205 / 328
concentration in spiked sample is calculated to be 35 and 75 mg/kg. In the absence of true
value of secalins in the fermented sourdough the spike values as well as the spike recoveries
calculated in these spike materials remain questionable.
ER 6
The method is a good initiative towards hydrolysed gluten detection in foods and uses well
characterized R5 antibody in the competitive ELISA.
Pros/Strengths of the Manuscript:
ER 1
Pro: uses the R5 monoclonal antibody. R-biopharm is a respectable company.
ER 2
ok
ER 3
While the authors clarify that the accuracy of the test cannot be determined, they indicate
however, that the method is precise and is therefore suitable and fit-for-purpose.
ER 4
The strength of the manuscript is that it shows that the R5 competitive ELISA is suited for
partially hydrolyzed gluten. The reported AACC collab study published in CFW also shows that
the method sufficiently met guidelines on recovery and LOD when challenging matrices were
used (incurred vs spiked). The manuscript is very technical, but correct. The manuscript
describes the current state-of-the-art in detecting hydrolyzed gluten. On the other hand the
collab challenge also revealed a weakness - the AACC collab demonstrated high RSDs.
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.
ER 6
The method shows good precision. Method will be helpful in hydrolyzed gluten (pepsin-
trypsin digested) detection since there is no currently validated method available.
Cons/Weaknesses of the Manuscript:
ER 1
Cons: work as presented is very misleading. Gluten has a specific target level making the
design of quantitative analytical methods straightforward. The validation should have focused
on 20 ppm and bracketed this concentration. The use of a mixture of wheat, barley, and rye
hydrolysate as a standard makes meaningful /accurate quantification impossible. The food
samples should have been made with incurred gluten and subjected to processing/hydrolysis
versus spiking with arbitrarily pre-hydrolyzed gluten. Discussion of these limitations might
help, but none presented.
ER 2
Data tables still need to be in units of mg/kg gluten, not prolamins.
ER 3
None that I can point out in the revised manuscript.
ER 4
The very technical style does make the manuscript not easy to read. Accuracy and/or high RSD
may be identified as weakness - a high lab to lab variation is there. There are however no strict
criteria for the allowed RSDs or Horrats of ELISA methods, by definition - e.g. due to the
complexity of the analyte (no single molecule) it will fall typically in a Type I. The AOAC
guidelines & best practices focus on LOD and recovery of allergen ELISAs. Concerns about
high RSD could be valid, but allowing a gluten ELISA with relatively high RSD in AOAC
methods is not unprecedented: AOAC 991.19 (2001) for intact gluten in foods.
ER 5
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
ERP PROFILE SUMMARIES
169