Sabine BaumgartnerIFA-Tulln, Rakousko

Antibodies and their suitability for food allergen detection

Alergeny v potravinách a jejich diagnostika27. duben 2012

Universität für Bodenkultur WienInteruniversitäres Department für Agrarbiotechnologie Tulln

Universität für Bodenkultur WienInteruniversitäres Department für Agrarbiotechnologie Tulln

Bezeichnung deutsch: ev. Untereinheit 2. Ebene I ev. Untereinheit 3. Ebene I Univ. Prof. DI. Dr. Max Mustermann 2

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

Campus Tulln 2011UFT

TZT

kindergartenDormitory

Natural MaterialsTechnology

Biotechnology inAnimal Production

Biotechnology inPlant Production

Environmental Biotechnology

Center for AnalyticalChemistry

Animal Nutrition, Products and Nutrition Physiology

Department for Agrobiotechnology,Department for Agrobiotechnology, IFAIFA--TullnTulln

21 Research Groups in 6 Institutes

Needle in the haystack – food allergens in food

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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

Baumgartner 61700 1800 1900 2000

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

Baumgartner 71700 1800 1900 2000

History of Food Allergen Labelling in the EU (2000/13/EC, 2003/89/EC, 2006/142/EC, 2007/68/EC, 1169/2011)

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CrustaceaenPeanut

Soy Milk TreenutsFishEggCereals

Celery, mustard, sesame, lupine, molluscs

1700 1800 1900 2000

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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

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

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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

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Peanut and products thereof

Composition

2,77,59,150,727,4Peanut

MineralsFibersCHLipidProtein[%]

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Peanut and products thereof Allergens

Albumins (2S) or Conglutinins: Ara h 2, 18 kDa, Xreacts - Ara h 6Ara 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

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Possible methods

Immunochemical methods ELISA Rapid tests – Lateral Flow

Devices, Dipsticks

DNA-based methods PCR rt-PCR PCR-ELISA

Upcoming MS-methods

© BarbaraCvak

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Immunochemical methods - Antibody based assays

Protein detection by highly specialised proteins

Polyclonale Abs (rabbit, chicken, sheep,...)

Monoclonale Abs (mouse, rat,…) Recombinant Abs

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Polyclonal egg yolk antibodies?

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Cryoconservation( - 70 °C, - 150 °C)

Immunisation

RepeatedCloning

Testing specific ABs

Cloning

Selection of hybridomas

Cell fusion

Myeloma cellsSpleen cells

Cell culture

Monoclonal antibodies

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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

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Challenges in LFD development

* sensitivity, specificity, yield

Antibody Production

Sample

StandardImmunogen

Test Performance

AntibodyQuality*

AntibodyPurification

Animals Colloidal gold

Conjugation

Membrane, Running buffer

+

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LFD principle – Sandwich formatReaction

batch Antibodies coupled to

colloidal gold

Wick pad

Backing cardNitrocellulose membrane

© Judith Rudolf

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Challenges in LFD development

* sensitivity, specificity, yield

Antibody Production

Sample

StandardImmunogen

Test Performance

AntibodyQuality*

AntibodyPurification

Animal Colloidal gold

Conjugation

Membrane, Running buffer

+

The extraction - example peanut

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Extraction of peanut proteinsRudolf et al., 2011, Food Add Contam

Buffer and signal intensity

Samples on HN-LFD

15 16

26

1513

21

0

26

30

0 0

30

0 0

30

0

32

0 0 0

32

0

32

0

14

15 14

4

1517

9

30

4

0

32 32

2

30 30

0

32

0

32 32 32

0

32

0

32

18

0

5

10

15

20

25

30

35

milk ch

ocolate

blank

milk ch

ocolate

10 m

g/kg PN

milk ch

ocolate

40 m

g/kg PN

dark

choc

olate blank

dark

choc

olate 10 m

g/kg PN

dark

choc

olate 40 m

g/kg PN

dark

choc

olate blank

dark

choc

olate 2,5

mg/kg H

N

dark

choc

olate 10 m

g/kg H

Nco

okie blank

cook

ie 5 mg/k

g PN

cook

ie 5 mg/k

g HN

ice cr

eam bl

ank

ice cr

eam 20

mg/k

g PN

ice cr

eam 20

mg/k

g HN

saus

age 8

mg/k

g PN

saus

age 8

mg/k

g HN

salad

dress

ing 4 m

g/kg PN

salad

dress

ing 4 m

g/kg H

N

instant

soup

15 m

g/kg P

N

instant

soup

15 m

g/kg H

Nco

rnflak

es 35

PN

cornf

lakes

35 H

Nyo

ghurt 6

PN

yogh

urt 6 H

Npo

sitiv

e / n

egat

ive

resu

lts

positve negative

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LFD – Hazelnut determination

Allergentest – QLRT-2000-01151

+

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Something new?

ELISA2007

ELISA2012

LFD2007

LFD2012

PCR2007

PCR2012

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 X

Example peanut – Kit comparison

Hurst et al., 2002 Koch et al., 2003 Poms et al., 200530 partners EC-ring

trialNumber of Kits 4 3 5Matrix Milk and dark

chocolateCookies Cookies, dark

chocolateMeasurement range 10-200 mg/kg 500-2500 mg/kg 2-10 mg/kgRecovery 3-250% 30-90% 44-191%

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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!!

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XIth ICAFA XIth International Conference on

AgriFood Antibodies Ibis Wien Mariahilf Hotel, Wien 3rd - 5th September 2012 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: c.x.smith@mmu.ac.uk

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St. Stephen´s Cathedral, Vienna

The funding and the people behind

Brigitte GallerParisa Ansari

Judith Rudolf

ChristophHasenhindl

ManuelaFührer

NorbertStoppacher

DeniseHuber

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Thank you for your attention!

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Same but different!