Rapid and Wash-Free Anti-Drug Antibody Assay Enabled by a Complementary Nanoluciferase Biosensor

Biotherapeutics have demonstrated remarkable therapeutic efficacy across a wide range of diseases; however, the desired clinical outcomes of biotherapeutics are sometimes hindered by the development of antidrug antibodies (ADAs). Traditionally, ADA monitoring relies heavily on ligand-binding assays (LBAs) such as enzyme-linked immunosorbent assays (ELISA) and electrochemiluminescence assays (ECLIA). Although effective, these methods require multiple washing and incubation steps, extending the data generation time to several hours or even days. In contrast, homogeneous immunoassays offer a promising alternative due to their "mix-and-read" feature, which significantly reduces operational complexity, time, and cost compared to conventional LBAs. Luciferase complementation represents an invaluable tool for visualizing protein-protein interactions in vitro, in cellulo, and in vivo. In this study, we explored the feasibility of rapid ADA detection using a wash-free homogeneous assay based on a split nanoluciferase (SplitNluc) biosensor system. Through case studies involving diverse therapeutic modalities, including antibody-drug conjugate (ADC), Fc-fusion protein, Fc-less multidomain biotherapeutic (MDB), and single-domain antibody (SdAb), we demonstrated that the SplitNluc platform is a versatile and efficient tool for ADA assay, offering ideal sensitivity, high precision and broad adaptability to various modalities. In summary, the SplitNluc assay represents an innovative and valuable platform for expedited and robust ADA detection. Moreover, our study highlighted the broader potential of luminescent biosensor-based approaches in advancing bioana-lytical workflows.