Gluten detection with a next generation of monoclonal antibody
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Gluten detection with a next generation of monoclonal antibody DI Elisabeth Hammer
Transcript of Gluten detection with a next generation of monoclonal antibody
Gluten detection with a next generation of monoclonal
antibody
DI Elisabeth Hammer
Gluten-free as trend…
Successful tennis players:– Novak Djokovic– Andy Murray– Sabine Lisicki
Sensitivity towards grains• Celiac Disease
auto-immune disease
• Wheat Allergyallergic reaction to wheat proteins
• Gluten Sensitivitynon-allergic and non-autoimmune condition, diagnosis by exclusion
• WDEIAWheat-dependent exercise-induced anaphylaxis
• …
CELIAC DISEASE
Celiac Disease (CD)• immune-mediated enteropathy
• caused by the ingestion of gluten
• occurs in genetically predisposed persons
• leads to the destruction of the microscopic finger-like projections of the small intestine, called villi
• roughly 1% of the world´s population is affected
• at present, the only effective treatment known is a life-long gluten-free diet
Celiac Disease - Prevalence
During the past 35 years the true prevalence of CD in USA doubled every 15 years
Catassi et al. Annal Med 2010
Gluten Digestion in CD
Gluten Proteins
Resistant proteinse.g. 33-mer
T-cellStimulation
Inflammatoryreaction
Truncationof villi
Digestion bydigestive enzymes
Deamidation byTransglutaminase
GLUTEN ANALYSIS
Gluten Analysis• Immunochemical
– ELISA– LFD
• LC-MS-MS
• PCR
Antibodies for Gluten Testing
Antibody G12 R5 Skerritt
Specificity QPQLPY(wheat gliadin)
QQPFP(rye secalin)
HMW (high molecular weight)
glutenins and ω-gliadin (wheat)
Detects also rye, barley wheat, barley rye, barley
oat (if potentiallytoxic)
Legislation and StandardsCOMMISSION REGULATION (EC) No 41/2009•Gluten-free (<20 ppm gluten)Very low gluten (<100 ppm gluten)
•No method(s) specified
Standards & Codes of Practise• Codex Standard 118-1979
Gluten-Free Labeling of Foods• Proposed rule for gluten-free labeling
Health Canada• Food Allergen Labeling Regulations in Canada Gazette, Part II (will come into force
August 2012)
Australia/New Zealand• Food Standards Code: gluten-free = “no detectable gluten” by the most sensitive
universally accepted test method
Codex Standard 118 – 1979for foods for special dietary use for persons intolerant to gluten
general outline of the methods:
The quantitative determination of gluten in foods and ingredients shall bebased on an immunologic method or other method providing at least equalsensitivity and specificity.
The antibody used should react with the cereal protein fractions thatare toxic for persons intolerant to gluten and should not cross-react withother cereal proteins or other constituents of the foods or ingredients.
Methods used for determination should be validated and calibrated against acertified reference material, if available.
The detection limit has to be appropriate according to the state of the art andthe technical standard. It should be 10 mg gluten/kg or below.
Gluten Methods
Toxicity of foodstuffs to Celiac patients
Detect the presence of toxic peptides
33-mer2-gliadin fragment of 33 amino acids (56–88) in lengthwas identified as the primary initiator of inflammatory responses in Celiac Disease (Shan et al., 2002)
Sequence of the immunotoxic 33-mer:LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF
(L = Leucin, Q = Glutamine, P = Proline, F = Phenylalanine, Y = Tyrosine)
Shan et al., Science 2002; 297: 2275-2279Anderson et al., Nat Med 2000;6:337-342Arentz-Hansen et al., J Exp Med 2000; 191:603-621
G12 Monoclonal Antibody
QPQLPY (gliadin - wheat)QPQQPY (secalin – rye)QPQLPF (hordein - barley)Oats (avenin – oats)
G12 Antibody Specificity
A peptide fragment of 33 residues
Remarkably potent T cell stimulator
Morón et al., PLoS ONE 2008 May 28;3(5):e2294.Morón et al., Am J Clin Nutr. 2008 Feb;87(2):405-14.
