AOAC ERP Gluten Assays - Dec 2018

Annex B

3.3

Apparatus

3.3.1 3.3.2 3.3.3 3.3.4 3.3.5

Microtiter plate spectrophotometer (450 nm)

Scale

Laboratory mincer/grinder, pestle and mortar, or Ultra-Turrax Shaker (e.g. Roto Shaker Genie, Scientific Industries Inc.) Centrifuge (e.g. Minifuge RF, Kendro, Hanau, Germany)

3.3.6 Temperature controlled water bath 50 °C/122°F (e.g. GFL, Burgwedel, Germany)

3.4

(Standard) Reference Material

Oat flour containing wheat or rye or barley gluten at a concentration of 10 mg/kg and 20 mg/kg as mentioned in AOAC SMPR ® 2017.021 (1). Additionally oat blank flour is provided.

3.5

Standard Solution and Spike Solution

The starting material for spiking consists of a mixture of four commercial wheat flours. It is stored at -20°C. For spiking purposes, the material is extracted with Cocktail (patented) and 80% ethanol. Briefly, 2.5 mL Cocktail (patented) is added to 250 mg of wheat flour mixture and incubated at 50°C for 40 min. Afterwards, 7.5 mL 80% ethanol is added, and the vial is rotated upside down for 60 min at room temperature. The solution is centrifuged at 2500 g for 10 min and filtrated. Based on HPLC analysis (Dr. Katharina Scherf, Leibniz-Institute for Food Systems Biology at the Technical University of Munich, Germany) the prolamin content of the mixture is 51.4 mg/g and the glutenin content is 24.9 mg/g, so in sum 76.3 mg/g total gluten. The total gluten content based on the total protein content multiplied by 80% (see SMPR ® 2017.021) is 75.2 mg/g. This value is used for further calculations, so that the spike solution described above has a concentration of 1.88 mg/mL total gluten. This solution is stable for at least 2 weeks at 20-25°C. The spike solution is diluted appropriately to the desired concentration with RIDASCREEN ® Total Gluten buffer. The starting material for preparation of standards was a mixture of four wheat cultivars (var. Akteur, var. Pamir, var. Julius, var. Tommi ). The materials were also characterized by Katharina Scherf for their gluten contents. 3.6.1 This test should only be carried out by trained laboratory employees. The instructions for use must be strictly followed. No quality guarantee is accepted after expiry of the kit (see expiry label). 3.6.2 Store the kit at 2-8°C (36-46°F). Let all kit components come to room temperature 20- 25°C (68-77°F) before use. Do not freeze any of the kit components. 3.6.3 Return any unused microwells to their original foil bag, reseal them together with the desiccant provided and further store at 2-8°C (36-46°F). The substrate/chromogen is light sensitive, therefore, avoid exposure to direct light. 3.6.4 Carefully dilute the components included in the kit as concentrates; avoid contaminations by airborne grain dust or dirty laboratory equipment. Wear gloves during the preparation and performance of the assay. Clean surfaces, glass vials, mincers and other equipment with 40% ethanol or 2-propanol. Carry out sample preparation in a room isolated from ELISA procedure. Check for gluten protein contamination of reagents and equipment. 3.6.5 Include ready to use standards in duplicate to each run of diluted sample extracts in duplicate. Do not reuse wells of the plate. Use separate pipet tips for each standard 3.6 General Preparation

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RIDASCREEN ® Total Gluten In-house validation report 2018-10-21