Rapid, Effective, and Versatile Extraction of Gluten in Food with Application on Different Immunological Methods

Abstract

One of the main concerns in gluten analysis is to achieve efficient extraction of gluten proteins. Conventional ethanol-based extraction solutions are inefficient and, because of this, it is necessary to use reducing agents or acids for proper solubilization. The extraction recommended by CODEX Standard 118-1979 (revised 2008) utilizes Cocktail solution (patent WO 02/092633 A1). However, it is harmful with a disgusting odor and is not compatible with some immunological techniques. Here, the versatility and extraction capacity of a new Universal Gluten Extraction Solution (UGES) (patent ES 2 392 412 A1) were evaluated using different methodological conditions, food matrices, and various immunological methods. UGES includes safer compounds for both the user and the environment, and it displayed similar extraction efficiency to that of the extraction method recommended for sandwich enzyme-linked immunosorbent assay (ELISA). The extraction time was significantly reduced from 100 to 40 min, depending on the type of the sample. Furthermore, unlike the currently used solution, UGES is compatible with competitive ELISA.

Keywords: celiac disease; gluten extraction solution; gluten proteins; gluten-free diet.

Figure 1

Optimization of time and temperature in the Universal Gluten Extraction Solution (UGES) protocol. (A) Effect of temperature in different types of samples. (B) Effect of time in simple and complex solid samples. (C) Effect of extraction time in liquid samples without suspended solids. The experiments were performed in duplicate, and the mean ± standard deviation (SD) is shown.

Figure 2

Statistical evaluation of the extraction procedure. (A) Comparison of agreement between pairs of assays for results of the food samples by sandwich ELISA using different extraction solutions different. Statistical analysis was performed by the Wilcoxon test. The p-values are given in the figure. (B) Scatter diagram with regression line and concordance bands for regression line; (b1) representation of the 60% ethanol solution versus Universal Gluten Extraction Solution (UGES); (b2) representation of the Cocktail solution versus UGES. The solid lines represent the regression line drawn for the food samples, and the dashed lines represent the 95% prediction interval.

Figure 3

Analysis of spiked samples by sandwich ELISA after extraction with Universal Gluten Extraction Solution (UGES) and Cocktail solutions. (A) Recovery of the different levels of gluten provided in the food samples. Results are expressed as ppm of gluten (mean ± SD) and percentage of gliadin recovery (R). N = number of analyses. (B) Comparison of agreement between pairs of assays for results of the food samples by sandwich ELISA with different extraction solutions. (C) Scatter diagram with regression line and concordance bands for regression line, indicative of commutability. <LOQ, less than the limit of quantification.

Figure 4

Analysis of spiked samples by competitive ELISA after extraction with Universal Gluten Extraction Solution (UGES) and 60% ethanol solutions. (A) Recovery of the different levels of gluten provided in the food samples. Results are expressed as ppm of gluten (mean ± SD) and percentage of gliadin recovery (R). N = number of analyses. (B) Statistical evaluation of the extraction procedure. (C) Scatter diagram with regression line and concordance bands for regression line. <LOQ, less than the limit of quantification; PWG, Prolamin Working Group.

Figure 5

Western blot analysis of gluten using different extraction procedures. (A) The efficiency in the extractability of Prolamin Working Group (PWG) gliadins with (a1) G12 monoclonal antibody (moAb) and (a2) A1 moAb. (B) The efficiency in the extractability of gluten from two commercial samples: (b1) biscuits and (b2) wheat flour (Triticum durum). MW, molecular weight marker.