interests :

– Precise identification of the allergens that cause disease. – Possibility to distinguish between patients who are truly

allergic to a given biological source and those with cross-reactivity to molecules shared among different biological sources.

– Identify the molecules to which specific immunotherapy must be targeted, with a view to developing optimised treatments.

• Investigations based on allergenic molecules or components with a broad range of aeroallergens, food allergens and stinging insects.

– Component-resolved IgE recognition patterns can Determine sensitisation patterns, Correlating them to the presence or absence of symptoms, the

severity of the latter, and the clinical phenotypes Contribute to the analysis of geographical differences.

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

related allergens.

Recombinant proteins

• Genetic engineering molecules in application to an allergy test requires careful validation, – Recombinant proteins can differ greatly from their natural

counterparts. Different expression systems (E. coli, Pichia pastoris, tobacco or insect

cells), different capacities to produce post-translation changes. – Prokaryotic cells such as E. coli do not produce the protein glycosylation seen

in the natural protein (affect the capacity to interact with Ig and compromise the usefulness of such proteins if these allergens require cross-reactive carbohydrate determinants (CCDs) for adequate recognition by IgE

» Bee venom hyaluronidase (Api m 2), major allergen of artemisia (Art v 1).

– Post-translation defects such as phosphorylation or the formation of disulphide bridges can cause recombinant allergens to differ in their folding or tertiary structure, thus producing variations in their conformational epitopes (importance for antigen recognition).

• Recombinant proteins should be validated, same allergenic characteristics as the natural allergen, potency in terms of mass units of allergen.

« specific » IgE for extracts

• From a laboratory point of view… – What does one positive sIgE for an allergen extract means ?

– 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 to make 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 ?”

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.

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. Gastro-intestinal disorders.

Clinical cases (III)

4. Eddy, M :

f13 (peanut) = 0,44 kUA/L SPT+ : birch, apple, peach, banana, garlic, peanut Swollen lips

Sensitization to birch…What can we conclude?

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 ?

?

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-conglycininnGly m glycinin

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 1Gly m 4

Bet v 1

Pru ar 1Pyr c 1 Pru av 1

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 3rCor a 8 rPar j 2nArt v 3Ara h 9Mal d 3Zea m 4Jug r 3

Pru ar 3

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 12rBet 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

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.

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

Maleki SJ et al, J Allergy Clin Immunol, 2003

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

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 severe reactions

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?

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

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?

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

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 ?– Multiple sensitizations to fruits and vegetables.

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/LCCD

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

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 ?– Sensitization to birch…

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

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..;

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 3), hazelnut (Cor a 8) and peanut (Ara h 9).

Peanut allergy study

62 Patients with positive sIgE f13 or f17 n= 38 (f13), n=24 (f17)

Patients with positive sIgE for PR-10 proteins : n = 36 Patients with positive sIgE

for nsLTP : n = 9Patients with positive sIgE for Storage proteins : n = 12

Patients with positive sIgE for CCD/MUXF3 : n =5

Cor a 1 n=14

rAra h 8 n=22

rAra h 9 n=5

rCor a 8 n=4

CCD/MUXF3 n=5

rAra h 3 n=4

rAra h 1 n=5

rAra h 2 n=3

OAS: n=11 Rh: n=18 AD: n=14 As: n=10 Asy: n=4 AO: n=2 U: n=3

OAS: n=2 Rh: n=3 AD: n=6 As: n=3 An: n=1 AO: n=3 U: n=5

OAS: n=1 Rh: n=1 AD: n=4 U: n=4 As: n=3

OAS: n=2 GIT: n=1 AD: n=1 U: n=1 Asy: n=5

Asy: Asymptomatic GIT: Gastrointestinal trouble OAS: Oral Allergy Syndorme Rh: rhinitis AD: Atopic Dermatitis As: asthma An: anaphylaxis AO: Angioedema U: Urticaria

R.Gadisseur, London, EAACI 2010.

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,

We would be able to explain everything !!

