Commercial Gluten Test Kits / Methods Jupiter Yeung, PhD Principal Scientist Food Products Association FDA Gluten Free Meeting, Aug 19, 2005

Gliadin 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 Antibody Detector Antibody Signal Sandwich 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 Gliadin Antibodies Specificity

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

Commercial Gliadin ELISA Kits * ppm gliadin ppm gluten = ppm gliadin x 2 Converted to ppm from  g/mL 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 wells 0.5 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 Kits 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

0 5 10 20 50 ppm gliadin controls Gliadin ELISA

Lateral Flow Device (Dipstick) Positive can mean less than LOD 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

Positive can mean less than LOD

Lateral Flow Device

Lateral Flow Device

Lateral Flow Device

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 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 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 18 th Meeting, WPGAT, 2004 Effects of Extraction Media

Hasselberg et al. Proceeding of the 18 th

Meeting, WPGAT, 2004

Effect of Partial Hydrolysis Ferre et al. Proceeding of the 18 th Meeting, WPGAT, 2004

Reference Materials IRMM-480 Wheat Gliadin NIST SRM 1567a Wheat Flour RM 8418 Wheat Gluten RM 8436, 8437, 8438, 8441a Others

IRMM-480 Wheat Gliadin

NIST

SRM 1567a Wheat Flour

RM 8418 Wheat Gluten

RM 8436, 8437, 8438, 8441a

Others

Confirmation 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 18 th Meeting, WPGAT, 2004

Detection & Speciation Wheat Gliadins Rye Scalins Barley Hordeins Oat Avenins Ab-based + - PCR + + + + MS + + + +