G12 Antibody
G12 antibody was raised against the immunotoxic 33-mer peptide
G12 targets the most immunotoxicproteins for those intolerant to gluten
G12 is capable of detecting potential immunotoxic varieties of oats
Products
• ELISA:AgraQuant® Gluten G12
• LFD:AgraStrip® Gluten G12
AgraQuant® Gluten G12
sandwich ELISA
5 standards (0, 4, 20, 80, 200 ppm)Standards were created using “Vital Wheat Gluten” (Roquette,UK), which was calibrated with the Prolamin Working Group (PWG) gliadin.
Limit of Detection: 2 ppm Gluten
Quantitation Range: 4 – 200 ppm Gluten
Calibration
0
0,5
1
1,5
2
2,5
0 100 200 300 400 500 600
O.D. (45
0nm)
ng/ml Gluten
WGPAT Standards
G12 Gluten Standards
Cro
ss r
eact
ivit
yFood Sample
AgraQuant®
Gluten G12[ppm]
Nuts
Pecan <4Walnut <4Almond <4Cashew <4Macadamia <4Peanut <4Hazelnut <4Pine Nuts <4Pistachio <4
Oils
Hazelnut Oil <4Walnut Oil <4Vegetable Oil <4Sunflower Oil <4
Seeds
Golden Linseed <4Brown Linseed <4Poppy <4Sesame <4Mustard <4
StarchesTapioca Starch <4Wheat Starch <4Potato Starch <4
Naturally gluten-free foods
Soya Mince <4Soy Bean <4Millet <4Buckwheat <4Rice Flour <4Quinoa <4
Gluten containing grains
Wheat Flour >200Rye >200Barley >200Firth Oat 7Oat sample – Spain 153GF Oat <4
Grains celiac patients have to avoid
Wheat
Barley
Rye
Oat
Controversy on oats
Studies demonstrating oatsafety in celiac patients diet:
• Srinivasan et al., 2006• Högberg et al., 2004• Kilmartin et al., 2003• Srinivasan et al., 1996• Janauinen et al., 1995• ...
Studies that show evidence of oat sensitivity in certain celiac patients:
• Silano et al., 2007• Hollén et al., 2006• Arentz-Hansen et al., 2004• Peräaho et al., 2004 • Lundin et al., 2003• ...
Romer G12 data on oatsOat Variety AgraQuant® Gluten G12 [ppm]
Bastion 4,304-704-Cn 7/2 4
Racoon 5,1Mason <4
00-61 Cn 7,4Buffalo 6Chris 4,2Fusion 6,6Becon <4
Hendon 5,6Brachan <4Lenon 6
Canyon 4,8Zuton 4,6Gerald <4
04-66Cn7 4,2Tardis <4
Dalguise <4Husky 6,3Balado <4
05-82ACn19 4,9Rhapsody 4,403AW24 4,703-37Cn 5,403-36Cn 5,5Mascani 5,9
Oat sample - Spain 153Firth 7
GF oat <4
Sp
ecif
icit
yI
Food SampleAgraQuant®
Gluten G12[ppm]
Food labelled as “Gluten-free”
Gluten Free Cookie Mix <4
Gluten Free Chocolate Bar <4
Gluten Free Cake Mix <4
Gluten Free Breadcrumbs <4
Gluten Free Pancake Mix <4
Gluten Free Bread Mix <4
Cherry Bakewell <4Lemon Shortbread <4Bread <4Shortbread <4Chocolate Brownie <4
Honey and Oat Cookie 26.3
Mini Muffin <4Custard Cream <4
Gluten Free Cake Mix <4
Sp
ecif
icit
yII Food Sample
AgraQuant®
Gluten G12[ppm]
Miscellaneous
Fennel <4Paprika <4Chickpea <4Soy Sauce <4Coriander <4Black Eyed Beans <4Puy Lentils <4Bread 139.0
Maize Flour (naturally contaminated with wheat)
6.7
Snack 90.7
Bisto Best (Gravy Granules) <4
Corn Flakes >200Multi Grain Pringles >200Cardamom <4
Baby Food
Apple and Banana Breakfast 4.7
Fruity Müsli 50.7Past Bolognese >200Mini Beef Casserole <4Creamed porridge 7.3
Rec
over
yMatrix
Spiking level [ppm]
Concentration [ppm]
Recovery [%]
Curry Sauce0 <4 -5 6.1 124.220 21.1 105.5
Crisps
0 <4 -5 4.2 84.010 8.1 81.020 18.1 90.5
