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Title: Investigation of the coeliac immunogenicity of cereal peptides and their quantification using monoclonal antibodies
Author: Abd Wahab, Widya
ISNI:       0000 0004 5989 8265
Awarding Body: King's College London
Current Institution: King's College London (University of London)
Date of Award: 2014
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Coeliac disease (CD) is a chronic, immune-mediated, small intestinal glutensensitive enteropathy affecting approximately 1% of individuals in the Europe and the U.S. The condition is exacerbated by the consumption of food containing wheat gluten, rye secalin and barley hordein. The keystone of CD treatment is strict compliance to a gluten-free diet. Designed foods for coeliac sufferers are mainly based on purified wheat starch that may contain traces of gluten. Hence, a sensitive and specific assay system is required to ensure the purity and safety of commercially available gluten-free foods. Such a system is based on the use of monoclonal antibodies (MAbs) raised against coeliac toxic gluten peptides. Several peptides have been identified in rye and barley that are thought to be potentially immunogenic and in turn CD toxic. An ω-gliadin/ C-hordein peptide (QPFPQPEQPFPW) and a rye secalin-derived peptide (QPFPQPQQPIPQ) have been identified from the literature as potentially CD toxic. They were assessed for CD in vitro immunogenicity and used to raise MAbs. We wished to investigate the in vitro responses of small intestinal gluten sensitive T cell lines to the described peptides. We have sought to develop MAbs to these coeliac toxic peptides as a prelude to developing improved assays to quantify the gluten content of foods for individuals with CD. In parallel to this work, using the available MAbs previously developed by our group, we proposed to design a specific Cocktail ELISA assay for gluten analysis. This would potentially provide an assay with improved specificity and sensitivity for CD toxic gluten proteins and peptides. The CD immunogenicity of ω-gliadin/ C-hordein peptide and a rye secalinderived peptide were assessed with T cell proliferation assays. These were undertaken by incorporation of 3H-thymidine to measure the immune response of the peptides and prolamin fractions to CD gluten sensitive small intestinal T cell lines. Balb c mice that received a gluten-free diet were immunised with PPD (tuberculin protein) conjugated to the coeliac toxic peptides described above. The gluten content in purified wheat starch which is used to make commercially available gluten-free foods was quantified using a unique cocktail ELISA by combining two of established MAbs raised against known CD toxic peptides. We found that the ω-gliadin/ C-hordein peptide and a rye secalin-derived peptide triggered positive responses in most of the CD small intestinal T cell lines specific to peptic-tryptic (PT) digested wheat gluten (n=9), barley hordein (n=7) and rye secalin (n=2), confirming their coeliac immunogenicity. The murine immunisations and fusions were successful. However, IgM antibodies were generated by the hybridomas to the gluten peptides that could not be used for gluten quantification. We found that we can quantify both CD toxic gliadin and glutenin in separate assays, simultaneously. Our method for gluten quantification could represent an improvement in the specificity of gluten quantification, since it measures CD toxic wheat proteins, including both gliadin and glutenin, rather than the currently accepted method of multiplying the gliadin content by two to yield a value for the total gluten content. However, we found that combining the MAbs in a single Cocktail assay yielded lower values compared to the summation of the individual quantified components. The ω-gliadin/ C-hordein peptide and a rye-derived peptide are immunogenic and were recognised by CD gluten sensitive T cell lines. Balb c mice were successfully immunised with these immunogenic peptides. The resultant hybridomas secreted IgM rather than IgG antibodies to the peptides. The former of which could not be used to develop immunological assays for gluten quantification. Characterisation of cocktail ELISAs with more than one MAb combination to gluten protein demonstrated lower values of gluten than the summation of simultaneous quantification of the individual components. We concluded that CD toxic gliadin and glutenin need to be measured individually and the value of both proteins be added together to quantify the gluten content of foods designed for individual with CD.
Supervisor: Ciclitira, Paul Jonathan ; Ellis, Heather Julia Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available