OMB Meeting Book - Jan. 11, 2018

5. Single Laboratory Validation An internal evaluation of spiked and incurred samples was conducted by Romer Labs ® in Tulln, Austria to confirm the performance of the G12 antibody-based sandwich ELISA test kit. Two different operators investigated heat-treated and non-heat treated samples with incurred gluten levels up to 124 mg/kg. In the validation it was shown that gluten could be accurately quantified by G12 Sandwich ELISA with reproducibility RSD(R) of 12.6% and repeatability RSD(r) of 10.8%. Tested samples showed a recovery between 89 % and 118%. Details are indicated in table 1.

Table 1: Performance statistics for overall G12 sandwich ELISA results

Gluten- free cookie

cookie powder mixture 1

cookie powder mixture 2

Incurred cookie

cookie dough

Nominal gluten content

0 mg/kg 106 mg/kg 102 mg/mg 124 mg/kg 124 mg/kg

No. of operators

2

2

2

2

2

Total No. of replicates Overall mean, mg/kg Repeatability SD, mg/kg Reproducibility SD, mg/kg Repeatability RSD, % Reproducibility RSD, %

18

17

18

18

18

1.63*

94.3

113.7

146.5

116.9

0.85 0.96 51.8 59.1

5.9 9.9 6.3

11.1 14.3

15.8 15.4 10.8 10.5

10.4 10.6

9.7

8.9 9.0

10.5

12.6

Bias (mg/kg) observed - nominal

1.63

-11.7

11.7

22.5

-7.1

Recovery, %=observed/nominal x 100

-

89.0

111.4

118.1

94.3

*result

Gluten-free blend flours were spiked with PWG gliadin delivered from the Working Group on Prolamin Analysis and Toxicity (Germany) at levels in the middle of the quantification range of the ELISA. The theoretical gluten concentration of the cookie materials was calculated according to the certified protein content of the PWG gliadin (van Eckert et al., 2006), considering the weight loss during heat treatment. The incurred materials were produced under the guidance of the Food Allergen Working Group of the Budapest University of Technology and Economics (Bugyi et al., 2012). Samples consisted of PWG gliadin, gluten-free blend flour, salt, sugar, sodium-bicarbonate, margarine and water. For measurements, two different cookie powder mixtures (without margarine), the lyophilized dough (with margarine, non-heat treated) and the baked cookies were used. Sample preparation, extraction, ELISA procedure and interpretation of results were conducted as described in the official method protocol.

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