AOAC ERP Gluten Assays - Dec 2018

Table 3. Performance characteristics using data from quadratic curve fitting; data from 19 participants with 21 different samples are divided into part A (samples 1-10) and part B (samples 11-21); for raw data from each participant including outlier calculation refer to table A-3 and table A-4 in Annex A

Part A / sample*

1

2

3

4

5

6

7

8

9

10 19 38

n Labs

19 38

18 36

18 36

18 36

18 36

19 38

16 32

19 38

19 38

N Replicates Mean; mg/kg s(r); mg/kg s(R); mg/kg

9.9

13.2 2.12 2.22 16.1 16.9

10.4 1.74 2.34

1.1

63.6 4.40 6.35

35.2 5.32 8.02 15.1 22.8

3.5

9.1

34.2 7.03 8.31

50.0 7.19 7.93 14.4 15.8

2.43 2.43 24.5 24.5

1.19 1.21

0.70 2.45

9.26 9.53

RSD(r), % RSD(R); %

16.7 111.1

6.9

20.0 102.2 20.5 70.4 105.2 24.3

22.4 113.6 10.0

*for decoding see table 2

Part B / sample*

11 19 38

12 19 38

13 19 38

14 19 38

15 17 34

16 19 38

17 19 38

18 19 38

19 19 38

20 19 38

21 17 34

n Labs

N Replicates Mean; mg/kg s(r); mg/kg s(R); mg/kg

2.5

21.2 6.44 6.92 30.4 32.7

15.8 4.46 5.52 28.3 35.0

28.1 6.47 7.80 23.0 27.8

1.7

5.5

12.4 5.76 6.59 46.6 53.3

22.6 6.64 7.78 29.4 34.4

6.4

13.0 3.09 3.39 23.8 26.2

19.7 1.98 3.15 10.1 16.0

1.37 1.74 54.5 69.1

1.99 1.99

2.92 3.39 52.6 61.0

1.90 2.31 29.9 36.3

RSD(r), % RSD(R); %

115.3 115.3

*for decoding see table 2

D. Principle

The basis of the test is the antigen-antibody reaction. The wells of the microtiter plate are coated with specific monoclonal antibodies against gluten proteins. By adding the standard or sample solution to the wells, present gluten proteins will bind to the specific antibodies. The result is an antibody-antigen complex. In a washing step components not bound are removed. Then antibodies conjugated to peroxidase (enzyme conjugate) are added. This antibody conjugate is bound to the antibody-antigen- complex. An antibody-antigen-antibody-complex (sandwich) is formed. Substrate/chromogen is added after removal of any unbound enzyme conjugate in a washing step. Bound enzyme conjugate converts the chromogen into a blue product. The addition of the stop solution leads to a color change from blue to yellow. The measurement is made photometrically at 450 nm against air. The absorption is proportional to the gluten protein concentration in the sample.

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