Presentation: 2025 ND EPSCoR Annual conference 

October 21, 2025, NDSU Memorial Union, Fargo, North Dakota

Development of a NanoLuciferase based live cell assay for the identification of ligand specific anti-RAGE antibodies

The receptor for advanced glycation end-products (RAGE) is a cell surface receptor with roles for physiological homeostasis and RAGE is upregulated in disease conditions including diabetes and cancer. The full-length RAGE protein comprises a variable-like (V) domain, two constant type domains (C1 and C2), a transmembrane segment, and a cytoplasmic tail. RAGE has multiple ligands such as AGEs, HMGB1, S100 proteins, amyloid β peptides, MAC-1, and nucleic acids. Targeting RAGE to block specific ligand interactions holds therapeutic potential across multiple diseases. This study aims to identify ligand-specific RAGE antagonists using a live-cell NanoLuciferase system (NanoBiT). For this purpose, HEK293 cells were stably transfected with an LgBiT-tagged human RAGE (hRAGE) receptor. To complement the LgBiT, SmBiT-tagged S100B and Sm-BiT tagged HMGB1 were employed, as both ligands are known to bind RAGE. Following complementation of LgBiT and SmBiT, luciferase activity was measured upon the addition of Furimazine substrate. Changes in luminescence signals were assessed to identify ligand displacement by anti-RAGE antibody binding. We successfully generated a stable LgBiT-RAGE expressing stable HEK293 cell line. SmBiT-tagged S100B and HMGB1 proteins were successfully constructed, expressed, and purified from E.coli. The interaction between LgBiT_hRAGE and SmBiT-tagged proteins effectively reconstituted the NanoLuciferase enzyme. The NanoBiT system demonstrated the ability to detect competition with untagged S100B, and antibody mediated ligand displacement of S100B. The assay was used to screen a panel of monoclonal antibodies for their ability to block binding of S100B and HMGB1, respectively, to RAGE.