Accuracy of ELISA Detection Methods for Gluten and Reference Materials: A Realistic Assessment

Abstract

The determination of prolamins by ELISA and subsequent conversion of the resulting concentration to gluten content in food appears to be a comparatively simple and straightforward process with which many laboratories have years-long experience. At the end of the process, a value of gluten, expressed in mg/kg or ppm, is obtained. This value often is the basis for the decision if a product can be labeled gluten-free or not. On the basis of currently available scientific information, the accuracy of the obtained values with commonly used commercial ELISA kits has to be questioned. Although recently several multilaboratory studies have been conducted in an attempt to emphasize and ensure the accuracy of the results, data suggest that it was the precision of these assays, not the accuracy, that was confirmed because some of the underlying assumptions for calculating the gluten content lack scientific data support as well as appropriate reference materials for comparison. This paper discusses the issues of gluten determination and quantification with respect to antibody specificity, extraction procedures, reference materials, and their commutability.

Determination of Gluten Peptides Associated with Celiac Disease by Mass Spectrometry

“…The accuracy of ELISA method is also compromised since the result is converted into gluten by multiplication by two, assuming that the gliadin/glutenin ratio is constant. Moreover, the current methods are not able to distinguish the cereal source (wheat, barley, rye) or cultivar [50,51].…”

Characterizing the Distribution of Ppm Gluten in Gluten Free Oatmeal Servings Contaminated with a Wheat Kernel

“…Glutenins can contain significant amounts of gluten as well (Huebner, 1970;Selmeier, Belitz & Wieser, 1991;Weiser & Koehler, 2009;Haraszi et al, 2011;Zilic, Barc, Pesic, Dodig & Ignjatovic-Micic, 2011;Diaz-Amigo & Popping, 2013;Khan et al, 2013).…”

“…For instance, the '% gluten that's gliadin' plays a big role as test kits measure only gliadin and assume a 1:1 ratio of gliadin to glutenin (Huebner, 1970). Doing a survey of research regarding gliadin/glutenin ratios though, shows this ratio can vary substantially (Huebner, 1970;Selmeier et al, 1991;Weiser & Koehler, 2009;Haraszi et al, 2011;Zilic et al, 2011;Diaz-Amigo & Popping, 2013;Khan et al, 2013), as illustrated in Table 4. Varying gliadin as a percent of total gluten, from 33% to 70%, produces average 'ppm/serving gluten' from 50 ppm to 110 ppm, and with stdevs from 37 ppm to 83 ppm respectively, as shown in Table 3.…”

Detection of Allergen Markers in Food: Analytical Methods

“…Since gluten is not soluble in common aqueous buffers, the extraction of ff ff gluten from foods for quantitation by ELISA is achieved by either aqueous ethanol alone or in combination with denaturing and reducing agents at high temperature. ELISA methods using aqueous alcohol alone may have significantly reduced gluten extraction effi fi ciency in thermally processed ffi foods, resulting in an underestimation of gluten [39,102]. Moreover, using gliadin as a calibrant may compromise gluten quantitation from rye and barley if the antibody affi nity to gluten varies with the grain source.…”