Antibodies and their suitability for food allergen detection

Alergeny v potravinách a jejich diagnostika 27. duben 2012

Sabine Baumgartner Universität für Bodenkultur Wien IFA-Tulln, Rakousko Interuniversitäres Department für Agrarbiotechnologie Tulln Universität für Bodenkultur Wien Interuniversitäres Department für Agrarbiotechnologie Tulln

Antibodies and their suitability for food allergen detection

Sabine Baumgartner

Christian-Doppler Laboratory for Rapid Test Systems for Allergenic Food Contaminants University of Natural Resources and Life Sciences Vienna, Dept. IFA-Tulln, Center for Analytical Chemistry Konrad Lorenz Straße 20 A-3430 Tulln, Austria Bezeichnung deutsch: ev. Untereinheit 2. Ebene I ev. Untereinheit 3. Ebene I Univ. Prof. DI. Dr. Max Mustermann 2 Campus Tulln 2011

UFT

TZT

kindergarten Dormitory Animal Nutrition, Products and Nutrition Physiology Biotechnology in Environmental Animal Production Biotechnology Biotechnology in Plant Production

Natural Materials Technology Center for Analytical Chemistry 21 Research Groups in 6 Institutes

Department for Agrobiotechnology, IFAIFA--TullnTulln Needle in the haystack – food allergens in food

Baumgartner 5 Timeline of Immunology . 1798 – Jenner - vaccination small pox . 1883 – 1905 development of cellular theory on immunity . 1890 – Von Behring/Shibasaburo Beginning of humoral theory of immunity . 1900 – Ehrlich Antibody formation theory . 1917 – Landsteiner Hapten . 1938 – Marrack Antigen-Antibodybinding hypothesis . 1942 – Landsteiner/Chase Anaphylaxis; Freund Adjuvants . 1948 – Antibody production in B-cells . 1964-68 T and B cell cooperation in immune response . 1965 – Secretory immunoglobulins . 1967 – Ishizaka Identification IgE . 1971 – Engvall/Perlmann – ELISA . 1972 – structure of antibody molecule . 1975 – Köhler/Milstein generation of first monoclonal antibody . 1980 - …IL receptor discoveries, T-cell receptor gene identification, Th1 vs Th2 model, T- cell activation, gene therapy, identification of toll-like receptors, vaccine development

1700 1800 1900 2000 Baumgartner 6 History of ELISA

. 1960 – Yalow/Berson First described radioimmunoassay . 1966 – Avrameas/Pierce linkage techniques Ab-enzyme; Wide/Porath immunosorbent technique . 1971 – Perlmann/Engvall and Schuurs/Van Weemen papers published to perform ELISA/EIA . Since now development of ELISA-like techniques utilizing fluorescence, electrochemiluminescence, real time PCR-reporters . 1980 – fully automated test instrumentation mainly diagnostic immunoassays . 1990 - first commercial available ELISAs for food allergen testing Lequin, 2005 Clin.Chem. 51, 2415-2418

1700 1800 1900 2000 Baumgartner 7 History of Food Allergen Labelling in the EU (2000/13/EC, 2003/89/EC, 2006/142/EC, 2007/68/EC, 1169/2011) Celery, mustard, sesame, lupine, molluscs

Crustaceaen Soy Cereals Fish Milk Treenuts Egg Peanut 1700 1800 1900 2000 Baumgartner 8 Demanding Food Allergen Detection . Detection of allergenic proteins in food, mainly processed . Detection - Proteinassay or Allergenassay . Extractable proteins . Markerproteins . Allergenic proteins . Sources different protein families . Protein specific extraction methods . Characterisation, Validation . No reference materials . Reference methods under development

Baumgartner 9 Protein classes . „Pathogenesis-related“ Proteins – PR-Proteins . Chitinases, Proteases, antifungal proteins, Bet v 1 homologes, Amylase inhibitors, Peroxidases, fruits (cherry, apple,..), barley, rye . Profilins . Actin-binding proteins, signalling: Celery, haselnut, soya, peanut . Parvalbumin . Ca2+-binding protein: cod, carp, salmon . Tropomyosin . Actin-binding protein: Shrimps, lobster, crab

Baumgartner 10 Protein classes . Seed storage proteins . Albumins (2S), Vicilins (7S), Legumins (11S), Glycinin (11S), ß- Conglycinin (7S): peanut, walnut, brazil nut, mustard, soya . LTP-Proteins (Lipid transfer proteins) . Lipidmetabolism: apple, peach, plum, corn . Proteases . Proteolysis: papaya, pineapple, kiwi . Peroxidases . Peroxidation: wheat, barley . Lectins . Carbohydrate binding: peanut agglutinin

Baumgartner 11 Peanut and products thereof

. Composition

[%] Protein Lipid CH Fibers Minerals

Peanut 27,4 50,7 9,1 7,5 2,7

Baumgartner 12 Peanut and products thereof . Allergens . Albumins (2S) or Conglutinins: Ara h 2, 18 kDa, Xreacts - Ara h 6 Ara h 6, 14 kDa, X - Ara h 2 . Vicilins (7S): Ara h 1, 71 kDa, Xreacts - walnut (Jug r 2), pea (Pis s 1) . Legumins or Glycinins (11S): Ara h 3, 14 kDa, Xreacts – Soya Ara h 4, 61 kDa . Profilins (Bet v 2 – Homologue): Ara h 5, 14 kDa, Xreacts - gras (Lol p 12), birch pollen (Bet v 2) . PR-Proteins: Ara h 8, 17 kDa, Xreacts – birch pollen (Bet v 1) . LTP-Proteins: Ara h LPT, Xreacts – peach (Pru p 3) . Oleosins: Ara h Oleosin, 17 kDa . Agglutinins: Ara h Agglutinin, 29 kDa

Baumgartner 13 Possible methods

©Barbara . Immunochemical methods Cvak . ELISA . Rapid tests – Lateral Flow Devices, Dipsticks

. DNA-based methods . PCR . rt-PCR . PCR-ELISA

. Upcoming MS-methods Immunochemical methods - Antibody based assays

. Protein detection by highly specialised proteins . Polyclonale Abs (rabbit, chicken, sheep,...) . Monoclonale Abs (mouse, rat,…) . Recombinant Abs

Baumgartner 15 Polyclonal egg yolk antibodies ?

