Detection and Quantitation of Gluten in Fermented-Hydrolyzed Foods by Antibody-Based Methods: Challenges, Progress, and a Potential Path Forward

Abstract

Celiac disease (CD) affects ~1 in 141 individuals in the United States, requiring adherence to a strict gluten-free diet. The Codex Standard and the European Commission states that gluten level of gluten-free foods must not exceed 20 ppm. The FDA requires food bearing the labeling claim “gluten-free” to contain <20 ppm gluten. Accurate quantitation of gluten in fermented-hydrolyzed foods by antibody-based methods is a challenge due to the lack of appropriate reference materials and variable proteolysis. The recent uses of proteases (e.g., proline endopeptidases or PEP) to hydrolyze immunopathogenic sequences of gluten proteins further complicates the quantitation of immunopathogenic gluten. The commercially available antibody-based methods routinely used to detect and quantitate gluten are not able to distinguish between different hydrolytic patterns arising from differences in fermentation processes. This is a severe limitation that makes accurate quantitation and, ultimately, a detailed evaluation of any potential health risk associated with consuming the food difficult. Utilizing gluten-specific antibodies, a recently developed multiplex-competitive ELISA along with western blot analysis provides a potential path forward in this direction. These complimentary antibody-based technologies provide insight into the extent of proteolysis resulting from various fermentation processes and have the potential to aid in the selection of appropriate hydrolytic calibration standards, leading to accurate gluten quantitation in fermented-hydrolyzed foods.

Immunogenic Potential of Beer Types Brewed With Hordeum and Triticum spp. Malt Disclosed by Proteomics

“…These latter authors also emphasized the possibility that additional immunotoxic peptides remained undetected due to several analytical drawbacks. The concerns related to the consumption of beer crafted with PEP from barley or wheat malt have been recently reviewed ( 14 ).…”

“…In general, quantification of gluten in hydrolyzed/fermented foods or beverages is hindered by a series of factors, especially related to the lack of appropriate reference standards ( 13 ). Multiplicity of barley varieties utilized for brewing, heterogeneity of the prolamins, variable degree of proteolysis, matrix interferences, ample dynamic range of proteolytic fragments, process induced modifications (e.g., non-enzymatic glycation) are additional issues complicating the accurate estimation of beer gluten content ( 14 ). Due to these drawbacks, it is clearly emerging that immunochemical methods such as both sandwich and competitive R5 ELISA, which are the most exploited and the unique recommended by food control authorities, could be unreliable for quantifying gluten in beer, because results are often affected by discrepancies spanning over several orders of magnitude ( 15 ).…”

Comparative Characterization of Gluten and Hydrolyzed Wheat Proteins

“…Additionally, the sensory properties were analyzed as well as the use of HWP as a nutritional additive. When characterizing the proteins and peptides in HWP, biochemical analyses, such as enzyme-linked immunosorbent assays (ELISAs) or Western blots, are widely used [ 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 ]. However, there are only few studies on commercially available HWP compared to native gluten.…”

From Polyclonal Sera to Recombinant Antibodies: A Review of Immunological Detection of Gluten in Foodstuff

“…Immunological techniques are based on the high affinity interaction of antibodies and antigens, which has led to the development of different vastly used applications, including: ELISA [ 41 ]: this is a quick, economic, versatile and robust method. ELISA presents a high sensitivity (in the low ppm range) and optical detection.…”

“…ELISA [ 41 ]: this is a quick, economic, versatile and robust method. ELISA presents a high sensitivity (in the low ppm range) and optical detection.…”