AOAC ERP Gluten Assays - Dec 2018

Annex B

3.9.10 Add 100 μL of the stop solution (see 3.1.8) to each well. Mix gently by shaking the plate manually.

3.10

Reading

3.10.1 Read the results with a microtiter plate reader. Measure the absorbance at 450 nm. Read within 10 minutes after addition of stop solution. 3.10.2 The dilution factor 1000 which results after sample preparation has already been considered for the standard concentrations. The concentration of the sample can directly be read from the standard curve. 3.10.3 A further dilution and new detection of samples is necessary for absorbance readings (A450 nm) > standard 6. Please follow instructions given in 3.8.8. Do not use diluted samples that were already measured for further dilution.

3.11 Calculations

3.11.1 Determine the gluten content of each duplicate sample wells by reference to a calibration curve measured by the actual test run utilizing special computer software; plot absorbance of standards vs gluten content of standards. It is recommended to using the RIDA ® SOFT Win (R-Biopharm AG, Z9999) with 4-parameter logistic regression analysis (5).

3.12 Criteria for Acceptance of Standard Curve

3.12.1 The course of the standard curve is shown in the Quality Assurance Certificate enclosed in the test kit. Absolute absorbances may vary between different runs (e.g. due to different temperatures or analysts). However, the shape of the standard curve should be similar to the one given in the Quality Assurance Certificate. Minimum requirements are: 3.12.2 OD at 450 nm for standard 6 higher than 1.2 3.12.3 OD values for standards should continuously increase with higher concentrations, especially when comparing standard 1 (0 mg/kg) and standard 2 (5 mg/kg) 3.12.4 An OD value for standard 1 which is much higher than the OD value stated in the certificate, could be an indication for errors during pipetting or incubation or contamination. 3.12.5 For a trained technician, coefficients of variations (CVs) for duplicates of standards and samples on the plate are usually less than 10% except for standard 1 (0 mg/kg) where CVs up to 30% can frequently occur. 3.12.6 All requirements should not be judged isolated but as a whole; if one criterion does not fit to the above-mentioned criteria, it is recommended not to repeat the analytical run directly but check for pipetting errors and results from control samples first.

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