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ER 5
RIDASCREEN Gliadin competitive assay can be used for the analysis in fermented and
hydrolyzed foods (e.g. beer, starch syrup, malt extract, sourdough, soy sauce etc. The method
is based on enzyme immunoassay format using a monoclonal antibody that can react gliadin
derived from wheat and related prolamins derived from rye and barley. The antibody binds to
the potentially immunostimulatory amino acid sequence QQWPFP which exists as motifs on
all the prolamin subunits. The antibody detects intact and partially hydrolyzed prolamins. No
cross reactivity has been observed with non-gluten cereals and millets. The calibration curve
covers gliadin concentration in a sample of 10 to 270 mg/kg.
ER 6
As mentioned previously, the applicability is limited to the enzyme used for hydrolysis since
other hydrolysates are not used in the method validation study.
General comments about the method:
ER 1
The method has the potential to be of considerable use to the scientific community.
However, as multi-laboratory examined, it is at best misleading and unacceptable. There is
also a serious problem associated with the lack of a proper standard material and the
approach taken of mixing three different grain digests. This counter-productive to meaningful
future interpretations.
ER 2
I think it is ok. Fundamentally I'm ok with it, just a few nagging issues with the manuscript and
the statistics tables.
ER 3
The authors have addressed the difficulties encountered in reading the original study report
in this revision.
ER 4
Looking at the SLV or in-house study of the method, the R5 competitive comes out “pretty
good". Just based on such an SLV one could already strongly consider the potential of the
method for a 1st action. Next to the in-house validation there is data of the AACC
collaborative study. This data is good to have, but it did reveal some of the weaknesses when
the method is strongly challenged with difficult matrices. We see more variation and higher
LOD, these are all logical outcomes and collaborative studies are needed for a "full
acceptance" of a new method in most organizations (AOAC, AACC, CEN). Another point that
can be considered is that the AACC results were obtained in 2011. When after a 1st action the
final action takes place 2 yrs further the AACC collab document is from > 6 years ago.
ER 5
The method is useful in detecting partially hydrolyzed gluten in foods. The other available test
kits for gluten don't have the ability of this analysis. The validation of the method could not
establish its accuracy in the lack of availability of a certified reference material. The possibility
do exist that the assay could be biased in the lack of proof of its accuracy. The accuracy of the
method can be reduced by potential specific enzymes (i.e., proline specific endpetidases)
which may be present in fermented and hydrolyzed foods samples. There is a possibility that
activities of these types of enzymes may cause false negative. The manuscript states that 90%
of the secalins in rye sour dough was not detectable by the assay after fermentation. The lack
of an alternate method to estimate secalins in the fermented rye doesn’t allow establishing a
true level of secalins in this sample. The secalins were spiked in gluten free quinoa sourdough
by fortifying this sample with the fermented sour dough at the levels so that secalin
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