AOAC Final Action Methods in 2018

0.07% Tween 80) containing 0.25% fish gelatin (Sigma) to 0, 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) .—One bottle containing 15 mL. ( e )  Stop solution (1 N H 2 SO 4 ) .—One bottle containing 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: ( j )  Distilled or deionized water . ( k )  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 90 mL distilled water). Wash buffer may be used within 1 week, if stored at 4°C.

Table 2014.03B. Cross-reactivity of the G12 antibody (G12 antibody shows no cross-reactivity to various nuts, oils, seeds, starches, or gluten-free grains)

Romer extraction solution, mg/kg gluten Gluten, %

Food category

Food sample

Gluten-containing  grains

Wheat flour

72222

7.2

Barley (Cumion) Durum wheat Spelt (Ostro) Rye (Capitan)

292390 15733 81926 41577

29.2

1.6 8.2 4.2

Naturally gluten-free  grains

Soya bean

<4

Soya mince Buckwheat

<4 <4 <4 <4 <4 <4 <4

Rice flour

Quinoa

Corn kernels

Teff flour

Millet

Oats

Bastion

4.3 7.4

00-61 Cn

Brachan

<4

Husky

6.3 6.6

Fusion Pecan Walnut Almond Cashew

Nuts

<4 <4 <4 <4 <4 <4 <4 <4 <4 <4 <4 <4 <4 <4 <4 <4 <4 <4 <4 <4 <4 <4

Macadamia

Peanut

Hazelnut Pine nut Pistachio

Seeds

Golden linseed Brown linseed

Poppy

Sesame Mustard

Oils

Hazelnut oil

Walnut oil

Vegetable oil Sunflower oil Tapioca starch Wheat starch Potato starch

Starches

Miscellaneous

Amaranth

© 2018 AOAC INTERNATIONAL