Validation of the GlutenTox(®) ELISA Rapid G12 Test Kit for Determination of Gluten in Select Non-Heat-Processed Matrixes and Heat-Processed Matrixes: AOAC Performance Tested Method(SM) 042301

BACKGROUND: The GlutenTox(®) ELISA Rapid G12 test kit is a quantitative method designed for the determination of the immunotoxic fraction of gluten in food samples. OBJECTIVE: To obtain AOAC Performance-Tested Methods(SM) certification for the method for the detection and quantification of gluten from wheat, barley, and rye flours in select foods (non-heat-processed) and incurred (heat-processed) matrixes. METHODS: The method was evaluated following the Guidelines for Validation of Quantitative Gluten Methods, with Specific Examples for ELISA Assays. The validation study was conducted at Hygiena Diagnóstica España using five food matrixes (soy flour, corn bread, seasoning mix, rolled oats, and evaporated milk) artificially contaminated with gluten from wheat, barley, or rye flour at different concentrations: 0, 5, 10, and 20 mg/kg. For each matrix and gluten contamination level, five or six individually extracted test portions were analyzed. A second bread matrix was prepared by baking a gluten-free bread mix spiked at 0, 20, and 30 mg/kg gluten from wheat, barley, or rye flour for incurred matrix testing. Ten individually extracted test portions were tested for each incurred bread and contamination level of gluten. RESULTS: The method met the AOAC performance requirements for detection and quantification of wheat gluten in the selected food matrixes, incurred bread sample, and spike levels of wheat gluten, showing an acceptable recovery. When tested with barley and rye flours, most of the results showed acceptable recoveries or a slight overestimation, depending on the matrix and gluten concentration. Method developer and independent laboratory results were comparable. CONCLUSIONS: The validation study demonstrated that the test kit is a reliable, accurate, quick, and easy-to-use method for the detection and quantification of gluten concentration in food and incurred matrixes from wheat, barley, and rye flours. HIGHLIGHTS: Most reagents provided in the kit are at ready-to-use concentrations.