Microarrays for allergy Immuno CAP ISAC Phn Romy

Microarrays for allergy - Immuno. CAP ISAC© - Phn Romy GADISSEUR, Prof. J-P. Chapelle, Phn. Biol. E. Cavalier 02/03/2009 1

Timeline…allergy diagnosis Characterization & designation of Ig. E Provocation tests (SPT) 1880 Standardization of allergen extracts RAST 1967 Recombinant allergen panels recreating allergen extracts First allergens cloned 1988 -91 Immuno. CAP ISAC (Phadia/ VBC Genomics) First allergen microarray 1995 -99 2000 2008 IN-VIVO testing IN-VITRO testing Component – Resolved -Diagnostics 2

Epidemiology of allergy n n n 4 th chronic disease (OMS). Child allergic asthma = 1 st chronic disease. +/- 30% allergic. Cost for healthcare systems +++. Allergies x 2 the last 15 years in developed nations !! Why ? An Occidentalized way of life. n More hygiene n n n ↓ infections => Different immune response against environmental allergens : development of allergy. Different alimentary diet. Sedentary life (hygrometry ++ & T°C ++) & Exposition to domestic allergens >> exposition to new allergens (confinement, air conditioning, domestic animals…). Passive smoking of children (allows Ig. E synthesis). Atmospheric pollution +++. 3

Allergène n n n Tout immunogène devient un allergène lorsque certaines conditions environnementales, associées à des facteurs génétiques individuels induisent une synthèse d’Ig. Es. Structure protéique complexe comprenant plusieurs déterminants allergéniques. La transformation de l’Antigène banal en allergène potentiellement nocif survient lors de la présentation de l’Antigène par les cellules dendritiques aux LT. Déterminant majeur = le + souvent responsable (vs mineur). Réactions croisées quand 2 allergènes ont des déterminants allergéniques communs. Pneumallergènes : allergène pénétrant dans l’organisme par voie respiratoire. n n Pollen > Acariens > Animaux > Blattes > Moisissures > … Trophallergènes : allergènes alimentaires (+produits exotiques). 4

Quantification des Ig. E spécifiques Ig. E Fce. RI B-cell Allergen Mast cell IL-4 IL-13 Th 2 -cell Antigen-presenting cell Histamine Leukotrienes Prostaglandins Cytokines Atopic disease IL-5 Eosinophil 5

Allergens nomenclature according to the IUIS (International Union of Immunological Societies) n n n t : tree f : food g : grass i : insect w : wheat c : cure n n n d : dust mite m : moisture e : epithelia p : parasite k : (professional allergens) Taxon n n t 3 : Betula verucosa g 6 : Phleum pratense d 1 : Dermatophag. pteronys. e 1 : Felis domesticus f 13 : Arachis hypogaea Empirical nomenclature genre 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, … « Recombinants » nomenclature (IUIS) Number according to the order of 6 purification of proteins

Dosage des Ig. Es au CHU 7

Components Production 8

Data Bases Allergenic molecules of Peanut Biochemical properties of allergens 9

Components - Resolved Diagnosis (CRD) n Interest & new applications of « recombinants » n More sensitive tests. n Help the decision to undergo a specific immunotherapy. Prediction and understanding of the cross-reactions. Prediction of the risks and severity of symptom. n n 10

« Boosted » extracts with recombinants n Latex : n n For 20 years, very important allergen. « The absence of Hev b 5 in Capture Antigen may cause falsenegative results in serologic assays for latex-specific Ig. E Antibodies » . (Z. Chen & al - The Journal of Allergy and Clinical Immunology, 2000). ØHev b 5 : labile protein. Ø Boosted extracts of latex with Hev b 5 for better results ? Ø Better sensitivity without loosing any specificity. Ø Less false negative results. Plus de patients + sensibilité + 11

Components - Resolved Diagnostics (CRD) n Interest & new applications of « recombinants » n More sensitive tests. n Help the decision to undergo a specific immunotherapy. n Prediction and understanding of the cross-reactions. Prediction of the risks and severity of symptom. n 12

Immunotherapy ? 13

Components - Resolved Diagnostics (CRD) n Interest & new applications of « recombinants » n n More sensitive tests. Help the decision to undergo a specific immunotherapy. n Prediction and understanding of the cross-reactions. n Prediction of the risks and severity of symptom. 14

Predict cross reactions n PR-10 protein, Bet v 1 homologue n n n 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. Cor a 1. 01 Cor a 1. 04 Gly m 4 Ara h 8 Dau c 1 Api g 1 Bet v 1 Pru p 1 Mal d 1 15

Oral Allergy Syndrome (OAS) n Oral Allergy Syndrome : allergic reactions that occur rapidly, within minutes of eating a trigger food. n n 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). o o o o Hazelnut, 46% Apple, 39% Peach, 24% Cherry, 22% Nut, 21% Pear, 20% Almond, 19% Plume, 17% o o o o Brazil nut, 16% Potato, 15% PR-10 Carrot, 13% r. Bet v 1 Peanut, 10% r. Pru p 1 Strawberry, 10% r. Gly m 4 r. Ara h 8 Orange, 9% r. Api g 1. 01 r. Aln g 1 Apricot, 7% r. Cor a 1 n. Act d 8 r. Mal d 1 r. Dau c 1 16

