Development of a Ready‐To‐Use Bioluminescence Immunosensor for the One‐Step Sensitive Detection of Antibodies Against African Swine Fever Virus

Rapid and reliable analytical techniques play important roles in various research fields and are particularly crucial for diagnosing infectious diseases in clinical settings. African swine fever (ASF) is a devastating viral pig disease for which no effective vaccine is available. The ongoing ASF pandemic has highlighted the importance of rapid and accurate diagnosis, which enables the timely implementation of control and eradication measures. In this study, a ready-to-use bioluminescence immunosensor based on a split-nanoluciferase (NanoLuc) reporter system was proposed for the one-step sensitive detection of ASF virus (ASFV) antibodies. Specifically, the NanoLuc subunits SmBiT/LgBiT were each genetically fused to the ASFV p30 protein and protein G and used as probes. The simultaneous binding of the probes to ASFV IgGs induced the reconstitution of functional NanoLuc, which can generate a strong bioluminescent signal output by catalysing the substrate furimazine. This immunosensor allows the rapid and homogeneous detection of ASFV antibodies in solution, requiring only one incubation step of 10 min. This immunosensor also has high sensitivity, high specificity, and a wide dynamic range and is particularly promising for point-of-care testing. Comparative analysis of clinical samples validated the reliability and robustness of this approach and demonstrated high consistency with enzyme-linked immunosorbent assay (ELISA) results (concordance rate: 98.71%). These results suggest that the proposed immunosensor provides an attractive alternative to conventional immunoassays and could be easily repurposed by generating specific probes for antibody detection in other diseases.