Monday, August 1, 2016
America's Center - St. Louis
Lora Benoit, IEH Laboratories & Consulting Group, Lake Forest Park, WA
David Cox, IEH Laboratories & Consulting Group, Lake Forest Park, WA
Madhu Katepalli, IEH Laboratories & Consulting Group, Lake Forest Park, WA
Jongkit Masiri, IEH Laboratories & Consulting Group, Lake Forest Park, WA
Cesar Nadala, IEH Laboratories & Consulting Group, Lake Forest Park, WA
Steven Gendel, IEH Laboratories & Consulting Group, Lake Forest Park, WA
Mansour Samadpour, IEH Laboratories & Consulting Group, Lake Forest Park, WA
Introduction: The prolamin fraction from wheat and related triticeae cereals exhibits immunopathogenic potential, and consumption of these grains is associated with coeliac disease. The “gold standard” used by the food industry relies on the use of immunodiagnostic tests based on the R5 monoclonal antibody (mAb). However, gluten detection systems based on this mAb have several shortcomings.
Purpose: In an effort to obtain novel serological reagents with improved specificity profiles, we generated monoclonal antibodies. Herein we describe the immuno-reactive profile of the candidate clones and compare their binding activity against the commercially available R5 mAb.
Methods: A synthetic peptide based on a duplicated and variably “deamidated” version of the canonical R5 binding site (L{Q/E}P{Q/E}{Q/E}PFP{Q/E}{Q/E}L{Q/E}P{Q/E} {Q/E}PFP{Q/E}{Q/E}A was used to immunize Balb/C mice. Tertiary boosts were performed using deamidated gliadin. Colonies were screened based on reactivity towards gliadin (wheat), deamidated gliadin (dg), hordein (barley), secalin (rye), and avenin (oat) using indirect ELISA. IgG+ clones with desirable reactivity profiles were studied by western blot analysis and used to develop sandwich ELISA.
Results: Using a combined vaccine approach, 4 candidate hybridoma clones were generated. Of these, 2D4 demonstrated high, and near equivalent activity against gliadin, hordein, and secalin; with no observed cross-reactivity against avenin (R5(-)), zein, orzenin, and soy. Western blot analysis of 2D4 demonstrated a pattern of reactivity that mirrored that of the R5 mAb. Competitive inhibition analysis suggested that 2D4 and R5 recognize similar epitopes. Sandwich ELISAs based on R5 and 2D4 showed that 2D4 reacted with deamidated gliadin more so than R5, had a more uniform reaction stoichiometry than did R5, and did not react with soy protein.
Significance: Of the clones tested, 2D4 demonstrated the most potential for use in future gluten detection systems, including use in sandwich ELISA for detection of wheat, barley, and rye-derived prolamin residues.