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 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 Mites d1: Dermatophagides pteronyssinus nDer p1 Dermatophagides pteronyssinus d202 rDer p2 Dermatophagides pteronyssinus d203 rDer p10 Tropomyosin Dermatophagides 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 e4: Cow Bos spp nBos d 6 BSA Serum albumin Bos spp. e204 e5: Dog Canis familiaris rCan f 1 Canis familiaris e101 rCan f 2 Canis familiaris e102 nCan f 3 Serum albumin Canis familiaris e221 e1: Cat Felis domesticus rFel d 1 Felis domesticus e94 nFel d 2 serum albumin Felis domesticus e220 e83: Swine Sus scrofa Swine serum albumin Sus scrofa e222 Foods f85: Celery Apium graveolens rApi g 1.01 PR10 Apium graveolens f417 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 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 f4: Wheat Triticum spp rTri a 19; Omega-5 Gliadin Triticum spp. f416

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 nAna c 2 Bromelin Ananas comosus k202 nApi m 1 Phospholipase A2 Apis mellifera k203 nCar p 1 Papain Carica papaya k201 nGal d 4 Lysozyme Gallus spp. k208 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 Carbohydrate Determinants (CCD) Bromelin k202 CCD; MUXF3 (from bromelin) Ro214 r = Recombinant n = Natif

Patient: Nom : Mister Albert Gik Adresse: Birch Street 1… Birth date: 01/01/1950

Date de demande: 19/11/2009Signature:

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, …

Evolution of in-vitro specific IgE diagnosis

• In the late 1980s, first miniaturised multianalyte immunoassays (microspots arranged in the form of microarrays in which miniaturised ligands were deposited)– Quantitative information and great sensitivity in application to a large

number of analytes in a single determination. – Thanks to developments in image analytical techniques and data

analysis.

• Allergenic component microarrays– Protein microarrays have been used to develop diagnostic panels and

biomarkers – extending the technique to clinical use in different areas such as autoimmunity, oncology, infectious diseases or allergy – the latter being the field where their applications will be reviewed.

Concept and development of allergen microarrays

• Definition– Small aqueous volumes of biomolecules, highly dense manner,

miniaturised device, hundreds or thousands of biological material probes, immobilised in a matrix component.

• Nanotechnology advances – Deposit, immobilise large number of protein aliquots on small

surfaces (spot sizes:micrometers) Miniaturised IgE measurement : patient specific IgE profile through

a single test, use purified natural and recombinant allergenic components, multianalyte panels

– determine the individual IgE antibody reactivity profile.

• Difficulties :– Antigens : intrinsic complexity (size, charge, solubility,

surface activity, three-dimensional structure) Must be carefully selected.

– Antigen spotting conditions : secure efficient immobilisation, prevent denaturalisation of the surface, maintain adequate accessibility to the allergen epitopes.

– Optimised test conditions and sensitive detection system. (sensitivity, specificity and reproducibility must be validated to ensure the accuracy of the results)

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.

Description of the technique

• Indirect solid-phase immunoassays.– Specific IgE semiquantitative indirect enzymoimmunoassay (EIA).

• The conventional tests use activated cellulose capsules as solid phase for fixing the allergens (UniCAP®, Phadia) while allergen microarrays generally use chemically modified glass as substrate.

• Allergenic proteins deposited in triplicate on the matrix to ensure the reproducibility or reliability of the test.

• Each glass slide contains four identical microarrays.• Each microarray is surrounded by a fine Teflon® layer to prevent

overflow of the sample during the test.

• Arbitrary units, ISAC Standardized Units (ISU), four classes (negative, low, medium and high), corresponding to the classes 0, 1–2, 3–4 and 5–6, respectively.

sIgE measurements

Immuno Solid-phase AllergenChip (ISAC)

Allergen-triplet afterfluorescence-reaction

Allergen molecule

Fluorescence-labeledsecondary anti-human

IgE

IgE-antibody (in the patient’s serum)

4 reaction sites

+/- 4 Hours

+/- 1 Hour

few minutes

Secondary fluorescence-labeled anti-human IgE

antibody

Wash buffer ImmunoCAP©ISAC

A two-step assay

IgE antibodies from the

patient serum bind to the

immobilized allergen

component.

Immuno Solid-phase Allergen Chip (ISAC)

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

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.

