AOAC ERP Gluten Assays - Dec 2018

Annex B

4.2 Independent Validation Studies

External lab not applicable. See OMA manuscript for data from collaborative test.

5 Conclusion The sandwich ELISA RIDASCREEN ® Total Gluten (R-Biopharm, R7041) was characterized during an in-house validation study at the method developer’s laboratory. This was part of the AOAC OMA process to obtain OMA First Action status. The accompanying collaborative test is described in a different document. The test kit employs a combination of four monoclonal antibodies including the R5 to quantify intact (non-hydrolyzed) gliadins and related prolamins from rye and barley, High-Molecular Weight (HMW) Glutenin-Subunits (GS) from wheat, HMW-secalins from rye and Low- Molecular-Weight (LMW)–GS from wheat. Samples are extracted by Cocktail solution/80% ethanol and analyzed within 50 minutes. The measurement range is between 5 mg/kg gluten and 80 mg/kg gluten using a calibrator made out of a gluten extract from a mixture of four wheat cultivars. To our knowledge this is the first commercial sandwich ELISA that not only detects all important gluten fractions from wheat, rye, and barley but quantifies these fractions with comparable dose-response curves. In consequence, the Codex Alimentarius factor of 2 is not necessary any longer and a change in the ratio of prolamins to glutelins (e.g. in starches) will led to a smaller analytical bias compared to existing commercial ELISA systems. The system showed no cross-reactivity against 83 different food commodities. LoD was found to be between 0.4 mg/kg and 1.9 mg/kg for oat-based food. LoQ was verified at a level of 5 mg/kg. AOAC reference materials mentioned in SMPR ® 2017.021 showed recoveries between 86% and 131% for wheat and barley, while rye showed higher recoveries between 137% and 170%. Recovery studies with incurred oat cookies and porridge revealed values between 55% and 122%. The precision of the whole procedure is mainly driven by inhomogeneity of samples which was proven by a precision of extraction experiment. A ruggedness study revealed no critical parameters for the extraction or ELISA procedure. The test kit is stable for at least 3 weeks at 37°C. The results of the in-house validation study confirmed that the method is accurate and suitable to measure gluten from all three grain sources with similar recoveries according to SMPR ® 2017.021. Together with the information obtained by the collaborative test and the in-house validation data, the RIDASCREEN ® Total Gluten should be recommended as Official Methods of Analysis SM First Action of AOAC INTERNATIONAL. (1) Boison, J., Allred, L., Almy, D., Anderson, L., Baumert, J., Bhandari, S., Cebolla, A., Chen, Y., Crowley, E., Diaz-Amigo, C., Doi, H., Don, C., Downs, M., Dubiel, N., Dyer, B., Emerson, L., Farrow, M., Fritz, R., Galera, C., Garber, E., Godefroy, S., Grace, T., Hochegger, R., Johnson, K., Kasturi, P., Koerner, T., Lacorn, M., Massong, F., Meinhardt, P., Mui, T., O’Meara, M., Pan, S.-J., Popping, B., Prinster, M., Quesada, E., Radcliffe, S., Scherf, K., Sharma, G., Shoji, M., Stoughton, M., Sweeney, L., Szpylka, J., Taylor, S., Tittlemier, S., Torgler, C., Wehling, P., Yeung, J., Zweigenbaum, J. (2018) J. AOAC Int. 101 , 1238-1242 (2) Ludvigsson, J.F., Leffler, D.A., Bai, J.C., Biagi, F., Fasano, A., Green, P.H.R., Hadjivassiliou, M., Kaukinen, K., Kelly, C.P., Leonard, J.N., Lundin, K.E.A., Murray, J.A., Sanders, D.S., Walker, M.M., Zingone, & F., Ciacci, C. (2013) Gut 62 , 43-52 6 References

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