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

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 1988-91 1995-99 2000 2008 Simplified overview of the historical development of allergy diagnosis and treatment. IN-VIVO testing IN-VITRO testing Component – Resolved -Diagnostics

Components : Production

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.

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

« 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 ?”

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 : 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.

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?

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 ? ?

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

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 1.01 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

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

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

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

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

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 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, 2006:395-463 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 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)

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?

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 !

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?

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

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.

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

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…

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

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 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

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 !!

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:

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.

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

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…

Immuno Solid-phase Allergen Chip (ISAC)

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 (> 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).

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 TotIgE, we didn’t observe any unspecific binding.

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 ®

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

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…

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 !

Thank you for your attention !!