Enzyme-Linked Immunosorbent Assays for the Detection and Quantitation of Gluten in Cereal-Based Foods
Enzyme-linked immunosorbent assays (ELISAs) for gluten quantitation use specific antibodies that interact with epitopes of gluten proteins that trigger an immune response in celiac disease patients. To date, four ELISA methods for gluten quantitation have been validated by international collaborative studies. The Skerritt ELISA method was validated in 2001. Three proprietary gluten ELISA methods have since been developed and were validated between 2010 and 2013: the R5 sandwich ELISA, R5 competitive ELISA, and G12 sandwich ELISA kits. In collaborative studies, the R5 sandwich ELISA had a limit of detection (LOD) of 3 mg of gluten/kg and a relative reproducibility standard deviation (RSDR) of 17 to 27% for samples containing intact gluten, whereas the G12 sandwich ELISA had an LOD of 4 mg of gluten/kg and an RSDR of 19 to 33% in 8 of 9 samples containing intact gluten. The R5 competitive assay targets hydrolyzed gluten and was less sensitive (LOD of 10 mg/kg) and showed higher variation (RSDR of 25 to 37%) than the sandwich ELISAs. Compared with the Skerritt ELISA method for quantitation of intact gluten (LOD of 20 mg/kg; RSDR of 23 to 56%) the more recently approved sandwich ELISAs showed better sensitivity and reproducibility and demonstrated that analytical progress has been achieved over time. Based on the collaborative studies conducted from 2010 to 2013 the R5 and G12 antibody-based proprietary test kits have been accepted as AACC International (AACCI) Approved Methods 38-50.01 and 38-52.01, respectively. They can be used to verify compliance with the Codex Alimentarius threshold for gluten-free food declarations, which has been set at 20 mg of gluten/kg of food (as-is basis) for several cereal-based matrices. Critical matrices to date, in particular, are cereal-based beverages. The R5 competitive ELISA assay (AACCI Approved Method 38-55.01 and American Society of Brewing Chemists [ASBC] Method Beer-49) should be used for fermented products and is the only collaboratively validated method currently available that meets the recommended detection limit of ≤10 mg of gluten/kg. Nevertheless, the gluten peptides formed during fermentation are different than those used in the kit for calibration; thus, further research on preparing more representative calibration materials for hydrolyzed gluten is necessary.