OMB First to Final Action Recommendation Checklist 11-12-18

ERP - GLUTEN ASSAYS: AOAC 2015.05 FAR

AOAC RESEARCH INSTITUTE AOAC OFFICIAL METHODS OF ANALYSIS (OMA) OMAMAN-19: R-Biopharm, Competitive Enzyme Immunoassay Based On The R5

Monoclonal Antibody To Determine Partially Hydrolysed Gluten In Foods Containing Wheat, Rye, And Barley Study Director: Dr. Markus Lacorn, R-Biopharm AG, An der neuen Bergstraße 17, 64297 Darmstadt, Germany/ Co- Study Director: Patricia Meinhardt, R-Biopharm Inc., 870 Vossbrink Drive, Washington, MO 63090 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 ER 3

ok

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. 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. 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 method shows good precision. Method will be helpful in hydrolyzed gluten (pepsin- trypsin digested) detection since there is no currently validated method available. 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. 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. 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 Page 4 of 10 Data tables still need to be in units of mg/kg gluten, not prolamins. None that I can point out in the revised manuscript.

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Cons/Weaknesses of the Manuscript: ER 1

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ER 5

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