A multiplexed immunoassay for simultaneous detection of antibodies to PRRSV, Actinobacillus pleuropneumoniae and Salmonella in pigs

To offer routine diagnostic analyses that are cheaper and less time-consuming than our current in-house ELISAs, we have developed and evaluated a Luminex-based multiplexed immunoassay that facilitates simultaneous detection and distinction between antibodies to porcine reproductive and respiratory syndrome virus (PRRSV) Type 1 and Type 2, Actinobacillus pleuropneumoniae (App) serovars 2, 6 and 12 as well as Salmonella Typhimurium and Salmonella Choleraesuis.

The multiplexed immunoassay employs the same antigens that are used for our in-house ELISAs, including crude viral lysates of PRRS strains as well as purified bacterial lipopolysaccharides from App serovars and Salmonella subtypes. Antigens were coupled separately to seven batches of magnetic beads with variant internal fluorescence, plexed and incubated with serum from infected or non-infected pigs. Bound serum antibodies were detected with layers of biotinylated anti-pig antibodies and fluorescently labelled streptavidin. Samples were analyzed in a Bio-Plex 200 reader, results were read as median fluorescence intensities and a sample-to-positive ratio was calculated. Receiver Operator Characteristic (ROC) curve analysis was used for comparing the quality of the assay with our in-house ELISAs.

Converting individual ELISAs with different assay conditions into a single multiplex analysis introduces the challenge of defining optimal assay conditions that can be applied to all antigen-antibody interactions included. By testing various assay conditions such as reactant concentrations, temperature, reaction time and buffer composition in singleplex assays, we succeeded in defining optimal assay conditions that could be applied to all analytes. Antigen-specific reactivities measured in a singleplex format were retained when combining beads coupled with antigens from the various pathogens in a multiplex format, indicating limited cross-reactivity. When validating the multiplex assay with a large number of sera from infected and noninfected pigs we observed a good correlation with our in-house ELISAs.

A bead-based multiplex assay was designed to simultaneously detect and distinguish antibodies in a single serum sample towards Type 1 and 2 strains of PRRSV, App 2, 6 and 12 as well as S. Typhimurium and S. Choleraesuis. The assay has high sensitivity and specificity for detection of antibodies in pig serum to all agents included and shows good overall agreement with our well-established in-house ELISAs. We expect that implementation of multiplex analysis in routine serological diagnostics in pigs will lower the cost of analyses and decrease response time.

Disclosure of Interest: None Declared.