Development of a conformation-specific nanobody-based ELISA for quantitative detection of intact Senecavirus A virions and virus-like particles.

Replication of Senecavirus A (SVA) generates intact virions and empty capsids, some of which disassemble into pentamers. To enable precise quality control of SVA intact viral antigen, we established a double-antibody sandwich ELISA to quantify intact SVA virions and virus-like particles (VLPs). The assay employs a conformation-specific nanobody (SV1) as the capture antibody, which binds both virions and VLPs, and a His-tagged nanobody (SVH1) as the detection antibody. Using purified virions as standards, the assay demonstrated a limit of detection (LOD) of 9.7 ng/mL, with a linear range spanning from 9.7 ng/mL to 1.25 μg/mL. Critically, it showed no cross-reactivity with pentamers and other viral particles, including foot-and-mouth disease virus (FMDV), porcine circovirus type 2 (PCV2), porcine parvovirus (PPV), classical swine fever virus (CSFV), and encephalomyocarditis virus (EMCV). The coefficients of variation (CV) were <10% for both intra-batch and inter-batch analyses. Validation against sucrose density gradient centrifugation (SDGC) confirmed concordant results. Applied to vaccine development, the ELISA quantified a 36% antigen loss in SVA VLPs after emulsification. In summary, this method provides a rapid, specific tool for SVA vaccine standardization.