AOAC ERP Gluten Assays - Dec 2018

Annex B

proteins contain the QQPFP epitope and are thus also detected by the R5 antibody. Barley glutelins, LMW-GS, HMW-GS from wheat and HMW-secalins from rye (attributed to the rye glutelins fraction) do not contain the QQPFP epitope. The low response to HMW-GS from wheat might be due to some impurity of the fraction or to a minor cross-reactivity. The monoclonal antibodies that were raised against wheat LMW-GS, show a high reactivity towards LMW-GS but also reactivity towards wheat prolamin (fig. 3) in the heterologous system. All other fractions were not detected (not shown). The exact epitopes of the LMW-GS antibodies are unknown. The protein sequences from LMW-GS are quite different to all other gluten proteins (Data base: UniProt). The low response to prolamins from wheat might be due to some impurity of the fraction or to a minor cross- reactivity. Figure 3. Reactivity of the LMW-GS antibody system against different fractions from wheat; all fractions were tested at concentrations between 5 ng/mL and 80 ng/mL; the LMW-GS antibody 1 was coated to microtiter plates and LMW-GS antibody 2 was used in the conjugate.

OD 450 nm

4.0

3.5

3.0

Wheat prolamins Wheat LMW-GS

2.5

2.0

1.5

1.0

0.5

0.0

0

20

40

60

80

100

Concentration (ng/ml)

The homologous system based on the HMW-GS monoclonal antibody shows the highest reactivity towards the wheat HMW-GS fraction, followed by rye glutelins and prolamins from wheat and rye (fig. 4). All other fractions were not detected (not shown). The HMW-GS antibody was raised against a peptide containing the GYYPTS sequence. It is assumed that this is also the main epitope of this antibody. This sequence is repetitively present on HMW-GS from wheat and HMW-secalins from rye (Data base: UniProt), which are attributed to the rye glutelins fraction. The low response to prolamins from wheat and rye might be due to some impurity of the fractions or to a minor cross-reactivity.

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