AOACRIGlutenMethods-2017Awards

Lacorn & Weiss.: J ournal of AOAC I nternational V ol. 98, N o . 5, 2015  1349

for the analysis of fermented and hydrolyzed food (e.g., beer, starch syrup, starch, malt extract, sourdough, and soy sauce) that are declared as “gluten-free.” The kit is not applicable for measurement of intact gluten.] Caution : Stop solution contains 0.5 M sulfuric acid; avoid skin and eye contact ( see Material Safety Data Sheet). The method is based on an enzyme immunoassay format using a monoclonal antibody that can determine hydrolyzed gluten derived from wheat, rye and barley. The antibody binds to the short amino acid sequence QQPFP and to related sequences, which exist as motifs on all the prolamin subunits (9). Some of these sequences are potentially celiac immuno-stimulatory (10, 11). Since the assay is calibrated to a prolamin hydrolysate mixture form wheat, rye, and barley, a conversion to “gluten” content is achieved by the conversion factor of 2 set by the Codex Alimentarius. No cross-reactivity has been observed to oats, maize, rice, millet, teff, buckwheat, quinoa, or amaranth. Protein fragments for gluten measurement from food are extracted by using ethanol. After centrifugation, the supernatant is used in a competitive method. The basis of the test is the antigen-antibody reaction. The microtiter wells are coated with a constant amount of gliadin. Standards (mixture of hydrolysates from wheat, rye, and barley prolamins) or sample solutions are pipetted, and peroxidase labeled antigliadin antibody (conjugate with monoclonal R5 antibodies) is added and incubated for 30 min. During incubation, free and immobilized analyte competes for the antibody binding sites (competitive enzyme immunoassay). Any unbound enzyme conjugate is then removed by a washing step. Substrate/chromogen is added to the wells and incubated for 10 min. Bound enzyme conjugate converts the chromogen into a blue product. Addition of the stop solution causes a color change from blue to yellow. The measurement is performed photometrically at 450 nm. The absorption is inversely proportional to the gluten concentration. The response of sample extracts is compared with response observed with calibrators. A. Principle

B. Apparatus Apparatus specified here has been tested in the laboratory; equivalent apparatus may be used. ( a )  Laboratory mincer/grinder, mortar and pestle, or Ultra- Turrax. —e.g., Mr. Magic, ds-produkte GmbH, Gallin, Germany. ( b )  Rotator or shaker. —e.g., Roto Shaker Genie (Scientific Industries Inc., Bohemia, NY). ( c )  Centrifuge .—e.g., Minifuge RF, Kendro, Hanau, Germany. ( d )  Microtiter plate reader .—e.g., Tecan Sunrise Remote (Tecan Group, Maennedorf, Switzerland). ( e )  Micropipets .—Variable 20–200 µL and 200–1000 µL. ( f )  Graduated pipets . ( g )  Graduated cylinders .—Up to 1000 mL, plastic or glass. ( h )  Centrifugal glass vials with screw tops . C. Reagents Items ( a )–( g ) are available as a test kit (RIDASCREEN ® Gliadin competitive, R-Biopharm AG). All reagents are stable at least over a period of 15 months at 2–8°C (36–46°F) from the date of manufacture. Please refer to the kit label for current expiration. ( a ) Microtiter plate .—Coated with gliadin (96 wells). ( b ) Five standard solutions .—Labeled 0, 20, 60, 180, and 540 ng/mLgluten, 1.3 mL each; ready to use, transparent-capped bottles. ( c ) Conjugate.— Horseradish peroxidase labeled R5 antibody; 0.7 mL, as an 11-fold concentrate, red-capped bottle. ( d )  Red Chromogen Pro.— Substrate/chromogen; 10 mL, ready to use, brown-capped bottle. ( e ) Stop solution .—14 mL, ready to use, yellow-capped bottle. ( f )  Sample diluent.— 60 mL, as a 5-fold concentrate, white-capped bottle. ( g )  Washing buffer .—100 mL, as a 10-fold concentrate, brown-capped bottle. Necessary or recommended but not provided with the test kit: ( h ) Distilled water . ( i ) Ethanol .—99% reagent grade. ( j ) Fish gelatin.— Sigma, St. Louis, MO; Part No. G-7765 or Serva, Heidelberg, Germany; Part No. 22156.

Table 2015.05. Performance statistics for overall competitive R5 ELISA results without outlier (gluten concentrations are shown) Sample ID a Symbol 1 2 3 4 5 6 7 Total No. of labs p 13 12 11 13 13 13 13 Total No. of replicates Sum(n(L)) 26 24 22 26 26 26 26 Overall mean of all data (grand mean), mg/kg XBARBAR 2.36 26.2 119.5 1.29 10.6 48.4 145.6 Repeatability SD, mg/kg s r 2.31 7.92 37.2 2.03 1.73 11.2 28.4 Reproducibility SD, mg/kg s R 2.98 9.67 37.2 3.05 3.65 12.5 40.0 Repeatability RSD, % RSD r 98.0 30.2 31.2 157.3 16.3 23.1 19.5 Reproducibility RSD, % RSD R 126.1 36.8 31.2 236.1 34.4 25.9 27.5 Recovery, % — b 87 119 — — 69 97 a See Table 1. b  — = Not applicable.