AOAC ERP Gluten Assays - Dec 2018

Annex B

In the following, these antibodies are characterized for their binding activities against all fractions from gluten. These fractions were also obtained from Katharina Scherf (Leibniz- Institute for Food Systems Biology at the Technical University of Munich, Germany). The preparation is described elsewhere (11). Figure 1 shows the reactivity of the combined system, where three monoclonal antibodies (R5, HMW-GS antibody and LMW-GS antibody 1) are bound to the microtiter plate and three antibodies (R5, HMW-GS antibody and LMW-GS antibody 2) are in the conjugate. With the exception of barley glutelins (mainly consisting of D-hordeins) all prolamins from wheat, rye, and barley as well as all important glutelins are detected at a comparable level. In detail, prolamins from wheat, rye, and barley as well as wheat HMW-GS, wheat LMW-GS, and rye glutelins are measured at a comparable OD value. To investigate the reactivity of each single antibody in the system towards the different gluten fractions, homologous systems were produced for this experiment. As can be seen in figure 2, the R5 monoclonal antibody reacted as expected from the literature with barley prolamins, rye prolamins, and rye glutelins at a higher dose-response compared to wheat prolamins (gliadins). HMW-GS from wheat were also detected at a low dose-response. Glutelins from barley and wheat LMW-GS were not detected (not shown).

Figure 2. Reactivity of the R5 antibody system against different fractions from wheat or rye or barley gluten; all fractions were tested at concentrations between 5 ng/mL and 80 ng/mL; the R5 was coated to microtiter plates and used in the conjugate.

OD 450 nm

4.0

3.5

Wheat prolamins

3.0

Wheat HMW-GS

2.5

Rye prolamins

2.0

Rye glutelins

Barley prolamins

1.5

1.0

0.5

0.0

0

20

40

60

80

100

Concentration (ng/ml)

The main epitope of the R5 antibody is the pentapeptide QQPFP (12), which is highly repetitive on prolamins from wheat, rye and barley (Data base: UniProt). The γ-40k-secalins and γ-75k-secalins which are usually attributed to rye prolamins are also present in the rye glutelins fraction in high abundance (personal communication Dr. Katharina Scherf, Leibniz- Institute for Food Systems Biology at the Technical University of Munich, Germany). These

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