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© Romer Labs, 2016
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
Incurred
cookie
cookie
dough
cookie
powder
mixture 1
cookie
powder
mixture 2
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
18
17
18
18
18
Overall mean, mg/kg
1.63*
94.3
113.7
146.5
116.9
Repeatability SD, mg/kg
0.85
5.9
11.1
15.8
10.4
Reproducibility SD, mg/kg
0.96
9.9
14.3
15.4
10.6
Repeatability RSD, %
51.8
6.3
9.7
10.8
8.9
Reproducibility RSD, %
59.1
10.5
12.6
10.5
9.0
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<LOD; results calculated by extrapolation of the calibration curve
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.