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.