An immunoassay based on bioluminescent sensors for rapid detection of African swine fever virus antibodies.

Serological assays for antibody detection have contributed significantly to the diagnosis and control of infectious diseases. African swine fever is the most devastating infectious disease of domestic pigs and wild boars, severely threatening the global pig industry in recent years. Here, we developed a rapid, simple, and sensitive immunoassay based on the split-luciferase system to detect IgG antibodies against African swine fever virus (ASFV). In this assay, the p30 protein of ASFV was genetically coupled to the LgBiT and SmBiT subunits of nanoluciferase, which were used as fusion probes for specific antibodies. Target engagement of the probes results in the reconstitution of a functional nanoluciferase, which further catalyzes bioluminescent reactions. Different orientations of the LgBiT and SmBiT-p30 fusion sensors were designed and investigated, and N-LgBiT/p30 and N-SmBiT/p30 were identified as a promising sensor pair for reforming active nanoluciferase in the presence of specific antibodies. After optimization, this split-luciferase complementation assay showed high sensitivity and specificity for the detection of ASFV antibodies. The analytical sensitivity of the assay was 16 times greater than that of the blocking enzyme-linked immunosorbent assay (ELISA) by the detection of serial dilutions of serum, and no cross-reaction was observed with other swine pathogens. As demonstrated in clinical samples, its performance is highly consistent with that of a commercial ELISA kit, with a concordance rate of 98.19%. This assay is simple and easy to perform, providing a more flexible and efficient approach for the measurement of ASFV antibodies in clinical applications.

Importance: The study is about a homogeneous split-luciferase assay for antibody detection. Split nanoluciferase biosensors for the detection of ASFV antibodies were designed. This sensor platform enables the sensitive and specific detection of antibodies. The split-luciferase assay is simple, rapid, and easy to use.