Validation of allergic component microarrays

• Allergen microarray tests must be validated and reproducible.– Must be evaluated independently of the manufacturer,

– Clinical studies in order to define the allergen panels that contain a repertoire of allergens similar or identical to that found in the natural sources, and which can offer data that are reproducible between laboratories, as well as between and within tests, with a view to establishing their robustness.

Validation of the method

• Method– Selection of 86 sera of patients (26 males/60 females)

+ sIgE (>0.10 kUA/L) for recombinant allergens (ImmunoCAP ®250 Phadia).

positive and negative sIgE for 55 recombinant or native purified allergen component of different kind of allergenic sources (latex, peanut, birch, Timothy grass, hazelnut, peach, soybean, cat, dog…).

In all, we compared the results of 555 sIgE measured in CAP (recombinant allergens) with the ISAC® determination.

We tested 2 samples with a high TotIgE rate (> 10.000 kU/L) on ISAC ®.

• R.Gadisseur et al, CCLM, in press.

(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).

Validation of the method

• Results:

– 384 results out of the 555 measurements were positive in CAP® and 171 were found negative.

302/384 positive results were also found Positive with ISAC (concordance 78,65%),

– 82 discrepant results : mean 0,67 kUA/L.

» 52 were below 0,35 kUA/L (former cut-off). New concordance 92,19%.

160/171 negative results were also found negative with ISAC (concordance 93,57%),

– 11 discrepant results : mean 1,57 ISU.

More frequently observed with some allergens (rAsp f 1, rPru p 3, nAna c 2, rApi g 1…)

– Until a rate of 150.000kU/L of TotIgE, we didn’t observe any unspecific binding.

Conclusions (I)

• Currently available standard products for allergy testing based on allergen extracts prepared from biological raw materials. – Natural mixtures of allergenic and non-allergenic molecules (not fully

standardized referring to their content of major/minor allergen components).• Increasing availability of allergen components,

– purified from their natural source or biotechnologically produced as recombinant proteins,

Revolution in allergy diagnosis : Molecular Allergology.• Highly cross-reactive allergen components,

– Panallergen : positive test results against numerous allergen extracts. Profilin : present in a broad variety of plant pollen and plant derived food.

– Consequently, when using extract based specific IgE testing it is difficult to identify the correct allergen source when only cross-reactive allergen components are involved.

– Availability of specific and cross-reactive marker components creates the platform for more informative diagnostics.

Advantages of allergic component microarrays

• In contrast to conventional diagnosis, microarrays allow us to investigate IgE reactivity to a large number of different molecules or allergenic components with a single and rapid test. – With the traditional methods it would be impossible to analyse the panel of

natural and recombinant allergens ensuring the presentation of a significant number of epitopes.

• The composition of the allergen panels can be expanded and improved continuously, depending on the availability of new components, with the incorporation of the most appropriate profiles defined by the clinical studies.

• The amount of patient serum consumed is far smaller than in the case of conventional immunoassaying. – 20 μl suffices to determine IgE for up to 400 individual allergens, while the

conventional tests consume 50μl for each allergen tested. Use of the technique in paediatric patients

– a minute amount of serum from a simple capillary blood sample, with highly reliable results versus venous serum sampling (global correlation coefficient 0.92), according to the studies of Ott et al.

ImmunoCAP ISAC

• ImmunoCAP ISAC Technology– Based on modern biochip technology, – sIgE antibodies : >100 purified natural or recombinant allergen

components, >40 common sources.– 20 μl of serum or plasma.

Capillary blood sampling can be used, enabling a less invasive procedure for testing young children

– High reliability by intrinsic replicate testing and quality controls.– Diagnosis and management 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.

– Personalised medicine, contribute to the diagnosis, prognosis and staging of disease and to treatment selection, facilitating preventive medicine, predictive diagnosis and individualised curative therapies.

Data interpretation: the power of numbers

• Vast amount of data that will be generated by future systems – Interpreted and managed in a structured and well-organised manner.

– Data base structure, Support the clinical scientist who needs to move the medical

diagnostic frontier forward Help the practicing physician both to treat the patient and cut

healthcare costs more effectively.

• Difficult interpretation of the results : – Should be limited to specialists !

Conclusion (II)

• 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…

Thank you for your attention !!