Development of Magnetic Particle–Based Chemiluminescence Immunoassays for Detecting PCV2 VLP‐ and Rep‐Specific Antibodies and Their Combined Use for Immune Evaluation and Replication‐Associated Exposure Assessment

Porcine circovirus 2 (PCV2) remains a major swine pathogen, and interpretation of capsid protein (Cap)–based serology is challenging in vaccinated populations because vaccine‐induced Cap antibodies overlap with replication‐associated serological signals. To address this issue, we developed two fully automated magnetic particle–based chemiluminescence immunoassays (CLIAs), including a Cap virus‐like particle (VLP)–based assay (VLPs‐CLIA) for evaluating Cap antibody response intensity and a replication‐associated protein (Rep)–based assay (rRep‐CLIA) for assessing replication‐associated serological exposure or pressure. We produced self‐assembled PCV2 Cap VLPs and recombinant Rep in a prokaryotic system and covalently coupled them to carboxylated magnetic beads for fully automated CLIAs. VLPs‐CLIA was calibrated and reported in activity units (U), whereas rRep‐CLIA was interpreted as a threshold‐based qualitative assay using a relative light unit (RLU) cut‐off. Analytical performance and agreement with reference enzyme‐linked immunosorbent assays (ELISAs) were evaluated using longitudinal and field serum samples. VLPs‐CLIA detected antibodies up to 1:25,600, approximately twofold higher than the reference ELISA (1:12,800), with 95.68% agreement (Kappa = 0.876, p < 0.001). rRep‐CLIA agreed well with Rep‐ELISA (94.86%; Kappa = 0.846, p < 0.001) and provided a complementary signal consistent with replication‐associated exposure. Both assays showed good precision (coefficients of variation [CVs] ranging from 2.83% to 6.94%) and good reagent stability. In field samples, VLPs‐CLIA tracked postvaccination Cap kinetics, whereas Rep seropositivity increased with age and exposure background, suggesting heterogeneous replication–associated pressure among herds. The two assays together support complementary herd‐level interpretation of vaccine‐induced Cap immunity and replication‐associated serological exposure, particularly in Cap‐vaccinated populations.