Food Allergen Library Improved component resolved diagnosis as a European joint initiative. from SAFE to Europrevall. Karin Hoffmann-Sommergruber
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1 Food Allergen Library Improved component resolved diagnosis as a European joint initiative from SAFE to Europrevall Karin Hoffmann-Sommergruber
2 SAFE Plant food allergies: field to table strategies for reducing their incidence in Europe K. Hoffmann-Sommergruber Medical University Vienna
3 Multicisciplinary Consortium 12 Partners including Clinical centers (4) Molecular biologists (6) plant breeders (2) Fruit juice company (1) Consumer scientist (1) Allergic patients interest group (3)
4 Apple as a model Well defined patients databank across Europe Screening techniques for allergen levels Allergen profiles of selected cultivars Identification of molecular markers for breeding techniques Improved diagnostics for fruit allergy component resolved diagnosis (CRD)
5 SAFE Achievements 38 Poster 53 oral presentations 45 articles /27 peer reviewed 3 PhD theses 2 workshops Cooperations established Blueprints and projects taken over by: other EUprojects EUROPREVALL ISAFRUIT
6 Framework 6 Integrated project (IP) Europrevall The Prevalence, Cost and Basis of Food Allergy across Europe
7 WP 3.1: Allergen Structure and Characterization WP 3.1.1: Preparation of natural and recombinant allergens (Karin Hoffmann-Sommergruber) WP 3.1.2: Physicochemical characterisation of natural and recombinant allergens (Ana Sancho) WP 3.1.3: Characterization of novel allergens (Thomas Holzhauser)
8 Aims of EuroPrevall Characterizing patterns and prevalence of food allergies across Europe in infants, children and adults Developing serological methods based on purified food allergens ( component resolved diagnosis ), including conventional and novel formats. Investigating environmental, dietary & genetic influences on food allergy. Delivering information (patterns & prevalence, socioeconomic cost) & new tools to improve management of this disease.
9 WP 1.3: Cross-Sectional Study Foods studied include those from Annex IIIa: Egg, milk, fish, shrimp, peanut, soybean, hazelnut, walnut, wheat, celery, sesame, mustard And selected additional foods: Known geographic distribution e.g. peach and lentil in southern Europe, Emerging allergenic foods e.g. kiwi fruit Potential allergenic foods in new EU member states e.g. buckwheat, poppy seeds Those linked to environmental allergies to pollen e.g. apple latex e.g. banana
10 Theme 3: Allergen Structure and the Food Matrix WP 3.1: Allergen structure and characterization Allergen library WP 3.2: Development and validation of novel diagnostic tools (component resolved diagnosis, CHIP, cellular assays) WP 3.3: Natural and fabricated food structures and component interactions in allergenic potential of foods
11 Preparation of natural and recombinant allergens When to use natural or recombinant allergens for a diagnostic setup? Which quality criteria to be met for a purified food allergen?
12 FOOD ALLERGEN abundant OCCURENCE low concentration stable throughout purification STABILITY instable range of various isoforms contributing to overall IgE response ISOFORMS high and low IgE binding protein glycosylation, SH-bridges,... relevant for IgE binding capacity POSTTRANSLATIONAL MODIFICATIONS posttranslational modifications don t effect IgE binding capacity Purification of NATURAL ALLERGEN Production of RECOMBINANT ALLERGEN
13 Physico-chemical Characterization of Allergens
14 Allergen library Source Apple Cow s milk Goat s milk casein Hazelnut Peach Allergen designation (IUIS) Mal d 1, Mal d 2, Mal d 3, Mal d 4, Bos d 4, Bos d 5, Bos d 8 Goat s milk casein Cor a 1.04, Cor a 9, Cor a 11, Cor a 2, rcor a 8 Pru p 1, Pru p 3
15 Source Peanut Allergen designation (IUIS) Ara h 1, Ara h 2,6, Ara h 3,4, Ara h 8, Celery Api g 1.01, Api g 4, Api g 5, Fish Egg Shrimp Cyp c 1, Gad m 1 Gal d 1, Gal d 2, Gal d 3, Gal d 4, Gal d 5 Pen a 1 Priority 2: Allergens from Kiwi, Mustard, Sesame, Soy, Walnut, Wheat Priority 3: Allergens from Sunflower, Carrot, Buckwheat, Banana, Lentil, Tomato
