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Antibodies and Their Suitability for Food Allergen Detection

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Antibodies and Their Suitability for Food Allergen Detection 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 35 positve 30 25 negative 20 15 4 14 15 positive / negative results 15 10 9 17 26 Samples on HN-LFD 15 5 4 0 16 0 milk chocolate blank 15 2 + milk chocolate 10 mg/kg PN 21 30 13 milk chocolate 40 mg/kg PN 26 30 dark chocolate blank 32 32 0 dark chocolate 10 mg/kg PN dark chocolate 40 mg/kg PN 30 0 0 dark chocolate blank dark chocolate 2,5 mg/kg HN 30 30 dark chocolate 10 mg/kg HN 30 00 cookie blank 32 0 cookie 5 mg/kg PN 32 cookie 5 mg/kg HN 00 32 32 32 0 ice cream blank ice cream 20 mg/kg PN ice cream 20 mg/kg HN 0 32 sausage 8 mg/kg PN sausage 8 mg/kg HN 32 00 0 32 18 salad dressing 4 mg/kg PN salad dressing 4 mg/kg HN 32 instant soup 15 mg/kg PN Allergentestinstant soup – QLRT-2000-01151 15 mg/kg HN 0 cornflakes 35 PN 14 cornflakes 35 HN 0 yoghurt 6 PN Baumgartner 23 yoghurt 6 HN 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.
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