Baumgartner 16 Monoclonal antibodies

Immunisation

Spleen cells Myeloma cells

Cell fusion

Selection of hybridomas Cryoconservation ( - 70 °C, - 150 °C) Cell culture

Cloning

Repeated Cloning

Testing specific ABs

Baumgartner 17 Immunoanalytical Solutions for Food Allergen Detection

Microtiterplates Lateral Flow Biosensors Micro Arrays ELISA’s Devices

96 wells 1 Assay 1 Assay Hundreds ½ h – 3 h Minutes Minutes Minutes Lab on-site Lab/on-site Lab

Baumgartner 18 Challenges in LFD development

Animals Colloidal gold Antibody Production Conjugation Antibody Purification

+ Standard Antibody Immunogen Quality* Sample

Test Performance Membrane, Running buffer

* sensitivity, specificity, yield Baumgartner 19 LFD principle – Sandwich format

Reaction batch Antibodies coupled to colloidal gold Wick pad

Nitrocellulose Backing card membrane

© Judith Rudolf Baumgartner 20 Challenges in LFD development

Animal Colloidal gold Antibody Production Conjugation Antibody Purification + Standard Antibody Immunogen Quality* Sample

Test Performance Membrane, Running buffer

* sensitivity, specificity, yield Baumgartner 21 The extraction - example peanut Rudolf et al., 2011, Food Add Contam Extraction of peanut proteins

Buffer and signal intensity

Baumgartner 22 +

LFD – Hazelnut determination

positve negative Samples on HN-LFD 35

0 0 0 2 30 0 0 4 4

25 9

15 14 15 18 17 20

32 32 32 32 32 32 32 32 32 32 32 15 30 30 30 30 30 30 26 26

positive / negative results 10 21

15 16 15 13 14 5

0 0 00 00 0 00 0 0 0

k k k N N N N N N N N N N N nk H PN PN H H la P HN P H P H P H blan g g g g g 6 /kg PN/kg PN k k /k /k k /kg P t te blang g te ie blank g/kg m b g g g/kg g s 35 s 35 urt 6 ur a ate blan k a e e h h mg/ mg/ mg/kg m m m m mg/kg mg/ k k g cola ol re 0 a a o 10 m 10 40 coo c 4 15 m yog y 2 rnfl rnfl chocolte te 40 m cho te te choc te 10 ing p a a a okie 5 ice age 8 u o o rk l o am 20 am s ssing 4ss c c cookie c5 mg/kg PN re re milk ark col col c re re ocola ocola d da sausagesau 8 mg/kg PN d h h ho ho hoco ce c ce d tant so c i i lad d la s stant soup 15 mg/kg HN a a in in Baumgartner 23 ilk c ark c ark c s s m milk c d d dark chocolatedark 2,5 mg/kg HN Allergentest – QLRT-2000-01151 Something new?

ELISA ELISA LFD LFD PCR PCR 2007 2012 2007 2012 2007 2012 Cereals X* X* X X X X Crustaceans X X X X X X Eggs X X X X X ? Fish ? ? ? ? X X Peanuts X* X* X X X X Soy X X ? X X X Milk X X* X X ? ? Treenuts X XXX* X XXX X XXX Celery X ? ? ? X X Mustard X X ? X X X Sesame X X* ? X X X Lupine X X ? X X X

Molluscs ? X ? ? X BaumgartnerX 24 Example peanut – Kit comparison

Hurst et al., 2002 Koch et al., 2003 Poms et al., 2005 30 partners EC-ring trial Number of Kits 4 3 5 Matrix Milk and dark Cookies Cookies, dark chocolate chocolate Measurement range 10-200 mg/kg 500-2500 mg/kg 2-10 mg/kg Recovery 3-250% 30-90% 44-191%

Baumgartner 25 Conclusion . …..what makes it so sophisticated?

. Find the appropriate analyte . Marker . Allergen . Whole protein . Referencematerials . Referencemethods . Thresholds/Limits . Nearly unlimited preparation and processing possibilities for food!!

Baumgartner 26 XIth ICAFA . XIth International Conference on AgriFood Antibodies . Ibis Wien Mariahilf Hotel, Wien

. 3rd - 5th September 2012 St. Stephen´s Cathedral, Vienna . http://sites.google.com/site/icafaconference/chain-act-10 . Dates for the Diary . 7th July 2012: Early registration and payment deadline . Download registration form: http://sites.google.com/site/icafaconference/album . Contact Prof. Chris Smith: . [email protected]

Baumgartner 27 The funding and the people behind

Christoph Parisa Ansari Hasenhindl Brigitte Galler

Denise Huber

Manuela Norbert Judith Rudolf Führer Stoppacher

Baumgartner 28 Thank you for your attention!

Same but different!

Baumgartner 29