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Testing For Glutens
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Commercial Gluten Test Kits / Methods
Commercial Gluten Test Kits / Methods
Jupiter Yeung, PhDPrincipal Scientist
Food Products Association
FDA Gluten Free Meeting, Aug 19, 2005
Jupiter Yeung, PhDPrincipal Scientist
Food Products Association
FDA Gluten Free Meeting, Aug 19, 2005
Gliadin Detection MethodsGliadin Detection Methods
• Immunochemical ELISA
Lateral Flow Device
Immunoblotting• PCR• MS
MALDI-TOF
LC/MS/MS
• Immunochemical ELISA
Lateral Flow Device
Immunoblotting• PCR• MS
MALDI-TOF
LC/MS/MS
22
Capture AntibodyCapture Antibody
DetectorDetectorAntibodyAntibody
SignalSignal
Sandwich ELISASandwich ELISA
• Skerritt mAb
– -gliadin (-gliadin)– wheat – 100% rye – 120% barley – 5%
• Mendez R5 mAb
– QXPFP toxic epitopes of ,,, -gliadins– Equally to wheat, rye and barley
• Both Ab recognize raw and cooked prolamins
• Skerritt mAb
– -gliadin (-gliadin)– wheat – 100% rye – 120% barley – 5%
• Mendez R5 mAb
– QXPFP toxic epitopes of ,,, -gliadins– Equally to wheat, rye and barley
• Both Ab recognize raw and cooked prolamins
Gliadin Antibodies SpecificityGliadin Antibodies Specificity
Company Product LOD* LOQ* Range* Replicates Costs
Neogen Alert (Qualitative) 10 1 $237/20Sampl
Veratox 5 5-50 1 $465/38Sampl
R-Biopharm RidaScreen 1.5 2.5 2.5-40 2 $425/96 wells
RidaScreen Fast 5 5 5-40 1 $400/48 wells
Tepnel Gluten Assay Kit, AOAC or High sensitivity
5 50 50-1000 2$425/96 wells0.5 5 5-100 2
Hallmark HAVen Gluten, AOAC
or High sensitivity
5 50 50-1000 2$425/96 wells
0.5 5 5-100 2
Diffchamb Transia Plate Gluten 5 5 5-100 2 $500/96 wells
Transia Plate Prolamins 1.5 0.8 0.8-12.5 2 $500/96 wells
Morinaga Wheat Protein 0.3* 3.12 3.12-20 2 $600/96 wells
Commercial Gliadin ELISA KitsCommercial Gliadin ELISA Kits
* ppm gliadin
ppm gluten = ppm gliadin x 2
* ppm gliadin
ppm gluten = ppm gliadin x 2
Converted to ppm from g/mLConverted to ppm from g/mL
Company Product Validation
Neogen Alert
Veratox
R-Biopharm RidaScreen
RidaScreen Fast PWG ring trial
Tepnel BioKit Gluten (AOAC OM)
High Sensitivity
Hallmark HAVen Gluten (AOAC OM)
High Sensitivity
Diffchamb Transia Plate Gluten NFA, Sweden
Transia Plate Prolamins
Morinaga Wheat Protein NIHS, Japan
Commercial Gliadin ELISA KitsCommercial Gliadin ELISA Kits
0 5 10 20 50
ppm gliadin controls
Gliadin ELISAGliadin ELISA
Company Product LOD
(ppm gluten)
Costs
R-Biopharm Rida Quick 10 $200 / 20 strips
Tepnel Gluten Rapid 100 $150 / 10 strips
Gluten Home 100 $ 75 / 5 strips
Lateral Flow Device(Dipstick)
Lateral Flow Device(Dipstick)
• Positive can mean less than LOD• Positive can mean less than LOD
Lateral Flow DeviceLateral Flow Device
Lateral Flow DeviceLateral Flow Device
Lateral Flow DeviceLateral Flow Device
Issues with Gluten Test Kits Issues with Gluten Test Kits
• Ab has different specificities to different prolamins, e.g. Skerritt Ab
• Various results from different extraction media: 40-60% EtOH, cocktails
• Different standards used from different kits
• False positives and negatives require a confirmatory test
• Limited availability of confirmatory test to most food lab
• No sampling plan
• Ab has different specificities to different prolamins, e.g. Skerritt Ab
• Various results from different extraction media: 40-60% EtOH, cocktails
• Different standards used from different kits
• False positives and negatives require a confirmatory test
• Limited availability of confirmatory test to most food lab
• No sampling plan
Extraction MediaExtraction Media
• Aq. Alcohol (40-60% EtOH)
• Cocktail (reducing agent + others)
- interaction of Ab and Ag
• Aq. Alcohol (40-60% EtOH)
• Cocktail (reducing agent + others)
- interaction of Ab and Ag
Matrix EffectMatrix Effect
• Cocktail (reducing agent + others)
- interference with complex food matrices, e.g. milk
- interaction of Ab and Ag
• Samples with high polyphenols, tannins and syrups require fish gelatin to improve recovery
• Cocktail (reducing agent + others)
- interference with complex food matrices, e.g. milk
- interaction of Ab and Ag
• Samples with high polyphenols, tannins and syrups require fish gelatin to improve recovery
Hasselberg et al. Proceeding of the 18th
Meeting, WPGAT, 2004
Hasselberg et al. Proceeding of the 18th
Meeting, WPGAT, 2004
Effects of Extraction MediaEffects of Extraction Media
Effect of Partial HydrolysisEffect of Partial Hydrolysis
Ferre et al. Proceeding of the 18th
Meeting, WPGAT, 2004
Ferre et al. Proceeding of the 18th
Meeting, WPGAT, 2004
Reference Materials Reference Materials
• IRMM-480 Wheat Gliadin• NIST
SRM 1567a Wheat FlourRM 8418 Wheat GlutenRM 8436, 8437, 8438, 8441a
• Others
• IRMM-480 Wheat Gliadin• NIST
SRM 1567a Wheat FlourRM 8418 Wheat GlutenRM 8436, 8437, 8438, 8441a
• Others
Confirmation MethodsConfirmation Methods
• Ab-Western blot• PCR – qualitative, quantitative• MS – MALDI-TOF, LC/MS/MS
• Ab-Western blot• PCR – qualitative, quantitative• MS – MALDI-TOF, LC/MS/MS
Garcia et al., Proceeding of the 18th Meeting, WPGAT, 2004
Garcia et al., Proceeding of the 18th Meeting, WPGAT, 2004
Detection & SpeciationDetection & SpeciationWheat
Gliadins
Rye
Scalins
Barley
Hordeins
Oat
Avenins
Ab-based + -
PCR + + + +
MS + + + +