Development and Validation of a Gluten-specific Sandwich ELISA based on a Novel Monoclonal Antibody, 2D4

Introduction: Cereal grains contain gluten, consisting of prolamin and glutelins. The prolamin fraction from wheat and related Triticeae can be immunopathogenic.  Consumption of these grains is associated with celiac disease (CD). Because CD manifests as debilitating symptoms and chronic complications, affected individuals must adopt a gluten-free diet. Avoiding gluten is challenging because foods labeled “gluten-free” are frequently contaminated with gluten. In response, regulatory authorities have established allowable gluten levels in foods labeled “gluten-free” to protect consumers from unanticipated exposures.

Purpose: We have generated a novel monoclonal antibody (mAb), 2D4, directed against the prolamins of Triticeae grains with improved characteristics relative to the R5 mAb.

Methods: A sandwich ELISA using 2D4 and denaturing buffer was validated using prolamin standards derived from wheat, barley, rye, oats, and deamidated wheat protein and 35 common commodities, for cross reactivity, interference, and LOD.  We also characterized prolamin extractability from complex foods and resistance of the assay to temperature deviations.

Results: The 2D4-based sandwich ELISA uses a 20 min extraction period, and ELISA incubation steps of 10 min, 10 min, and 5 min.  There was no cross-reactivity with any of the commodities tested. Spike analysis averaged 85% recovery. The LOD was established at 1 ppm prolamin or ~2 ppm gluten. Analysis of complex foods demonstrated improved recovery in thermally processed foods using the denaturing buffer system as compared to standard 60% ethanol extraction. Assessment of the assay at 18, 22, and 25°C showed resistance to temperature deviations.

Significance: The development of a highly sensitive and rapid test method capable of accurately detecting trace amounts of prolamin residues (modified and native) in under 45 min should aid food manufacturers and regulatory entities in monitoring for gluten and gluten derivatives that have previously proved challenging.