Optimizing bead based multiplex immunoassays for detection of piscine orthoreovirus specific antibodies in Atlantic salmon (Salmo salar)

Introduction Future growth in aquaculture relies strongly on the control of diseases and pathogens. Effective long-term protection against viral infections is not yet fully understood for fish, and tools to monitor adaptive immune responses are few. Assays that can detect specific antibodies produced in response to viral infection in fish are still in their early development. Multiplex bead based assays are sensitive, and allow detection of multiple antigen-specific antibodies simultaneously in very small amounts of plasma or serum. Lipid modification of antigens to increase their antibody binding is a potential strategy to further increase assay sensitivity. Here, we aim to optimize assays for detection of antibodies targeting Piscine orthoreovirus (PRV), the virus associated with the disease Heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon. Methodology A bead-based multiplex assay has previously been developed for detection of PRV-specific antibodies. Here, PRV antigens and other salmonid virus antigens were lipid modified using the Twin Arginine Translocase (Tat) pathway in recombinant E. coli to increase their binding to antigen-specific IgM. Results Using the bead based immunoassay we detected plasma IgM directed against recombinant outer capsid protein µ1 and the non-structural protein µNS of PRV. Results from using this assay on plasma from a PRV cohabitation challenge trial indicated that antibody production was initiated approximately two weeks after the peak phase of PRV infection, coinciding with typical HSMI pathology. Other outer capsid proteins from PRV and other salmonid virus antigens were tested in the assay, but failed to bind specific antibodies. Lipid modified versions of these antigens were tested for increased binding of specific IgM. Conclusion Sensitive bead based multiplex immunoassays combining several viral antigens would be a valuable tool for disease control in salmon aquaculture. Antigen modification may further increase the sensitivity of such assays.