Microsphere immunoassay (MIA)

Detection of NHP and dog IgG was conducted as previously described [15]. Briefly, microspheres coupled with EBOV VP40, NP, GP, SUDV GP, MARV GP, LASV GP, and control beads coupled to PBS and BSA were combined and diluted in PBS-1% BSA at a dilution of 1/200. Fifty μL (containing approximately 1,250 beads of each type) of the microsphere suspension were added to each well of black-sided 96-well plates. Serum samples were diluted 1:1000 (NHP) or 1:200 (dog) in PBS-BSA and dog samples were serially diluted two-fold for titrations. Fifty μL of diluted serum were added to the microspheres in duplicate and incubated for 30 min on a plate shaker set at 700 rpm in the dark at room temperature. The plates were then washed twice with 200 μL of PBS-BSA using a magnetic plate separator (Millipore Corp., Billerica, MA). Fifty μL of red-phycoerythrin (R-PE) conjugated F(ab’)2 fragment rabbit anti-dog IgG (H+L) (Rockland Immunochemicals, Inc) were added at 1 μg/mL to the plates and incubated on a 96-well plate shaker for another 45 min. The plates were washed twice, as above, and microspheres were then resuspended in 100 μL of sheath fluid and analyzed on the MAGPIX Instrument (MilliporeSigma). Data acquisition detecting the median fluorescence intensity (MFI) was set to 50 beads per spectral region. Antigen-coupled beads were recognized and quantified based on their spectral signature and signal intensity, respectively.

Assay cutoff values were calculated first by taking the mean of technical duplicate values using the average MFI (indicated as a solid black line). Cut-offs were generated by determining the mean of 1/3^rd serum samples (22) showing the lowest MFI values plus three standard deviations as determined by Microsoft Office Excel program. Serum samples showing MFI values greater than the cutoff value were considered positive. If the cut-off value fell below the internal control BSA cutoff, the highest BSA-bead reading of 45 MFI was used (indicated by a dotted line). Graphical representation of the data was done using Prism, Graphpad Software (San Diego, CA).