Exploring Gluten Assessment in Marketed Products through a Sandwich ELISA Methodology Based on Novel Recombinant Antibodies

This study presents the development of a sandwich ELISA method for gluten detection in foods, using recombinant Fab antibody fragments against gliadin. The Fabs were chemically biotinylated and immobilized on streptavidin-coated plates as capture antibodies, while alkaline phosphatase-conjugated Fabs were used as detection antibodies. Four different gliadin-binding Fabs were tested and the Fab pair Fab8E-4 and Fab-C showed the best compatibility. An indirect sandwich immunoassay, using unmodified Fab8E-4 for capture and Fab-C as the detection antibody, achieved a detection limit of 26 ng/mL of gliadin, corresponding to 10 mg/kg of gluten in foods. No cross-reactivity was observed against 60 gluten-free species commonly used in the food industry. Analysis of 50 commercial products demonstrated consistent results compared to the standard method for gluten detection. The complete lack of cross-reactivity of the developed immunoassay with oat products potentially provides an advantage over other gluten detection systems.