AOAC ERP Gluten Assays - Dec 2018

Annex B

Table 3 shows that there is no consistent tendency or trend in the results when extraction parameters were varied within an expectable range, regardless of the contaminating grain Table 4 shows that there is no consistent tendency or trend in the results when ELISA parameters were varied within an expectable range. The reaction of every sandwich ELISA starts with the addition of the first standard / sample. In case that many samples are pipetted onto the same plate, a considerable time shift (different effective incubation times on the plate) can occur between the first well and the last well. For the plate shift experiment, the same sample extract was pipetted on a whole plate, including a standard curve. Standards and the sample extract were pipetted using a normal pipet. The tip for the pipet was exchanged and flushed after every two wells, simulating the pipetting of different sample extracts and standards in duplicates. Each single well with sample extract was evaluated separately (see table 5). Table 5. Pipetting scheme (vertical) for characterization of a shift of results due to pipetting velocity. 1 2 3 4 …. 12 A STD 1 STD 1 sample 5 sample 6 ….. sample 70 B STD 2 STD 2 sample 7 sample 8 …. sample 72 C STD 3 STD 3 sample 9 sample 10 …. sample 74 D STD 4 STD 4 sample 11 sample 12 …. sample 76 E STD 5 STD 5 sample 13 sample 14 …. sample 78 F STD 6 STD 6 sample 15 sample 16 …. sample 80 G sample 1 sample 2 sample 17 sample 18 …. sample 82 H sample 3 sample 4 sample 19 sample 20 …. sample 84 Figure 5 shows the graphical analysis of the plate shift experiment. Overall the plate shift due to the pipetting velocity is quite low. From the formula within figure 5, it can be calculated that 17 mg/kg gluten is achieved after about 42 replicates when the true concentration of the sample was 20. Due to the fact that the method developers advise to use a pre-plate in case of more than 3 stripes, the plate shift effect is negligible. The same is true for an experiment with a horizontal pipetting direction (results not shown).

mg/kg gluten

30

25

y = -0.0727x + 20.045

20

15

10

5

0

0

12

24

36

48

60

72

84

replicate (no.)

Figure 5. Results (mg/kg) from vertical pipetting scheme for one sample extract in 84 single replicates using a micropipette with tip exchange and tip flushing before each duplicate.

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