AOAC ERP Gluten Assays - Dec 2018

Annex B

and each sample extract to avoid cross-contamination and pre-flush the tip before pipetting standard or sample extract. Use a multistepper pipet for adding the conjugate, substrate/chromogen and stop solution. Use a single tip for each of these components. 3.6.6 In case that more than three microtiterplate strips (24 wells) are necessary, a second uncoated plate should be used as a pre-plate. Standards and samples are quickly transferred to the coated microtiter plate with an 8-channel pipette. 3.6.7 Components and procedures of the test kit have been standardized for use in this procedure. Do not interchange components between kits of different batches (lot numbers). 3.6.8 Store samples in a cold and dry room protected from light. Ensure that no cross- contamination takes place. 3.6.9 If necessary, check for gliadin contamination of reagents and equipment with the RIDA ® QUICK Gliadin (Art. No. R7003). 3.6.10 Keep in mind that solid samples can be inhomogeneous, therefore ground a representative part of the samples very well and homogenize before weighting. 3.7.3 Buffer -- The buffer (see 3.1.9) is ready to use. Bring the solution to room temperature (20-25 °C; 68-77 °F) before use. Make sure that the buffer is not contaminated with gluten during use. 3.7.4 Sample dilution buffer for further dilution -- Use a solution consisting of 1% Cocktail (patented), 3% of 80% ethanol, and 96% buffer, e.g. 50 µL Cocktail (patented), 150 µL 80% ethanol and 4800 µL buffer. Do not use the diluted samples that were already measured for further dilution since the diluted samples are stable for 30 min only. Re- start the dilution using the extract obtained after filtration. Dilute this extract afresh 1:25 (see 3.8.7) with buffer. For additional dilution, use the sample dilution buffer for further dilution. 3.7.5 Washing buffer -- Provided as a 10-fold concentrate (see 3.1.10). Before use, the buffer has to be diluted 1:10 (1+9) with distilled water (i.e. 100 mL buffer concentrate + 900 mL dist. water). Prior to dilution, dissolve possibly formed crystals by incubating the buffer in a water bath at 37 °C (99 °F). The diluted buffer is stable at 20 - 25 °C (68 - 77 °F) for four weeks. 3.7 Preparation of components 3.7.1 80% aqueous ethanol -- Add 200 mL ethanol to 50 mL distilled water and shake well. 3.7.2 Cocktail (patented) -- The Cocktail is ready to use (see 3.2.8)

3.8

Sample Preparation

3.8.1 Weigh a representative amount (200 g) of oats or oat products and homogenize.

3.8.2 Weigh 1 g ± 0.01 g of homogenized sample to a 50 mL centrifuge tube. Add 10 mL Cocktail (patented), cap the tube vial, mix vigorously, and pay attention to obtain a homogenous suspension. For tannin- and polyphenol-containing matrices (e.g. chocolate, coffee, cocoa, chestnut flour, buckwheat, teff flour, millet or spices) add 1 g gluten-free skim milk powder prior to addition of the 10 mL Cocktail (patented).

3.8.3 Add 30 mL 80 % ethanol, close the tube and mix well. Incubate for 40 min at 50 °C in a water bath.

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