Purpose: In an effort to generate novel monoclonal antibodies with improved specificity profiles relative to the R5 system, we generated anti-prolamin monoclonals in mice. Herein, we describe the immuno-reactive profile of 8 candidate clones and compare activity against that of R5.
Methods: Prolamins from wheat, barley, and rye were purified using standard techniques and 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 selected based on reactivity towards gliadin, hordein, secalin, and avenin using indirect ELISA. IgG+ clones that retained high activity were raised and purified from ascites and further studied by ELISA and western blot analysis in conjunction with the R5 mAb.
Results: The hybridomas were functionally grouped as 1) Skerrit-like (3C1, 3G9, 10C10, and 7B5) by their preferential ability to detect gluten from wheat and rye over barley, 2) R5-like (28A4 and 25A5) based on their ability to pan detect gluten from wheat and rye over barley, and 3) unique (7E3, which equally detects gluten from barley and wheat, and 4F7, which preferentially detects barley over wheat). None of the clones demonstrated meaningful reactivity against corn, rice, and soy. Of the R5-like mAbs, 25A5 demonstrated essentially equal detection of gluten from wheat, rye, barley, and oats. In comparison, 28A4 demonstrated greatly diminished activity towards avenins.
Significance: Owing to limitations with the existing gluten-detection systems, we have generated a series of novel monoclonal antibodies directed against the prolamin fraction of triticeae cereals. Of the 8 clones tested, 28A4 and 25A5 demonstrated potential for use in future gluten detection systems, with divergent applications with respect to avenin detection.