Chocolate(+ gelatine)
0 <4 -5 5.0 100.010 6.9 69.020 16.3 81.5
Corn Snack0 <4 -5 5.4 108.010 8.5 85.0
Rice Flour0 <4 -5 5.0 10020 16.9 84.5
Yogurt0 <4 -5 5.5 110.020 20.8 104
Paprika
0 <4 -5 5.3 106.010 10.1 101.020 16.3 81.5
Comparative dataSample
AgraQuant® Gluten G12 ELISA
Gluten [ppm]
R5 ELISAGluten [ppm]
Fruity Muesli (Baby Food) 50.7 54.9
Maize flour (naturally contaminated) 6.7 8.2
Honey and Oat Cookie (gluten free) 26.3 28.9
Pita Bread (gluten free) 4.3 <5
English Muffins (gluten free) 4.8 <5
Spaghetti (gluten free) 6.9 <5
Shortbread (gluten free) <4 <5
Chocolate Brownie (gluten free) <4 <5
Cherry Bakewell (gluten free) <4 <5
Bread 139.0 >80
Corn Flakes >200 >80
Crisps (5 ppm spike) 4.2 <5
Chocolate (5 ppm spike) 5.0 <5
FAPAS samplesFAPAS Round 2781
cake mix
AgraQuant Gluten G12 ELISA
(mg/kg Gluten)
Assigned Value R5 ELISA – R-Biopharm R7001(mg/kg Gluten)
Assigned Value Veratox ELISA –Neogen(mg/kg Gluten)
FAPAS 2781 A <4 Negative Negative
FAPAS 2781 B 22.6 27.4 42.6
FAPAS 2781 C 95.6 91.6 120.7
FAPAS Round 2792infant soy formula
AgraQuant GlutenG12 ELISA
(mg/kg Gluten)
Assigned Value R5 ELISA – R-Biopharm R7001(mg/kg Gluten)
Assigned Value R5 ELISA – R-Biopharm R7002(mg/kg Gluten)
FAPAS 2792 A 119.8 134.2 141.0
FAPAS 2792 B <4 Negative Negative
FAPAS Round 2795cake mix
AgraQuant Gluten G12 ELISA
(mg/kg Gluten)
Assigned Value R5 ELISA – R-Biopharm R7001(mg/kg Gluten)
Assigned Value R5 ELISA – R-Biopharm R7002(mg/kg Gluten)
Assigned Value Ingenasa -R5 ELISA 30.GLU.K2(mg/kg Gluten)
FAPAS 2795 A 51.3 58.5 43.4 71.4
FAPAS 2795 B <4 Negative Negative Negative
FARRP
No. Sample
R-BiopharmRIDASCREEN®
Gliadin kit
NeogenVeratox-Gliadin R5
kit
Romer LabsAgraQuant®
Gluten G12 kit
Morinaga Wheat Protein ELISA kit
Gluten, ppm
Standard Deviation
Gluten, ppm
Standard Deviation
Gluten, ppm
Standard Deviation
Wheatprotein,
ppm
Standard Deviation
1 Ground raw meat 1690 310 2038 35.0 1762 90.7 1972 4882 Cocoa powder 229 71.6 244 57.6 142 6.30 134 36
3Organic Kamut® grass juice powder 78.9 3.22 67.1 19.27 59.8 1.92 BLQ
4 Red lentil flour 73.6 9.57 63.8 5.59 49.8 6.97 37.5 20.45 Brown rice syrup 131 9.50 92.7 9.48 128 15.1 63.9 17.6
6Organic wheat grass juice powder 18.9 0.11 35.0 12.6 6.80 1.23 BLQ
7 Sugar cookie* BLQ BLQ BLQ BLQ8 Oat flour BLQ BLQ 18.6 3.54 BLQ9 Yeast extract powder 891 83.8 1539 818 176 28.5 500 21110 Corn flour 185 24.2 154 28.4 118 24.5 204 34.511 Sorghum powder 36.7 15.7 BLQ BLQ 7.67 2.96
12Organic Alfalfa grass juice powder 121 6.49 67.7 2.47 80.2 6.02 BLQ
13 Malt drink 19.4 0.686 25.4 2.79 17.0 0.94 17.9 5.4314 Nutty rice bar 16.3 2.12 16.1 1.10 11.1 1.48 22.7 0.95215 Green pea flour 13.7 1.59 15.8 0.389 11.4 2.58 16.7 7.2216 Resistant wheat starch 8.19 1.52 BLQ 5.76 0.90 14.9 0.005
Plaimein Amnuaycheewa
*Sugar cookies originally tested positive in r-Biopharm Ridascreen Gliadin kit at a rather low but detectable level. Failure to detect gluten residues by any method in this comparative analysis is likely attributable to sub-sampling differences or perhaps a laboratory error in the initial analysis.
