AOACRIGlutenMethods-2017Awards

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

90 mL distilled water). Wash buffer may be used within 1 week, if stored at 4°C. F. Sample and Test Portion Preparation Obtain a representative sample and homogenize a minimum of 5 g in a mortar or blender as fine as possible. Weigh out 0.25 g of homogenized sample into a vial with a minimum 10 mL capacity, which can be tightly sealed. For chocolate-containing samples, additionally add 0.25 g of powdered fish gelatin. Add 2.5 mL extraction solution (under a fume/chemical hood), close vials, and mix vigorously on a vortex. Visually check for clumps, and continue mixing until samples are well dispersed in the extraction solution. Incubate at 50°C (122°F) for 40 min in a water bath. Allow the extracts to cool to room temperature and add 7.5 mL of 80% ethanol; mix well. Shake for a total of 60 min at room temperature (20–25°C/68–77°F) with a rotary shaker. (After about 30 min in the rotator, check the vials visually if all sample material has suspended in the liquid. If clumps have formed, vortex and let the vials rotate for the second 30 min to complete the extraction procedure). Centrifuge samples for 10 min at 2000 × g to obtain a clear aqueous layer between the particulate sediment and supernatant. Note, in some cases, a thin fatty layer creaming on top of the supernatant. Collect the aqueous supernatant (extract) and transfer into a new vial. Dilute supernatant at least 1:10 (0.1 + 0.9 mL) with prediluted sample dilution buffer (depending on the expected prolamin content of the sample). If prediluted samples are not immediately used for determination by ELISA, close vials and keep in the dark at room temperature (20–25°C/68–77°F) for a maximum of 7 days until ELISA experiments. Bring all reagents to room temperature (20–25°C, 68–77°F) before use. Use dilution of the sample extract to carry out ELISA experiments. Run standards and diluted sample extracts in duplicate. Place an appropriate number of antibody-coated microwells in a microwell strip holder. Record standard and sample positions. Using a single-channel pipettor, add 100 µL of each ready-to- use standard or prepared sample into the appropriate well. Use a fresh pipet tip for each standard or sample. Make sure the pipet tip has been completely emptied. 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 conjugate from the green-capped bottle (about 120 µL/well or 1 mL/strip) and place in a separate container (e.g., reagent boat when using the G. Determination (Assay)

10, 50, 200 and 500 ng/mL gluten, calibrated to the WGPAT gliadin (86% highly purified gliadin from 40 different European wheat varieties). ( c )  Conjugate solution (peroxidase-labeled antibody, ready- to-use) .—One bottle containing 13 mL. ( d )  Substrate solution (stabilized peroxide substrate and 3,3 ′ ,5,5 ′ -tetramethyl-benzidine in a dilute buffer solution) .— 15 mL. ( f )  Diluent buffer .—One bottle containing 20 mL of 5× concentrated diluent buffer. Contains a final concentration of 20 mM PBS-Tween (0.9% sodium chloride, 0.07% Tween 80) with 0.25% fish gelatin (Sigma) and 0.01% Proclin as a preservative. ( g )  Wash buffer .—One bottle containing 60 mL of 10× concentrated wash buffer. Contains a final concentration of 20 mM PBS-Tween (0.9% sodium chloride, 0.05% Tween 20) with 0.01% Proclin as preservative. ( h )  Extraction solution .—One bottle containing 105 mL of ready-to-use proprietary extraction solution containing reducing agents. ( i )  Fish gelatin .—One sachet containing 10 g. Additional reagents needed, but not provided with the test kit: ( a )  Distilled or deionized water . ( b )  Ethanol .—80% (v/v). D. General Instructions Due to the sensitivity of the assay, a gluten-free environment must be maintained. It is preferable to perform the assay in a separate room from that used for sample preparation and extraction. Make sure balance and the surrounding space, as well as equipment such as spatulas, are clean. Cleaning can be done by using a 70% alcoholic solution. Spatula should be cleaned after each sample weighing by a 70% alcoholic solution. Store kit at 2–8°C (35–46°F) and let all components equilibrate to 20–25°C (68–77°F) before use. Include ready-to-use standards in duplicates to each run of samples. Use separate pipet tips for each standard and each sample extract to avoid cross-contamination. It is recommended that an eight-channel pipettor is used to perform the assay. No more than 48 samples and standards total should be run in one experiment when using an eight-channel pipettor (24 when samples and standards are added in duplicate, e.g., six test strips). If using only single-channel pipets, it is recommended that no more than a total of 16 samples and standards are analyzed in one experiment (eight when standards and samples are added in duplicate, e.g., two test strips). E. Preparation of Components Delivered with the Kit ( a )  Sample dilution buffer .—Dilute diluent buffer concentrate 1:5 with distilled water (e.g., add 20 mL of concentrated diluent buffer to 80 mL distilled water). Dilution buffer may be used within 24 h, if stored at 4°C. ( b )  Wash buffer .—If a precipitate is formed during storage of the wash buffer concentrate, the concentrate should be warmed up until it is dissolved. Dilute wash buffer concentrate 1:10 with distilled water (e.g., add 10 mL of concentrated wash buffer to One bottle containing 15 mL. ( e )  Stop solution (1 N H 2 SO 4 ) .—One bottle containing