Purpose: In an effort to obtain novel serological reagents with improved specificity profiles relative to the R5 system, we generated monoclonal antibodies in mice. Herein, we describe the immuno-reactive profile of the candidate clones and compare their binding activity against that of the R5 mAb.
Methods: A combination of deamidated gliadin and synthetic R5-peptide (LQPQQPFPQQLQPQQPFPQQA) was used to immunize female Balb/C mice. Spleens from seroconverted mice were fused with SP2/0-Ag14 cells and cultured using HAT techniques. Colonies were screened and initially selected based on reactivity towards gliadin, hordein, secalin, and avenin using indirect ELISA. IgG+ clones that retained high activity were raised and IgG purified from ascites was further studied by ELISA and western blot analysis and used to develop a sandwich ELISA.
Results: Using a combined vaccine approach, two hybridoma clones were generated, 2B9 and 1A11, which demonstrated very high, and near equal activity against gliadin, hordein, and secalin, and no cross-reactivity against avenin, zein, orzenin, or soy as determined by indirect ELISA. Western blot analysis of these two clones demonstrated a pattern of reactivity that mirrored that of the R5 mAb. Both clones were conjugated to HRP and used to develop sandwich ELISAs.
Significance: Of the clones tested, 1A11 and 2B9 demonstrated the most potential for use in future gluten detection systems, including use in sandwich ELISA for detection of wheat, barley, and rye-derived gluten residues.