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This assay could aid in the rapid detection of subclinical shedders of S. equi, enabling quicker treatment and helping to limit the spread of strangles in equine populations.

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Abstract.A multiplex enzyme-linked immunosorbent assay (ELISA) to detect antibodies to Bovine viral diarrhea virus (BVDV), Bovine herpesvirus 1 (BHV-1), Bovine parainfluenza virus 3 (BPV-3), and Bovine respiratory syncytial virus (BRSV) was developed on the commercially available Meso Scale Discovery (MSD) platform. The multiplex ELISA used the same antigens (relatively crude lysates of cultured virus) that are used in the current in-house separate ELISAs. Testing a panel of field samples demonstrated the multiplex assay could simultaneously detect the presence of several antibodies in a single assay and that there was good agreement between the multiplex ELISA and the in-house ELISAs. The use of a multiplex ELISA could offer significant benefits for routine serological testing through reduced staff time, reduced reagent cost, and streamlined laboratory operation.Key words: Bovine respiratory disease; Meso Scale Discovery; multiplex enzyme-linked immunosorbent assay; serology. Mayers and SawyerThe MSD platform combines electrochemiluminescence (ECL) and microarray-style spatial gridding technology to allow the monitoring and detection of up to 10 separate assays within a single well of a 96-well microtiter plate. 5 The MSD 96-well plates are fabricated to contain separate carbon electrodes (from 1 to 10) within each well, which can be coated with different biological molecules. For the current study, the electrodes were coated with the different antigens used in the individual ELISAs. The fundamental basis of the assay is the same as a typical serology ELISA in that a series of incubation and wash steps in 96-well plate format result in the capture of antibodies in a serum sample and subsequent detection by addition of a labeled anti-species secondary antibody. In this case, the label is a proprietary MSD electrochemiluminescent molecule, SULFO-Tag™.a Generation and measurement of the electrochemiluminescent signal are achieved through electrical stimulation of the SULFO-Tag a -labeled antibodies, where only labels in close proximity to the carbon electrodes are detected. Measurement of the resulting light signal from around the individual electrodes occurs in the MSD Sector Imager instrument, which effectively performs the same function as a microtiter plate reader. 8Initial assay development and optimization of parameters such as antigen concentration, serum dilution, and washing conditions were carried out at the AHVLA laboratories in a single-plex format on the MSD platform, where the wells of the MSD 96-well plates contained a single carbon electrode. This provided the opportunity to optimize each assay individually before combining in a multiplex format.Multiplex plates, where different antigens were adsorbed onto the individual carbon electrodes within each well, were printed by MSD using proprietary robotic printing systems. In the current study, 7-plex plates were printed, which consisted of a carbon electrode coated in each of the 4 positive antigens, and 3 electrodes coated in negative antigen (lysate...