Commercial gluten test kits methods
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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

Commercial Gluten Test Kits / Methods

Jupiter Yeung, PhD

Principal Scientist

Food Products Association

FDA Gluten Free Meeting, Aug 19, 2005


Gliadin detection methods

Gliadin Detection Methods

  • Immunochemical ELISA

    Lateral Flow Device

    Immunoblotting

  • PCR

  • MS

    MALDI-TOF

    LC/MS/MS


Commercial gluten test kits methods

Sandwich ELISA

Signal

DetectorAntibody

Capture Antibody

22


Gliadin antibodies specificity

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 gluten test kits methods

Commercial Gliadin ELISA Kits

* ppm gliadin

ppm gluten = ppm gliadin x 2

Converted to ppm from g/mL


Commercial gluten test kits methods

Commercial Gliadin ELISA Kits


Commercial gluten test kits methods

Gliadin ELISA

0 5 10 20 50

ppm gliadin controls


Commercial gluten test kits methods

Lateral Flow Device

(Dipstick)

  • Positive can mean less than LOD


Commercial gluten test kits methods

Lateral Flow Device


Commercial gluten test kits methods

Lateral Flow Device


Commercial gluten test kits methods

Lateral 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


Extraction media

Extraction Media

  • Aq. Alcohol (40-60% EtOH)

  • Cocktail (reducing agent + others)

    - interaction of Ab and Ag


Matrix effect

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

Effects of Extraction Media

Hasselberg et al. Proceeding of the 18th

Meeting, WPGAT, 2004


Effect of partial hydrolysis

Effect of Partial Hydrolysis

Ferre et al. Proceeding of the 18th

Meeting, WPGAT, 2004


Reference materials

Reference Materials

  • IRMM-480 Wheat Gliadin

  • NIST

    SRM 1567a Wheat Flour

    RM 8418 Wheat Gluten

    RM 8436, 8437, 8438, 8441a

  • Others


Confirmation methods

Confirmation Methods

  • Ab-Western blot

  • PCR – qualitative, quantitative

  • MS – MALDI-TOF, LC/MS/MS


Commercial gluten test kits methods

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


Detection speciation

Detection & Speciation


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