AOAC ERP Gluten Assays - Dec 2018

Annex B

Egg powder

0.143 0.106 0.161 0.161 0.161 0.161 0.106 0.228 0.228 0.143

0.414 0.429 0.426 0.426 0.426 0.426 0.429 0.445 0.445 0.414

0.208 0.133 0.142 0.181 0.171 0.175 0.172 0.208 0.166 0.191

Fig, dried fruit Orange juice

Pineapple, dried fruit

Papaya, dried fruit

Potato, flour

Skim milk powder Stabilizer xanthan Sugar beet sirup

Tea, black*

4.1.4 Ruggedness Study These experiments were undertaken to show the influence of parameters on test kit results. These parameters are known to be subject of variation during use of the test kit. The parameters tested for their ruggedness were, [ 1] extraction time at 50°C (first step in water bath), [2] water bath temperature for first incubation step, [3] incubation time for extraction for the second incubation step at room temperature, [4] pipetted volumes for standards and samples, [5] incubation time for all ELISA steps, [6] incubation temperature of the ELISA procedure, and [7] plate shift due to pipetting velocity. Table 3. Variation of water bath temperature from 40°C up to 60°C for extraction at 40 min extraction time (first extraction step); variation of incubation time from 30 min up to 50 min at 50°C extraction temperature (first extraction step); variation of incubation time from 50 min up to 70 min at room temperature (second extraction step); 3 different incurred and processed samples were tested; samples were incurred with wheat, or rye, or barley; results are given in mg/kg gluten. Temperature water bath 1 st Incubation time 2 nd Incubation time Control 40°C 60°C 30 min 50 min 50 min 70 min Sample A (wheat) 33.9 31.2 33.3 29.2 28.7 28.2 30.1 Sample B (rye) 39.0 34.4 36.6 35.4 36.7 38.7 38.6 Sample C (barley) 34.3 31.8 33.8 36.4 41.4 33.3 37.7 Table 4. Variation of pipetted volumes for all pipetting steps from 90 µl to 110 µL; variation of incubation time from 18 min to 22 min for the first and second ELISA incubation step combined with variation of incubation time from 9 min to 11 min for the third ELISA incubation step; variation of incubation temperature from 20°C to 30°C for the whole ELISA procedure; 5 different samples were tested; samples contained wheat, or rye, or barley; results are given in mg/kg gluten. Control 90 µl 110 µl 18 min 22 min 20 °C 30°C Sample A (blank) 2.24 3.29 2.9 2.42 2.9 2.68 1.09 Sample B (wheat) 23.6 28.4 24.5 22.0 24.5 23.3 24.0 Sample C (barley) 31.2 27.7 27.9 27.6 27.9 24.8 42.2 Sample D (wheat) 38.1 39.1 38.1 33.0 38.1 39.6 40.1 Sample E (rye) 33.0 31.9 32.7 29.0 32.7 32.2 39.7

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RIDASCREEN ® Total Gluten In-house validation report 2018-10-21