Commercial Gluten Test Kits Methods Jupiter Yeung

Commercial Gluten Test Kits / Methods Jupiter Yeung, Ph. D 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

Sandwich ELISA Signal Detector Antibody Capture Antibody 22

Gliadin Antibodies Specificity • Skerritt m. Ab – -gliadin ( -gliadin) – wheat – 100% rye – 120% barley – 5% • Mendez R 5 m. Ab – QXPFP toxic epitopes of , , , -gliadins – Equally to wheat, rye and barley • Both Ab recognize raw and cooked prolamins

Commercial Gliadin ELISA Kits Company Neogen Product Alert (Qualitative) Veratox R-Biopharm Hallmark LOQ* Range* Costs 1 10 Replicates $237/20 Sampl 5 5 -50 1 $465/38 Sampl 1. 5 2. 5 -40 2 $425/96 wells 5 5 5 -40 1 $400/48 wells Gluten Assay Kit, AOAC 5 50 or High sensitivity 0. 5 5 50 -1000 2 5 -100 2 HAVen Gluten, AOAC or High sensitivity Rida. Screen Fast Tepnel LOD* $425/96 wells Morinaga 50 50 -1000 2 0. 5 5 5 -100 2 $500/96 wells Transia Plate Prolamins Diffchamb 5 1. 5 0. 8 -12. 5 2 $500/96 wells Wheat Protein 0. 3* 3. 12 -20 2 $600/96 wells Transia Plate Gluten * ppm gliadin ppm gluten = ppm gliadin x 2 Converted to ppm from g/m. L $425/96 wells

Commercial Gliadin ELISA Kits Company Neogen Product Validation Alert Veratox R-Biopharm Rida. Screen Fast Tepnel Bio. Kit Gluten PWG ring trial (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

Gliadin ELISA 0 5 10 20 50 ppm gliadin controls

Lateral Flow Device (Dipstick) Company Product LOD (ppm gluten) Costs R-Biopharm Rida Quick 10 Tepnel Gluten Rapid 100 $150 / 10 strips Gluten Home 100 $ 75 / 5 strips • Positive can mean less than LOD $200 / 20 strips

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% Et. OH, 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% Et. OH) • 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

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 1567 a Wheat Flour RM 8418 Wheat Gluten RM 8436, 8437, 8438, 8441 a • Others

Confirmation Methods • 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 Ab-based Wheat Rye Barley Oat Gliadins Scalins Hordeins Avenins + - PCR + + MS + +