16 Allergen library: Interactions Purified allergen Datasheet Ref. serum NMR P 28 Allergen Library P16 CHIP P 26 CAP P 34 Hist. Rel P 35 Allergen characterisation Database P1 Feedback needed Clinical Partners Feedback
17 RNA Extraction Expression and Purification of rpru p 1 cdna Synthesis TA Cloning / Sequencing Expression System petblue-2 in E. coli Tuner (DE3) placi Cell disruption (French Press) Anion exchange chromatography (Q Sepharose) Hydrophobic interaction chromatography (Phenyl Sepharose) NHSB 33 kd - 24 kd - Bet v 1 homologue From peach 17 kd - 11 kd -
18 Data from mass spectrometry rpru p 1 M+Na + Theoretical: Da Measured: Da Sequenced peptides after tryptic digest: -GVFTYESEFTSEIPPPRLFKAFVLDADNLVPKIAPQAIKHSEILEGDGGPGTIKKITFG 60 EGSQYGYVKHKIDSIDKENHSYSYTLIEGDALGDNLEKISYETKLVASPSGGSIIKSTSH 120 YHTKGDVEIKEEHVKAGKEKASNLFKLIETYLKGHPDAYN 159
19 CD spectrum of rpru p 1 CD at ph CD at ph 3.0
20 Purification protocol (Mal d 2) Thaumatin Extraction of proteins from peeled ripe apples Precipitation of proteins with 80% ammonium sulfate Dialysis against 20 mm Tris/HCl, ph 8 Precipitation of pectin by 40 mm CaCl 2 Dialysis against 20 mm Tris/HCl, ph 8 Precipitation of proteins by aceton Concanavalin A Unbound proteins: Mal d 2 Anion exchange chromatography, Resource Q
21 Mal d 2 allergenic activity OD (405 nm) Patient No. Mal d 2 reduced kd Mal d 2 non-reduced kd
22 Ara h 1-7S Globulin
23 Ara h 3 /4-11S globulins
24 Ara h 2/6 2S albumins
25 Comparison of CAP and Allergen Chip
26 Comparison of CAP and Allergen Chip I IgE reactivities in both CAP and Chip are similar (plus/plus, minus/minus) shrimp nmal d 3 (n = 20) rmal d 4 (n = 20) rapi g 1 (n = 20) rapi g 4 (n = 20) rapi g 5 (n = 20) rcor a 1 (n = 20) rcor a 2 (n = 20) rcor a 8 (n = 20) rpru p 1 (n = 20) npru p 3 (n = 20) ngal d 1 (n = 20) ngal d 2 (n = 20) ngal d 3 (n = 20) ngal d 4 (n = 20) ngal d 5 (n = 20) Bos d 4 (n = 17) Bos d 5 (n = 17) Bos d 8 (n = 17) ngad c 1 (n = 20) rcyp c 1 (n = 20) Pen a 1 (n = 20) apple celery hazelnut peach egg milk fish CAP and chip different (+/-, -/+) CAP and chip identical (+/+, -/-) 2007, Madrid, Spain nmal d 2 (n = 20) number rmal d 1 (n = 20)
27 NEW Api g 1 and Cor a 2 Comparison of CAP and Allergen Chip I IgE reactivities in both CAP and Chip are similar (plus/plus, minus/minus) shrimp 20 apple celery hazelnut peach egg milk fish number 5 rmal d 1 (n = 20) nmal d 2 (n = 20) nmal d 3 (n = 20) rmal d 4 (n = 20) rapi g 1 (n = 20) rapi g 4 (n = 20) rapi g 5 (n = 20) rcor a 1 (n = 20) rcor a 2 (n = 20) rcor a 8 (n = 20) rpru p 1 (n = 20) npru p 3 (n = 20) ngal d 1 (n = 20) ngal d 2 (n = 20) ngal d 3 (n = 20) ngal d 4 (n = 20) ngal d 5 (n = 20) Bos d 4 (n = 17) Bos d 5 (n = 17) Bos d 8 (n = 17) ngad c 1 (n = 20) rcyp c 1 (n = 20) Pen a 1 (n = 20) 0 CAP and chip different (+/-, -/+) CAP and chip identical (+/+, -/-) 2007, Warsaw, Poland
28 Latex - k82 Sensitization Pattern - CAP (All Centers, 1122 Sera Tested, 24/05/07) Peanut - f13 Soybean - f14 Hazelnut - f17 Walnut - f256 Celery - f85 Kiwi - f84 Apple - f49 Peach - f95 Sesame seed - f10 Mustard seed - f89 Wheat - f4 Fish - f3 Shrimp - f24 Buckwheat - f11 Corn - f8 Carrot - f31 Tomato - f25 Melon - f87 Banana - f92 Lentil - f235 Sunflower Seed - k84 Poppy Seed - f224 Timothy Grass - g6 Birch - t3 Olive - t9 Cypress - t23 Plane Tree - t11 Mugwort - w6 Parietaria - w21 Chenopodium - w10 Ragweed - w1 House Dust Mite - d1 Cat - e1 Dog - e Class 6 Class 5 Class 4 Class 3 Class 2 Class 1 Allergen CAP Hen's Egg - f1 Cow's Milk - f2 subjects
29 Summary Quality criteria for allergens either recombinant or natural - need to be established These well characterized allergens are the basis of the concept of component resolved diagnosis. This will contribute to identify relevant sensitization patterns and to improve dietary recommendations for the food allergic patient in the future. However this needs future efforts as well as financial support from the EC fo the benefit of the European society.
30 Thanks to IFR C. Mills, A. Sancho, N. Rigby PEI S. Vieths, G. Reese,T. Holzhauser RRes P. Shewry, J. Marsh INRA J.M. Wal, K. Adel-Patient CERM S. Alessandri MUW S. Gaier, M. Bublin, H. Breiteneder Univ. Salzburg P.Briza DTU V. Barkholt REFLAB P. Skov VBC Genomics Biomay Phadia
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