Fluorescence Microsphere Immunoassay for Detection of Antibodies to Porcine Reproductive and Respiratory Syndrome Virus and Porcine Circovirus type 2 using Protein A, Protein G, and Protein A/G

The purpose of this study was the development of multiplex fluorescence microsphere immunoassay (FMIA) for the detection of porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) specific IgG antibodies by incorporation of non-species-specific conjugates Protein A, G, and A/G in place of the secondary antibody. A total of 205 serum samples obtained from pigs experimentally infected with PRRSV and/or PCV2 were tested. For the production of recombinant antigens, PRRSV nucleoprotein (N) and PCV2 capsid protein (CP) were expressed in Escherichia coli and purified on a nickel affinity column. The purified proteins were covalently coupled to carboxylated microsphere beads. Four target antigens were assembled into a single multiplex and tested against sera from swine infected or co-infected with PRRSV or PCV2. Protein A, G, and A/G have been tested in place of secondary conjugate porcine IgG. All conjugates were capable of detecting antibody and the detection of IgG responses against PRRSV and PCV2 antigen targets were varied with A> A/G> G.In the absence of species specific reagents the incorporation of Protein A, G, and A/G provide a suitable substitutes.