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Chapter 18 : Critical Evaluation of Uncertainties of Gluten Testing: Issues and Solutions for Food Allergen Detection
Category: Food Microbiology; Applied and Industrial Microbiology
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This chapter focuses on a specific wheat allergen and the appropriate methods for its detection in food products to demonstrate the complexity of analytical challenges that the scientific community is facing. Such challenges apply to food allergen detection in general. In this chapter, the Osborne nomenclature is used because it is most cited among immunoassay developers. In the U.S. market, gluten commercial kit providers include Neogen Corp., whose enzyme-linked immunosorbent assay (ELISA) system based on two monoclonal antibody (mAbs), for rye and wheat, and Morinaga, whose ELISA uses polyclonal antibodies for wheat proteins. The gluten test is used to demonstrate the complexity of detection methods for food allergens in processed samples and the uncertainties and implications of results. Gluten content in foods can be evaluated by either commercial ELISA kits or lateral flow devices (LFD). Four gluten levels were evaluated in this study (50, 25, 10, and 5 ppm gluten) in two different matrices, gluten-free high-fiber bread mix and matrix-free 60% ethanol. The R-Biopharm kit results can be used to illustrate the problems associated with the selection and use of reference materials. The authors have used NIST gluten SRM 8418 to evaluate the kits, and have provided a detailed discussion on the characteristics of R5 (R-Biopharm) and Skerritt (Tepnel) monoclonal Abs (mAbs) based on findings and published information. The detection of gluten proteins is quite difficult due to the complexity of the targets and specificities of the Ab.
Wheat protein fractions. Gn, glutenin; Gl, gliadin; A/G, albumin/globulin. (A) Gluten fraction recognition by detector antibody from commercial kits. MW Std, molecular weight standards (in thousands). (B) Image analysis of gluten fractions separated by SDS-PAGE.
Detection of gluten proteins in bread crumb and crust extracted with cocktail (C), 60% ethanol (A), cocktail and aqueous ethanol (B), and Morinaga (M) buffers by detector antibody from commercial ELISAs. Protein profiles from SDS-PAGE of bread samples are compared to those of wheat flour (APF). MW, molecular weight (in thousands).
SDS-PAGE of wheat (W), barley (B), and rye (R) extracted with cocktail (C) and 60% ethanol (A). Specificity and cross-reactivity of detector antibodies from commercial ELISA evaluated by immunoblot. MW Std, molecular weight standards (in thousands).
Classification of gluten proteins
Commercial gluten test kits d
Effects of food matrices, extraction methods, and ELISA types in determination of gluten levels in spiked samples a
Detection of wheat protein fractions by commercial ELISAs
Protein composition of cereals b