Multiplex testing for PRRSV, SIV, and PCV2 antibodies using a Multiplexed Fluorometric Immuno Assay (MFIA)

Porcine Respiratory Disease Complex is a significant problem for the swine industry. It is caused by the interaction of multiple non-infectious and infectious factors, including PRRSV, SIV, and PCV2. Measurement of antibodies to these agents is routinely performed for monitoring herd status or optimizing vaccination protocols. Testing for all these agents at the same time in a single assay could potentially save a lot of labor, time, and cost compared to traditional serological methods.

Antigens (ag): recombinant proteins from PRRSV type 1 and 2, AIV, and PCV2 were expressed in E. coli (PRRSV, PCV2) or using Baculovirus (AIV). The purity of the recombinant proteins was evaluated using SDS-PAGE. The identity of each protein was further confirmed by Western blot analysis with anti-His, PRRSV, AIV or PCV2 antibodies.

Ag coupling: ag were coupled to 4 different sets of magnetic beads (Magplex®, Luminex) using proprietary methods. For each ag, the optimal conditions providing the highest signal-to-noise ratio were determined.

1-plex MFIA: all incubations were done at room temperature in the dark on a shaker. Diluted samples were incubated for 1 hour with relevant antigencoated bead suspension. After serial washings biotinylated goat anti-swine IgG was added and the plates incubated for 30 minutes. The plates were further washed prior to adding streptavidin – phycoerythrin and incubated for 30 minutes. After final washings the beads were suspended in assay buffer and the plates were read using a dual-laser instrument (LX200®, Luminex). Assay parameters were optimized to provide the most consistent and reproducible results.

4-plex MFIA: once the four 1-plex assays worked properly the 4 bead suspensions were mixed and used in a 4-plex assay. Possible interaction between ag and non-immune binding were examined using selected non-immune, homologous and heterologous immune serum controls.

ELISAs: samples were also tested using the PRRS X3 Ab, the Influenza A Ab (IDEXX) and the Ingezym Circo IgG (Ingenasa), .

Test evaluation: test sensitivity and specificity were evaluated using serum samples of known status.

Results obtained with the 4-plex MFIA were very similar to those obtained with the corresponding ELISAs for PRRSV type 1 and 2 and SIV. By contrast a poor correlation was noticed for PCV2.

This MFIA appears to be an interesting alternative to traditional ELISAs for PRRSV and SIV as it offers similar diagnostic performances while saving labor, time, and cost.

Disclosure of Interest: A. Broes Conflict with: Biovet, I. Caya Conflict with: Biovet, M. Bertrand Conflict with: Biovet.