# Presentation: 874981

## 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

**Capture Antibody**

**Detector****Antibody**

**Signal**

**Sandwich ELISA**

## 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

- 		–   -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

**Gliadin Antibodies Specificity**

## Commercial Gliadin ELISA Kits

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

**Commercial Gliadin ELISA Kits**

- * 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 | (AOAC OM) |
|  |        High Sensitivity |  |
| Diffchamb | Transia Plate Gluten | NFA, Sweden |
|  | Transia Plate Prolamins |  |
| Morinaga | Wheat Protein | NIHS, Japan |

**Commercial Gliadin ELISA Kits**

## Gliadin ELISA

**    ****0         5        10        20      50**
**  ****ppm gliadin controls**

**Gliadin ELISA**

## Lateral Flow Device

| Company | Product | LOD 
(ppm gluten) | Costs |
| --- | --- | --- | --- |
| R-Biopharm | Rida Quick | 10 |  $200 / 20 strips |
| Tepnel | Gluten Rapid | 100 | $150 / 10 strips |
|  | Gluten Home | 100 | $ 75 / 5 strips |

**Lateral Flow Device**
**(Dipstick)**

-   Positive can mean less than LOD

## Lateral Flow Device

**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

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

- Meeting, WPGAT, 2004

**Effects of Extraction Media**

## 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 | + | + | + | + |