Detection Of Salmonid Igm Specific To The Piscine Orthoreovirus Outer Capsid Spike Protein Sigma 1 Using Lipid-Modified Antigens In A Bead-Based Antibody Detection Assay

Bead-based multiplex immunoassays are promising tools for determination of the specific humoral immune response. In this study, we developed a multiplexed bead-based immunoassay for the detection of Atlantic salmon (Salmo salar) antibodies against Piscine orthoreovirus (PRV). Three different genotypes of PRV (PRV-1, PRV-2, and PRV-3) cause disease in farmed salmonids. The PRV outer capsid spike protein sigma 1 is predicted to be a host receptor binding protein and a target for neutralizing and protective antibodies. While recombinant sigma 1 performed poorly as an antigen to detect specific antibodies, N-terminal lipidmodification of recombinant PRV-1 sigma 1 enabled sensitive detection of specific IgM in the bead-based assay. The specificity of anti-PRV-1 sigma 1 antibodies was confirmed by western blotting and pre-adsorption of plasma. Binding of non-specific IgM to beads coated with control antigens also increased after PRV infection, indicating a release of polyreactive antibodies. This non-specific binding was reduced by heat treatment of plasma. The same immunoassay also detected anti-PRV-3 sigma 1 antibodies from infected rainbow trout. In summary, a refined bead based immunoassay created by N-terminal lipid-modification of the PRV-1 sigma 1 antigen allowed sensitive detection of anti-PRV-1 and anti-PRV-3 antibodies from salmonids.