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Predict cross reactions n LTP (non-specific Lipid Transfer Protein, ns. LTP) n n n 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 Often associated with allergic reactions to fruit and vegetables in southern Europe Zea m 4 LTP Cor a 8 Pru p 3 Pru av 3 Mal d 3 Ara h 9 n. Pru p 3 r. Cor a 8 r. Par j 2 n. Art v 3 Ara h 9 Mal d 3 Zea m 4 Jug r 3 18

Predict cross reactions n Profilin n Seldom associated with clinical symptoms but may cause demonstrable or even severe reactions in a small minority of patients PFL r. Mer a 1 r. Bet v 2 r. Phl p 12 r. Hev b 8 r. Mal d 4 r. Phl p 12 r. Bet v 2 n. Ole e 2 r. Mer a 1 r. Hev b 8 r. Mal d 4 r. Pru p 4 n. Act d 9 n. Ana c 1 n. Ara h 5 19

Predict cross reactions n Storage protein n n 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 r. Ses i 3 Cor a 9 r. Ber e 1 Ara h 2 Ara h 3 n. Gly mβ-conglycinin n. Gly m glycinin 20

Predict cross reactions n Lipocalins n n Very stable proteins Allergen components displaying limited cross-reactivity between species r. Fel d 4 r. Can f 1 r. Can f 2 n. Mus m 1 Lipocalins r. Can f 1 r. Can f 2 r. Fel d 4 n. Mus m 21

Predict cross reactions n Parvalbumin n A major allergen in fish A marker for cross-reactivity among different species of fish and amphibians A protein remarkable resistance to heat and digestion causing reactions also to cooked foods Ø sensitizing agents for > 95% of fish allergic patients Parvalbumin r. Cyp c 1 r. Gad c 1 CARP r. Cyp c 1 COD r. Gad c 1 22

Predict cross reactions n Serum albumin n A common protein present in different biological fluids and solids e. g. cows milk and beef, eggs and chicken. Heat labile protein. Cross-reactions between albumins from different animal species are well known, for example between cat and dog and cat and pork. BSA n. Bos d 6 n. Can f 3 n. Gal d 5 n Equ c 3 Serum albumin n. Fel d 2 n. Bos d 6 n. Can f 3 n. Equ c 3 n. Gal d 5 Cav p GSA Gal d (meat) Cav p GSA n. Fel d 2 Gal d (meat) 23

Predict cross reactions n Tropomyosin n n An actin-binding protein in muscle fibers A marker for cross-reactivity between crustaceans, mites and cockroach Tropomyosin r. Pen a 1 n. Pen i 1 n. Pen m 1 r. Der p 10 n. Bla g 7 r. Pen a 1 n Pen i 1 n Pen m 1 r. Der p 10 n. Bla g 7 r. Ani s 3 24

t 224 n. Ole e 1 ou t 25 (European Ash) New s. Ig. E available !! Olive tree n n n. Ole e 1 represents the best the ash allergy in Europe. In the North of Europe, No Olive tree : If Ole e 1 +++ : Allergy to ash, Ligustrum, Forcicia… Ash Ligustrum Forcicia 25

Components - Resolved Diagnostics (CRD) n Interest & new applications of « recombinants » n n More sensitive tests. Help the decision to undergo a specific immunotherapy. Prediction and understanding of the cross -reactions. Prediction of the risks and severity of symptom. 26

Microarrays n VBC Genomics (expert in microarrays) and Phadia (expert in allergy tests) have combined “innovative biochip technology” with “cutting-edge research in molecular allergology” to develop Immuno. CAP® ISAC. n n Modern multiplex in vitro diagnostic tool for the allergy specialist, based exclusively on allergen components. Miniaturized immunoassay platform for measurement of s. Ig. E 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. 27

Allergen Components available on Immuno. CAP® ISAC Native components Recombinant components 28

Allergen Components available on Immuno. CAP® ISAC 29

Allergen Components available on Immuno. CAP® ISAC 30

Allergen Components available on Immuno. CAP® ISAC 31

Allergen molecule Immuno Solid-phase Allergen. Chip Fluorescence-labeled secondary anti-human (ISAC) Ig. E Allergen-triplet after fluorescence-reaction Ig. E-antibody (in the patient’s serum) 4 reaction sites 32

Wash buffer Immuno. CAP©ISAC A two-step assay +/- 1 Hour Ig. E antibodies from the patient serum bind to the immobilized allergen component. +/- 4 Hours Secondary fluorescencelabeled antihuman Ig. E antibody few minutes 33

