Western blot analysis of fermented-hydrolyzed foods utilizing gluten-specific antibodies employed in a novel multiplex competitive ELISA

Horseradish peroxidase (HRP) conjugated gluten-specific antibodies (G12, R5, 2D4, MIoBS, and Skerritt), from nine commercial gluten ELISA test kits, previously utilized in the development of a multiplex competitive ELISA for the detection of fermented-hydrolyzed gluten, were utilized in western blot analyses of 59 fermented-hydrolyzed foods from four food groups (beer, soy-based sauces, vinegar, and sourdough bread). The protein/peptide profiles generated by the nine gluten-specific antibodies varied in size distribution and intensity dependent on the type of food, with minor differences between related products. Cluster analysis of the estimated gluten concentration values (based on western blot band intensities relative to intact gluten standards at 2.5 mu g/mL and 100 mu g/mL) and that of the relative response of the nine gluten-specific antibodies to different gluten proteins/peptides, distinguished among the different categories of fermented-hydrolyzed foods; comparable to what was observed in the multiplex competitive ELISA. Further, unlike the competitive ELISA, the western blot analyses distinguished between the presence of antigenic proteinaceous materials and false positives due to the presence of binding inhibitors (as observed with four soy-based sauces and one vinegar). Limitations of western blot analysis often include lower sensitivity than the comparable competitive ELISA and problems quantitating gluten-derived peptides and proteins. As a result, western blot analysis provides an orthogonal approach that can be used to both confirm the multiplex competitive ELISA while also providing additional insight into the protein/peptide profile of fermented-hydrolyzed foods.