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Commercial Gluten Test Kits / Methods

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.

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Commercial Gluten Test Kits / Methods

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

  2. Gliadin Detection Methods • Immunochemical ELISA Lateral Flow Device Immunoblotting • PCR • MS MALDI-TOF LC/MS/MS

  3. Sandwich ELISA Signal DetectorAntibody Capture Antibody 22

  4. 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

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

  6. Commercial Gliadin ELISA Kits

  7. Gliadin ELISA 0 5 10 20 50 ppm gliadin controls

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

  9. Lateral Flow Device

  10. Lateral Flow Device

  11. Lateral Flow Device

  12. 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

  13. Extraction Media • Aq. Alcohol (40-60% EtOH) • Cocktail (reducing agent + others) - interaction of Ab and Ag

  14. 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

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

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

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

  18. Confirmation Methods • Ab-Western blot • PCR – qualitative, quantitative • MS – MALDI-TOF, LC/MS/MS

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

  20. Detection & Speciation

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