Gluten-Check ELISAs

True gluten intolerance or Coeliac Disease (CD) affects 1% or more of the EC & US populations; it is caused when toxic proteins in cereal gluten damage the absorptive areas of the small intestine. CD manifests itself as e.g. intestinal pain, bloating and cramps, and in children as a failure to thrive. CD can only be reversed by strict adherence to a diet low in gluten.

Increasingly many people also perceive themselves to be food intolerant and may avoid wheat/gluten as a result. These phenomena have led to a rise in the availability of “gluten-free” food products and the consequent requirement to monitor gluten levels in such products more closely.

Codex Standard 118 and recent EC Regulations define “gluten free” foods for PARticular NUTritional use (PARNUTS) as containing less than 20 Parts Per Million (PPM or mg/kg). PARNUTS foods above 20 PPM but below 100 PPM must be labelled “very low gluten”.

Imutest Gluten-Check kits utilise ELISA techniques and are designed for the measurement of cereal gluten at low levels - the assay range is nominally between 5 - 110 mg/kg (PPM) if the stated extraction and dilution ratios are used. The range can be extended upwards by amending these ratios. Some matrices require less preparation, thereby lowering the assayrange and LOD. The nominal Limit Of Detection (LOD) of the assay is below 0.5PPM gluten. The assay can be used to test e.g. food raw materials/ingredients, environmental swab samples and in-process/finished food products.

The assay detects the omega gliadin fraction of wheat as a marker of total gluten and was originally developed by Skerritt & Hill for higher level gluten detection (AOAC Final Action 991.19).

Food samples are firstly prepared e.g. by milling/grinding/chopping etc to make them homogeneous.

Any gluten in the sample is then dissolved using an extraction solution containing protein and a tannin binding component to maximise gluten recovery.

This extraction solution is also available stabilised & ready-to-use in the Imutest Gluten Extraction kit (Cat. No. A6017), which saves both time and money; the inclusion of yellow dye in pre-filled extraction tubes helps confirm that samples have been diluted prior to testing.

This extract is then diluted and added to microwells coated with an antibody to cereal gliadin; any gluten present binds tightly to these antibodies.

After this first incubation the microwells are washed to remove unwanted proteins/food components.

Gluten is then detected by adding anti-gliadin attached to an enzyme molecule (HRP).

After this incubation the microwells are again washed to remove unbound enzyme.

A colourimetric substrate (TMB) is then added; this reacts with any HRP enzyme present to form a bright blue colour. The more colour that develops, the more gluten was present in the original sample.

The substrate reaction is then stopped using dilute acid and the colour in the microwells is measured in a plate reader (450nm).

The gluten content of the original sample is then calculated with reference to a Standard Curve of PPM gluten versus OD450nm.

Food Sample Preparation

• Prepare food samples/Kit Controls by milling/grinding/chopping until homogeneous.
• Add 1 part Test Portion to 9 parts of Extraction Solution
• Extract by shaking for 2 mins; incubate for 45 minutes at 55°C, mixing during the extraction.
• Shake again for 2 mins; centrifuge for 10 mins. or allow 30 mins. for the extract to settle.
• Remove a portion of the extract above the food pellet.
• Dilute extracts in the Diluent Solution provided.

ELISA Protocol: All active reagents are ready to use; Diluent and Wash solutions require only a simple dilution step.

• Pipette diluted Sample/Control Extracts & Ready to Use Standards into anti-gliadin coated microwells.
• Mix. Incubate at room temperature for 40 minutes.
• Wash wells THREE times with Working Wash Solution.
• Pipette Anti-Gliadin HRP into wells.
• Mix. Incubate at room temperature for 40 minutes.
• Wash wells FOUR times with Working Wash Solution.
• Pipette TMB Substrate reagent into wells.
• Mix. Incubate at room temperature for 15 minutes.
• Pipette Acid Stop Solution into wells.
• Mix. Read wells at 450nm wavelength.
• Draw a standard curve and calculate PPM Gluten results for all Controls/Samples.