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

Sandwich ELISA Signal Detector Antibody Capture Antibody 22 22

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 Company Product LOD* LOQ* Range* Replicates Costs Neogen Alert (Qualitative) 10 1 $237/20Sampl Veratox 5 5-50 $465/38Sampl R-Biopharm RidaScreen 1.5 2.5 2.5-40 2 $425/96 wells RidaScreen Fast 5-40 $400/48 wells Tepnel Gluten Assay Kit, AOAC or High sensitivity 50 50-1000 0.5 5-100 Hallmark HAVen Gluten, AOAC or High sensitivity Diffchamb Transia Plate Gluten $500/96 wells Transia Plate Prolamins 0.8 0.8-12.5 Morinaga Wheat Protein 0.3* 3.12 3.12-20 $600/96 wells * ppm gliadin ppm gluten = ppm gliadin x 2 Converted to ppm from g/mL

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 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) 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 $ 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

Extraction Media 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

Effects of Extraction Media Hasselberg et al. Proceeding of the 18th Meeting, WPGAT, 2004

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

Reference Materials 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

Garcia et al., Proceeding of the 18th Meeting, WPGAT, 2004

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