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A novel and sensitive method for the detection of T cell stimulatory epitopes of α/β- and γ-gliadin
  1. E H A Spaenij-Dekking1,
  2. E M C Kooy-Winkelaar1,
  3. W F Nieuwenhuizen2,
  4. J W Drijfhout1,
  5. F Koning1
  1. 1Department of Immunohematology and Blood Transfusion, Leiden University Medical Centre, Leiden, the Netherlands
  2. 2Department of Innovative Ingredients and Products, TNO Nutrition and Food Research, Zeist, the Netherlands
  1. Correspondence to:
    Dr E H A Spaenij-Dekking
    Department of Immunohematology and Blood Transfusion, E3-Q, Leiden University Medical Centre, PO Box 9600, 2300 RC Leiden, the Netherlands; e.h.a.spaenij-dekkinglumc.nl

Abstract

Background: It is now generally accepted that coeliac disease (CD) is caused by inflammatory T cell responses to gluten peptides bound to HLA-DQ2 or -DQ8 molecules. There is overwhelming evidence that CD patients can mount T cell responses to peptides found in both α-gliadin and γ-gliadin molecules. Assays that would detect the presence or absence of such peptides in food would thus be accurate indicators of safety for consumption by CD patients.

Aims: The development of a sensitive method to detect T cell stimulatory epitopes of α-gliadin and γ-gliadin molecules in food products.

Methods: Monoclonal antibodies (mAb) were raised against peptides encoding the T cell stimulatory epitopes of α-gliadin (amino acids (aa) 59–71) and aa γ-gliadin (aa 142–153 and aa 147–159). These mAb competition assays were developed that quantitatively detect T cell stimulatory epitopes present on both intact proteins and peptides of sizes recognisable by CD4+ T cells.

Results: With the mAb based competition assays, T cell epitopes were detected in pepsin/trypsin digests of wheat proteins and ethanol extracts of various food products, with detection levels lower than those reached with gluten specific T cells. Moreover, the presence of T cell stimulatory epitopes was also detected in preparations of barley, rye, and triticale, other cereals known to be toxic for CD patients.

Conclusions: A new antibody based method has been developed, detecting the presence of T cell stimulatory gluten peptides. This can be used to further ensure the safety of food consumed by CD patients.

  • aa, amino acids
  • HLA, human leucocyte antigen
  • CD, coeliac disease
  • mAb, monoclonal antibody
  • tTG, tissue transglutaminase
  • HMW, high molecular weight
  • LMW, low molecular weight
  • ppm, parts per million
  • TTd, tetanus toxoid
  • BSA, bovine serum albumin
  • PBS, phosphate buffered saline
  • TMB, 3,3′,5,5′-tetramethylbenzidine
  • ELISA, enzyme linked immunosorbent assay
  • SAMA, S-acetyl-mercaptoacetic acid
  • coeliac disease
  • T cell epitopes
  • α-gliadin
  • γ-gliadin
  • monoclonal antibodies
  • competition assay
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