FARRP
No. Sample
R-BiopharmRIDASCREEN®
Gliadin kit
NeogenVeratox-Gliadin R5
kit
Romer LabsAgraQuant®
Gluten G12 kit
Morinaga WheatProtein ELISA kit
Gluten, ppm
Standard Deviation
Gluten, ppm
Standard Deviation
Gluten, ppm
Standard Deviation
Wheatprotein,
ppm
Standard Deviation
17 Tortilla chips 494 53.5 468 104 650 24.9 1000 23318 Organic oat flakes 158 41.9 173 118 72.1 7.96 119 70.119 Bar flavor 152 2.63 134 18.8 64.6 9.60 45.9 0.60420 Cereal powder 69.4 2.72 46.7 8.49 69.6 15.9 30.1 7.0621 Wheat starch powder 41.0 0.057 21.8 5.23 19.9 3.55 53.0 19.1
22Org. creamy buckwheat powder 31.6 18.7 26.5 21.1 20.9 9.12 2.08 1.34
23 Ground rice crisp 14.0 0.438 15.4 0.247 9.00 1.01 12.5 3.75
24Barley grass juice powder 7.47 2.31 5.15 0.636 BLQ BLQ
25 Hemp protein powder 466 133 433 92.0 104 25.3 97.3 31.726 Cookie 277 3.31 226 20.4 149 31.3 83.7 32.127 Navy Bean flour 86.8 3.50 110 14.4 65.5 18.2 70.6 8.3328 White rice flour 92.0 4.39 79.5 27.5 106 24.0 35.8 9.22
29Barley grass juice powder 65.5 9.34 43.2 5.23 62.6 9.17 6.11 2.08
30 Gluten free oats flour 10.7 6.15 14.1 4.14 9.25 6.96 12.4 0.88431 Seasoning powder 5.73 0.120 BLQ BLQ 6.57 0.37032 Resistant wheat starch 6.41 0.262 BLQ BLQ 7.87 6.30
Plaimein Amnuaycheewa
AgraStrip® Gluten G12
• 3 different cutoff levels:5, 10 and 20 ppm
• Result within 11 minutes
• Specificity: Cross reactivity with Rye, Spelt, Barley and some oat varieties. Good correlation with AgraQuant® Gluten G12 ELISA
AgraStrip® Gluten G12Procedure:
- Extraction - Detection
• 0.2 g of sample
• 1 min Extraction
• Dilution
10 minutes
Validation Data
AgraStrip® Gluten G12
Yogurt 5 ppm
Dairy Free Spread 5 ppm
Chocolate 10 ppm
Curry Sauce 5 ppm
Rice Flour 5 ppm
LOD in spiked commodities
Rinse water:within a pH range of 5 to 9 there is no effect on the results obtained with AgraStrip® Gluten G12
Validation Data
Gluten on surface [mg] Stainless Steel Plastic
0 - -0,2 - -0,4 - -1 - -2 - -4 + +10 + +20 + +
Swabbing
The AgraStrip® Gluten G12 Kit was able to recover 4 mg Gluten when swabbing both a stainless steel and a plastic surface.
G12 based MethodsLink established between Celiac Disease and detection of immunotoxic peptides
Methods target a potent & robust immunotoxic peptide
An attractive antibody for improving gluten detection
