AOACRIGlutenMethods-2017Awards

H albmayr -J ech et al .: J ournal of AOAC I nternational V ol . 98, N o . 1, 2015  109

eight-channel pipettor). Using an eight-channel pipet, dispense 100 µL of conjugate into each well. Incubate at room temperature (20–25°C, 68–77°F) for 20 min. Empty the contents of the microwell strips into a waste container. Wash by filling each microwell with diluted wash buffer, and then emptying the buffer from the microwell strips. Repeat this step four times for a total of five washes. Take care not to dislodge the strips from the holder during the wash procedure. Lay several layers of absorbent paper towels on a flat surface and tap microwell strips on towels to expel all of the residual buffer after the fifth wash. Dry the bottom of the microwells with a dry cloth or towel. Measure the required amount of substrate from the blue-capped bottle (about 120 µL/well or 1 mL/strip) and dispense into a separate container (e.g., reagent boat for an eight-channel pipettor). Pipet 100 µL of the substrate into each microwell using an eight-channel pipettor. Incubate at room temperature (20–25°C, 68–77°F) for 20 min in the dark. Measure the required amount of stop solution from the red-capped bottle (about 120 µL/well or 1 mL/strip) and dispense into a separate container (e.g., reagent boat for an eight-channel pipet). Pipet 100 µL of stop solution into each microwell using an eight-channel pipettor. The color should change from blue to yellow. Eliminate air bubbles prior to reading wells as they are likely to affect analytical results. Read the absorbance of wells with a microwell reader using a 450 nm filter. Record OD readings for each microwell. I. Calculations Use unmodified OD values or OD values expressed as a percentage of the OD of the 200 ppm standard to construct a dose-response curve using the five standards (0, 4, 20, 40, and 200 ppm gluten). Gluten concentration given for the standards already consider sample preparation and 1:10 dilution according to method protocol. Gluten concentrations of samples can be calculated by interpolation from this standard curve using a point-to-point calculation. If a sample contains gluten levels higher than the highest standard (>200 ppm), the sample extract should be further diluted with dilution buffer such that the diluted sample results are in the range of 4 to 200 ppm and reanalyzed to obtain accurate results. The dilution factor must be included when the final result is calculated. J. Criteria for Acceptance of Standard Curve An example for the calibration curve is shown in the Certificate of Analysis included in each test kit. Higher OD values of the absorbance at 450 nm compared to the certificate may indicate insufficient washing or gluten contamination. For samples showing OD values higher than the 200 ppm standard, a further dilution and repeated analysis is recommended. The additional dilution factor must be taken into consideration during calculation. H. Reading

Any coloration of the substrate solution prior to the analysis or OD value of less than 1.1 absorbance units for 200 ppm standard may indicate instability or deterioration of reagents.

Collaborator´s Comments

Participants were following the instructions and the study coordinator did not receive any comments that changes to the procedure had been made. One laboratory reported that the test kit was not cold on arrival, but the results could still be used.

Results and Discussion

Two laboratories returned result sheets that could not be used. This was due to high CV in calibration duplicates and incomplete result sheets. Negative results that were reported