Development of motif-specific monoclonal antibodies for global protein citrullination detection with minimal cross-reactivity to homocitrullination

Protein citrullination plays critical roles in biological processes such as immunity, gene regulation, and inflammation. Dysregulated citrullination is implicated in diseases including rheumatoid arthritis, multiple sclerosis, and cancers, making it a potential biomarker and therapeutic target. To overcome current challenges in citrullination detection using immunodetection, we developed motif-based monoclonal antibodies against citrullination. We immunized rats with a pool of over 490,000 citrullinated peptides designed to represent common citrullination motifs in human tissue proteomes. Two monoclonal antibody clones were established and validated for sensitivity and specificity, using ELISA and western blot against in vitro citrullinated and homocitrullinated proteomes, as well as ionomycin-activated human neutrophils. Both clones demonstrated great sensitivity to diverse citrullinated proteins, robust discrimination against homocitrullination, and quantitative readout in biological samples. These antibodies provide powerful tools for studying global citrullination dynamics and hold promise for biomarker discovery and diagnostic applications in diseases involving peptidylarginine deiminase (PAD) dysregulation.