Immuno Solid-phase Allergen Chip (ISAC) n Test results are measured with a biochip scanner and evaluated using proprietary software. n n Capital. Bio´s Lux. Scan dual laser scanner. No other special laboratory equipment needed. Immuno. CAP ISAC is a semi-quantitative test and results are reported in ISAC Standardized Units (ISU). Medical report generated n Sent to the contracting medical practitioner. 34

Immuno Solid-phase Allergen Chip (ISAC) 35

Medical report generated Qualitative & Semi. In northern and central Europe birch and related tree pollens quantitative Classification by results category of proteins are considered to be the main sensitizer and are often causing respiratory symptoms. The presence of PR-10 proteins in many plant foods often cause symptoms due to cross-reactivity. Many of these proteins are heat labile and cooked foods are often tolerated. Often associated with local symptoms such as oral allergy Commentaries syndrome (OAS). for a better interpretation Often associated with allergic reactions to fruit and vegetables in northern Europe. 36

Medical report generated 37

Our 1 st validation of the method n Method n 22 sera of patients n Positive (>0. 35 k. UI/L) specific Ig. E (s. Ig. E) tests for recombinant allergens (Immuno. CAP© 250 Phadia) and/or a clinical anamnesis of allergy and/or SPT. n n 17 sera : n Positive s. Ig. E for different kind of sources (latex, peanut, birch, timothy grass, hazelnut, peach, soja, cat or dog). n In all, 136 s. Ig. E for recombinant had been performed. 16 sera : n Clinical history of the allergy thanks to an anamnesis of a clinician. n Rhinitis, asthma, conjunctivitis, oral allergy syndrome, oedema, dermatitis, hives, colic or diarrhea. 9 sera : n Skin Prick Tests (SPT). We managed a microarray determination on each serum. Then, we compared the results obtained with the s. Ig. E, the SPT or the anamnesis. 38

Our 1 st validation of the method n Results: n The results of the Immuno. CAP©ISAC were similar to those of the SPT. n Majority of the allergens tested had the same results on ISAC and on SPT (regarding positive and negative tests). Amongst the 136 s. Ig. E (recombinant allergens) tested, the Immuno. CAP© ISAC found 132 times concordant results. n The Immuno. CAP© ISAC provided results in agreement with the anamnesis in all the 16 cases. n 39

Validation & Clinical Case n U. M. (27 years old) n n Follow-up allergies. Symptoms : n n SPT : n Positive Colic & diarrhea n +++ Dust mites, grass, birch n After drinking milk or eating n + Pollens of ash and poplar, fat ? hazelnut, mustard n No problem with bread, fish n Suspicious n Don’t eat any shellfish n Peanut, soja, nuts. Rhinitis, prutitus, urticaria n No problem with latex n s. Ig. E Dyspnea during exercise n Positive n Peanut, soja, shrimp, wheat. 40

Clinical Case n Clinician’s diagnosis & his decisions : n Allergic rhinitis n n n Due to acarians all the year and increased during birch pollinisation. Polysensitized to grass and to ash and birch pollens Diarhaea ? n Suspiciously sensitized to Peanut, soja, shrimps, wheat and mustard. n n n Probably due to “Cross-reactions” with birch (hazelnut, peanut, nuts & soja) n No eviction needed. Real allergy to shrimps, mustard and wheat : n Eviction ! Next step n Oral provocation tests peanut, hazelnut. 41

Clinical Case Results of Immuno. CAP© ISAC n PR-10 (Bet v 1 -like) : n n Dust Mites : n n Cor a 1, 04 (PR-10) – Cor a 8 (ns. LTP) Peanut n n Sal k 1 Hazelnut n n Gly m conglutin, Gly m 4 (PR-10) Saltwort n n Cyn d 1 - Phl p 4 - Ole e 1 - Pla a 2 - Cry j 1 - Cup a 1 - Mer a 1 Soja n n Der p 1 - Der f 1 - Der p 2 - Eur m 2 -Der f 2 Pollens : n n Bet v 1 - Pru p 1 - Aln g 1 - Cor a 1, 04 - Mal d 1 - Ara h 8 - Gly m 4 Ara h 8 (PR-10) – Ara h 1 (storage protein) Ana c 2 (CCD marker) Cor a 8 Ara h 1 42

Conclusions of our first validation n The Immuno. CAP© ISAC permitted to find out an allergy to a major allergen of peanut and of hazelnut (Ara h 1 & Cor a 8). Ø Immediately, the clinician cancelled the planned oral provocation tests for peanut and hazelnut. n It permitted to avoid a hazardous oral provocation test in an allergic patient. n Very positive feedback of clinicians. 43

Advantages of Immuno. CAP© ISAC n The results provided by the Immuno. CAP© ISAC allowed us to see, in a single analytical step, the allergen sensitization profile of the patients tested. n Replicate (triplicate) array layout. CRD rapidly and cost-effectively. 20 μL of patient serum/plasma give complete results. n n We think that the Immuno. CAP© ISAC will have an essential role to play in the diagnosis and the management of complex patterns of sensitization. 44

Thank you for your attention !! 45