Purpose: In an effort to obtain novel serological reagents with specificity for avenins, we generated monoclonal antibodies in mice. Herein, we describe the immuno-reactive profile of candidate clones.
Methods: Avenins were isolated from R5 (-) oats using a modified Osbourne fractionation technique and subsequently 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 against avenins alone were raised, subcloned, and IgG purified from ascites was further studied by ELISA and Western blot analysis.
Results: Subclones of 3G5, 3G7, 5B6, and 11E7 demonstrated very high activity against avenin with no cross-reactivity towards gliadin, hordein, and secalin, zein, orzenin, or soy as determined by indirect ELISA. Western blot analysis using these reagents established protein binding profile and was used to inform on the development of a sandwich ELISA for avenin that was capable of detecting avenin in food at levels lower than 10 ppm.
Significance: These 4 mAb reagents demonstrated potential for use in future avenin detection systems, including use in sandwich ELISA. The development of avenin detection systems should provide the celiac community with improved diagnostic tools to better assist in disease management.