Gluten Test Kit Methods Usda

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 + + + +