An innovative sandwich ELISA system based on an antibody cocktail for gluten analysis

A cocktail sandwich ELISA based on the employ of two monoclonal antibodies (MAbs) as coating antibodies and a third MAb conjugated to horseradish peroxidase has been developed for the analysis of gluten in foods. Given that each MAb displays a wide specificity spectrum for wheat, barley, rye and oats prolamins, their combination for ELISA ensures a high crossreactivity with most of the potentially toxic gliadin, hordein, secalin and avenin protein family. One of the unprecedented features of the cocktail sandwich ELISA is that it permits for the first time analysis of barley hordeins in foods, which is unattainable using conventional or commercial ELISA kits. Besides, gliadins, hordeins and secalins are recognised to the same extent. The system provides a high detection sensitivity for gliadins, hordeins, secalins and avenins (1.5, 0.05, 0.15 and 12 ng/ml, respectively). The working linear range comprises 3-100 ng/ml with a gliadin detection limit of 1.5 ppm. This limit of detection is even better than that demanded in the latest Codex recommendation, 10 ppm. Cocktail ELISA data were contrasted with those of commercial ELISA kits and confirmed by mass spectrometry, a non-immunological technique which provides evidence for the occurrence of false positive results with the commercial kits.