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
Detector Antibody
Capture Antibody 22 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
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
0.5
5-100
Hallmark
HAVen Gluten, AOAC
or High sensitivity
Diffchamb
Transia Plate Gluten
$500/96 wells
Transia Plate Prolamins
0.8
Morinaga
Wheat Protein
0.3*
3.12
$600/96 wells
* ppm gliadin
ppm gluten = ppm gliadin x 2
Converted to ppm from g/mL

6. 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

7. Gliadin ELISA
ppm gliadin controls

8. 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

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: % 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
Wheat
Gliadins
Rye
Scalins
Barley
Hordeins
Oat
Avenins
Ab-based + -
PCR MS