Validated multiplex-competitive ELISA using gluten-incurred yogurt calibrant for the quantitation of wheat gluten in fermented dairy products

Currently, there are no available methods for accurate quantitation of gluten in fermented or hydrolyzed foods. In this study, gluten-incurred yogurt was used as a calibrant with a multiplex-competitive ELISA to quantitate gluten in fermented dairy products such as yogurt, kefir, and buttermilk, followed by a single-laboratory validation of the method. Four-parameter logistic calibration curves using five gluten-specific antibodies (R5, G12, 2D4, MIoBS, and Skerrit) were constructed, and averaging of the antibody responses was used as a strategy to get a single quantitative value. The lower limits of detection (LLOD) and quantitation (LLOQ) of the method were 1.9 and 5.5 µg/mL (ppm), respectively. Analysis of wheat gluten-incurred fermented dairy products (5, 8, 20, 100, and 500 µg/mL) prepared with multiple starter cultures and fermented for 24 or 48 h resulted in average gluten recoveries of 69-165%, 57-167%, and 54-148% for yogurt, kefir, and buttermilk, respectively. Only a few samples exceeded 150% recovery. The average coefficient of variation (CV) ranged from 10 to 34%, with the majority of the samples having a CV of < 30%. Experimental variations such as long-term refrigerated storage, spiking gluten after initial fermentation, using higher than recommended starter culture concentrations, or using wheat flour for contamination resulted in acceptable gluten recovery (50-150%) for the majority of the samples. Comparison of the performance of this method with a commercial competitive ELISA showed that the method has greater quantitative accuracy. This newly developed and validated method appears sufficiently sensitive and accurate to quantitate the amount of wheat gluten before fermentation, in select fermented dairy products.