AOAC ERP Gluten Assays - Dec 2018

Annex B

4 Summary of Results and Discussion

4.1

Manufacturer’s In-house Study

4.1.1 General remarks The manufacturer’s in-house validation scheme followed the AOAC SMPR ® 2017.021, AOAC Appendix M (6), CLSI guideline EP25-A for stability testing of in-vitro diagnostics (7) and long- lasting practical experiences of the method developers from other AOAC approvals e.g. AOAC Performance Tested Method ℠ no. 101501 for RIDASCREEN ® FAST Milk (8). Selectivity Study: Antibodies and Analytical Targets The sandwich ELISA RIDASCREEN ® Total Gluten contains four different monoclonal antibodies. One of them is the R5 monoclonal which was raised against secalins. The specificity of this antibody is described elsewhere (9). The second antibody was raised against a HMW-GS peptide which contains the toxic sequence GYYPTS (10). The third and fourth antibodies were raised against an LMW-GS extract that was obtained from Katharina Scherf (Leibniz-Institute for Food Systems Biology at the Technical University of Munich, Germany). While the R5 and the HMW-GS antibody are coated on the microtiterplate and are in the conjugate (homologous system), two LMW-GS antibodies were used, because it was not possible to set up a homologous system. One of them is coated on the plate while the other one is in the conjugate. 4.1.2

Figure 1. Reactivity of the combination of all four antibodies against different fractions from wheat or rye or barley gluten; all fractions were tested at concentrations between 5 ng/mL and 80 ng/mL.

OD 450 nm

3.0

2.5

Wheat prolamins

Wheat LMW-GS

2.0

Wheat HMW-GS

Rye prolamins

1.5

Rye glutelins

Barley prolamins

1.0

Barley glutelins

0.5

0.0

0

20

40

60

80

100

Concentration (ng/ml)

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