AOAC ISPAM Stakeholder Panel Meeting Book (August 26, 2018)

Our concept is that this experiment will be performed by the kit developer as part of the single-laboratory validation (SLV) and tested independently by a third-party laboratory. This would be performed on oat flour samples only as a test of kit/antibody response. We anticipate method precision will need to be demonstrated on each claimed matrix by SLV and multilaboratory experiments as required by the SMPR. In addition, developers will also be required to show ruggedness, inclusivity, and exclusivity as described in the references (1, 2). We anticipate procurement of materials for spiking here at General Mills for gluten-free oat flour and also most contaminant grains. We hope to develop a series of test materials which will be made available to all kit developers to evaluate proposed kit response. We should note here that, in the past, it was common to use purified protein materials as spiking agents to assess the recovery of kits. Previous Official Methods SM had used the PWG gliadin standard as spiking material, with good success. In this project, it will be difficult to obtain similar materials for hordein and secalin, but in this case, we feel it appropriate to use unextracted grains as spiking materials. We feel this recovery experiment should be a demonstration of both the antibody response and the extraction efficiency—it is a validation of the entire kit, not just the antibodies. As such, it is better to demonstrate the ability of the method to extract and detect the proteins from grain materials in their native and cooked states. Gluten-Free Oat Flour We have obtained very low gluten-free oat flour for spiking materials by mechanically cleaning oats and selecting the fraction expected to be lowest in gluten. For spiking purposes, we look for flour lots with an observed analytical mean <1 ppm gluten as tested by the R5 ELISA method, 18 replicates at 5 g test portion. Flour at this level is deemed low enough to be used in spiking experiments. Contaminant Grains As we have encountered in previous work, the decision on what contaminant grains to use in spiking studies is controversial. The age-old question is whether or not to use a single cultivar, or use a blend of common cultivars which are widely planted in the region. We have obtained several common rye and barley cultivars currently planted in North American oat regions for study. At the guidance of the working group, we are willing to blend these cultivars or use them unblended to make spiking materials. See section below for Spiking Materials for Barley and Rye for a list of cultivars and how they were chosen. For wheat spiking material, we were anticipating using the MoniQA wheat gluten flour material, but as yet, that has not been made available to us. If time is critical, we would be willing to source common wheat cultivars from our wheat breeder contacts and perform similar operations to develop a blend of wheats to use as spiking materials. Characterizing the Contaminant Grains We anticipate grinding each cultivar independently, and then weighing together appropriate masses of flour to make the spiking blend. After the composite blend is made, it will need to be tested for gluten content. The gluten estimate of the spiked material will be necessary to calculate recovery. We have several ideas on how to do this. We hesitate to refer to this step as a “reference” method in fear that whatever the working group decides as the best way to accomplish this will enshrine this method as the best or only way to do this type of characterization. We don’t want to establish a precedent on the ideal method, and would only use this as a way of calculating recovery. Possible methods for estimating gluten in grain samples: ( 1 ) Method of Wieser et al. (3) using RP-HPLC after selective Osborne-like extraction. Katharina Scherf, research fellow at the German Research Center for Food Chemistry, Leibniz Institute, Germany, has been using this method to characterize grains. The method is well-established, but requires HPLC. ( 2 ) We have tried water and NaCl extraction to remove soluble proteins (by definition non-gluten by Codex) followed by centrifugation, and then Dumas nitrogen on the remaining pellet. Our results have been similar to the Wieser results. Gluten = Dumas N 2 *5.68

Table 1. Spike data for C series samples

Gluten spike ppm Dumas 0.4 M NaCl in PBS

Gluten spike ppm Dumas water/NaCl

Gluten spike ppm HPLC R5 ELISA ppm

Sample Grain Cultivar Mass flour, g Mass spike, g Calc ppm grain

B100C Barley Jalpur B200C Barley Jalpur

1000 1000 1000 1000 1000 1000

0.1014 0.2014 0.1027

101.4 201.4 102.7 201.0 102.2 201.9

5.88

5.67

3.96 7.87 7.83

23.91 39.23 11.17 33.57 16.34 29.86 0.958

11.68

11.26

W100C Wheat W200C Wheat R100C Rye R200C Rye

Mix Mix

8.22

7.52

0.201

16.08

14.71

15.33

Holo Holo

0.1022 0.2019

3.98 7.87

3.34 6.60

2.36 4.66

Unspiked

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