A broadly applicable split-luciferase biosensor approach for rapid antibody detection in emerging infectious diseases.

Conventional immunoassays such as ELISA are widely used in serological testing, yet their reliance on multi-step workflows and labeled reagents limits diagnostic scalability and speed. Bioluminescence-based biosensors are attractive alternatives that offer high sensitivity, operational simplicity, and cost efficiency for detecting diverse analytes. Here, we present a broadly compatible bioluminescent biosensor for antibody detection based on analyte-mediated reconstitution of split-Nanoluciferase (NanoLuc). The platform utilizes engineered bifunctional probes, comprising the protein G C2 domain fused to split-NanoLuc subunits (LgBiT and SmBiT), which serve dual functions in antibody binding and signal generation. Upon immunocomplex formation with multi-epitope antigens, the probes colocalize LgBiT and SmBiT, reconstituting NanoLuc activity and producing a quantifiable bioluminescent signal. We implemented this Fc-binding split-NanoLuc complementation assay to detect antibodies against African swine fever virus (ASFV). The optimized system showed high sensitivity, a wide linear dynamic range, and no cross-reactivity with sera positive for other common swine viruses. Clinical validation exhibited 97.11% agreement with a commercial ELISA kit, confirming its practicality and reliability. Furthermore, the sensor platform was seamlessly adapted to detect antibodies against SARS-CoV-2 and Chikungunya virus (CHIKV) without requiring additional molecular design or reconfiguration, highlighting its inherent versatility. By leveraging the broadly applicable Fc-binding capacity of protein G and the intrinsic modularity of split-NanoLuc, this strategy streamlines assay development and eliminates the need for species-specific secondary antibodies. Together, our findings demonstrate a proof-of-concept biosensor approach that could be further developed into a useful tool for rapid antibody detection in emerging infectious disease settings. KEY POINTS: • Fungal biological processes alter upon illumination, also under the microscope • Red shifted fluorescent protein toolboxes decrease interference by illumination • Innovations like two-photon, lightsheet, and near IR microscopy reduce phototoxicity.