Multiplex method for simultaneous serological detection of porcine reproductive and respiratory syndrome virus and porcine circovirus type 2

Porcine circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV) are major contributors to the porcine respiratory disease complex (PRDC). Routine serological diagnosis and surveillance play an important role in the prevention of PRDC, as it is a leading cause of economic losses to the swine industry. We herein describe an advanced microsphere-based immunoassay that permits the simultaneous detection of antibodies to PCV2 and PRRSV, thereby reducing the time and effort involved in testing. Recombinant PRRSV nucleoprotein antigen and the PCV2 capsid antigen were coupled to fluorophore-dyed beads with distinct spectral addresses. Weekly serum samples from 72 pigs that were experimentally exposed to either PCV2, PRRSV, or both PCV2 and PRRSV were used to validate the microbead assay (MBA) in comparison with the "gold standard" enzyme-linked immunosorbent assays. The kinetics of the PCV2- and PRRSV-specific antibody responses measured by the microbead assay were comparable to those of the standard assays; Spearman's rank correlations were 0.72 (P < 0.001) for PRRSV and 0.80 (P < 0.001) for PCV2. Diagnostic sensitivity and specificity were determined using field sera whose positive or negative status was determined by the standard tests. The diagnostic sensitivity and specificity were both 98% for PCV2 and were 91% and 93%, respectively, for PRRSV (kappa coefficients, 0.85 and 0.67 for PCV2 and PRRSV, respectively). Multiplexing did not interfere with assay performance or diagnostic sensitivity. Therefore, the described study demonstrates proof of concept for the development of more versatile and economical microbead array-based multiplex serological test panels for veterinary use.