AOAC ERP Gluten Assays - Dec 2018

Annex B

2

Introduction

2.1

Principle

The basis of the test is the antigen-antibody reaction. The wells of the microtiter plate are coated with specific monoclonal antibodies against gluten proteins. By adding the standard or sample solution to the wells, present gluten proteins will bind to the specific antibodies. The result is an antibody-antigen complex. In a washing step components not bound are removed. Then antibodies conjugated to peroxidase (enzyme conjugate) are added. This antibody conjugate is bound to the antibody-antigen-complex. An antibody-antigen- antibody-complex (sandwich) is formed. Substrate/chromogen is added after removing of any unbound enzyme conjugate in a washing step. Bound enzyme conjugate converts the chromogen into a blue product. The addition of the stop solution leads to a color change from blue to yellow. The measurement is made photometrically at 450 nm against air. The absorption is proportional to the gluten protein concentration in the sample. The use of wheat flour and gluten in foodstuff is extremely common because of their useful effects on e.g. texture, moisture retention and flavor. Gluten is a mixture of prolamin and glutelin proteins present in wheat, rye and barley. Coeliac disease (CD) is a permanent intolerance to gluten that results in damage to the small intestine and is reversible when gluten is avoided by diet (2). The Codex Alimentarius Commission has stipulated in the „Codex Standard for Foods for Special Dietary Use for Persons Intolerant to Gluten” (3) the limit value for gluten-free food at 20 mg/kg gluten. The official type I method for quantitative gluten determination according to the Codex Alimentarius is an ELISA which uses the R5 antibody (Mendez). This requirement is fulfilled by the sandwich ELISA RIDASCREEN ® Gliadin (Art. Nr. R7001). Since its introduction to the analytical community, the R5 method to quantify gluten led to a strong improvement of the situation for the food industry and CD patients. During the last years some questions arose on the use of the Codex Alimentarius factor of 2 to recalculate from prolamins to gluten, an overestimation of rye and barley, inadequate detection of glutelins, and the inhomogeneous distribution of gluten in oats (4). Especially the inhomogeneity of gluten from wheat, rye or barley in oats led to AOAC Standard Method Performance Requirement (SMPR ® ) 2017.021 which was set up by stakeholders by vote in 2017. RIDASCREEN ® Total Gluten was validated in-house following the AOAC SMPR ® 2017.021 and AOAC Appendix M. Due to the long-lasting experience of the method developer additional experiments will be presented that show a deeper insight into the performance of the method. 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%. Recoveries 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 significant parameters for the extraction or ELISA procedure. The test kit is stable for at least 3 weeks at 37°C. 2.3 Summary of Results 2.2 General Information

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