Preparation and characterization of enzymatically hydrolyzed prolamins from wheat, rye, and barley as references for the immunochemical quantitation of partially hydrolyzed gluten

  • Benedict Gessendorfer
  • Peter Koehler
  • Herbert Wieser
Original Paper

DOI: 10.1007/s00216-009-3080-6

Cite this article as:
Gessendorfer, B., Koehler, P. & Wieser, H. Anal Bioanal Chem (2009) 395: 1721. doi:10.1007/s00216-009-3080-6

Abstract

Celiac disease (CD) is a permanent gastrointestinal disorder characterized by the intolerance to a group of proteins called gluten present in wheat, rye, barley, and possibly oats. The only therapy is a strict lifelong gluten-free diet. The standard method for gluten determination in foods produced for CD patients is the R5-enzyme-linked immunosorbent assay (ELISA) as proposed by the recent Codex Alimentarius Draft Revised Standard. This test is based on the determination of prolamins, the alcohol-soluble proteins of gluten, and is available as a sandwich ELISA for intact proteins and as a competitive ELISA for gluten-derived peptides. While the suitability of the sandwich ELISA including a wheat prolamin (gliadin) reference for calibration has been shown by various studies and a ring test, the competitive ELISA still lacks a convenient reference for the quantitation of gluten peptides in fermented cereal foods (e.g., sourdough products, starch syrup, malt extracts, beer). Therefore, the aim of the present study was to prepare a suitable reference for the quantitation of partially hydrolyzed gluten in fermented wheat, rye, and barley products. The prolamin fractions from barley (hordein) and rye (secalin) were isolated from corresponding flours by means of a modified preparative Osborne fractionation. The prolamin fraction from wheat was obtained as reference gliadin from the Prolamin Working Group. The prolamin fractions were successively digested by pepsin and trypsin or pepsin and chymotrypsin procedures, which have been used for CD-specific toxicity tests on cereal storage proteins for many years. The protein/peptide content (N × 5.7) of the prolamin fractions and digests, which was the basis for the calculation of the gluten content by means of ELISA, varied between 67.1% and 96.0%. The prolamin fractions and enzymatic digests were then tested for their response in both sandwich and competitive assays. Intact prolamins responded similarly in both ELISA showing no important differences between the cereals. In the case of digested proteins, however, the sandwich ELISA was considerably less sensitive than the competitive ELISA. The former provided approximately 40% and the latter 70% of the signal intensity obtained with the intact prolamins. Thus, the combination of the competitive ELISA and the enzymatic digests of prolamin fractions as reference was considered to be an adequate system for the analysis of partially hydrolyzed gluten. The limit of detection using a peptic-tryptic hordein digest as reference was 2.3 µg prolamin equivalent per milliliter, and the limit of quantitation was 6.7 µg prolamin equivalent per milliliter. This system was applied for the determination of gluten equivalents in five commercial beverages based on fermented cereals.

Figure

Schematic representation of a competitive ELISA (top) and examples for the measurement of the prolamin content of different samples and references (bottom)

Keywords

Celiac disease Gluten-free diet Gluten content ELISA 

Abbreviations

CD

Celiac disease

ELISA

Enzyme-linked immunosorbent assay

HMM

High molecular mass

HPLC

High-performance liquid chromatography

LOD

Limit of detection

LOQ

Limit of quantitation

Mr

Molecular mass (relative)

PAGE

Polyacrylamidegel electrophoresis

PC

Peptic-chymotryptic

PT

Peptic-tryptic

PWG

Prolamin working group

RP

Reversed-phase

RT

Room temperature

SDS

Sodium docecylsulfate

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Benedict Gessendorfer
    • 1
  • Peter Koehler
    • 1
  • Herbert Wieser
    • 1
  1. 1.Deutsche Forschungsanstalt für LebensmittelchemieFreisingGermany