Development of Motif-Specific Monoclonal Antibodies for Global Protein Citrullination Detection with Minimal Cross-Reactivity to Homocitrullination

Protein citrullination, a post-translational modification (PTM) catalyzed by peptidylarginine deiminases (PADs), 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 cancer, making it a potential biomarker and therapeutic target. Immunodetection is the most commonly used technique to study citrullination. However, most commercially available antibodies against citrullination are either protein-specific or lack the sensitivity and specificity of the broad variety of modified proteins. In addition, existing anti-pan citrullination antibodies often fail to distinguish citrullination from homocitrullination, a chemically similar modification on lysine. This cross-reactivity limits their utility in deciphering the distinct biological roles of these PTMs. To address these challenges, we employed a motif-based strategy to generate monoclonal antibodies against citrullination. This approach leverages PAD enzyme sequence preferences from human proteome, enhancing specificity for citrullination while minimizing cross-reactivity with homocitrullination. 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 novel antibodies provide powerful tools for studying global citrullination dynamics and hold promise for biomarker discovery and diagnostic applications in diseases involving PAD dysregulation.