Reliable luciferase immunoprecipitation system assays for the detection of porcine reproductive and respiratory...

Porcine reproductive and respiratory syndrome virus (PRRSV) is a major infectious pathogen that poses a significant threat to the global pork industry and causes substantial economic losses. Vaccination remains one of the most important strategies for controlling PRRS; however, the protective efficacy of current commercial vaccines is often limited against highly diverse emerging field strains. In addition to vaccination, rapid and accurate diagnosis of PRRSV infection is essential for implementing effective biosecurity measures. In this study, we developed two luciferase immunoprecipitation system (LIPS) assays for detecting PRRSV antibodies, targeting the nsp7 protein. These assays utilize Gaussia luciferase and Nano luciferase, the latter supplemented with an N-terminal signal peptide, as reporter proteins. Two fusion antigens, GLuc-nsp7 and GNLuc-nsp7, were prepared for the LIPS development. Following optimization of reaction conditions, we determined that an input of 10⁷ luminance units of fusion antigen and a serum dilution of 1:50 yielded the highest positive-to-negative (P/N) ratio. Both LIPS assays showed no cross-reactivity with sera from pigs infected with other common porcine pathogens. These assays detected PRRSV-specific antibodies as early as 3 days post-challenge (dpc), whereas the commercial IDEXX ELISA did not detect antibodies until 8 dpc, demonstrating the superior diagnostic sensitivity of the LIPS platform. Furthermore, the GNLuc-nsp7-based LIPS assay, which does not require automated substrate addition, may be particularly suitable for on-farm diagnostic applications. In summary, we developed two reliable LIPS assays for PRRSV antibody detection, demonstrating strong potential for both laboratory and field applications.