Immunospot s6 macroanalyzer

Virus samples were titered on Vero cells using infectious center assays (ICA) or focus-forming assays (FFAs). Vero cells were seeded at 1.2 × 10⁴ cells/well in 96-well plates, cultured overnight, and infected with 100 µL of serial dilutions of virus samples for 4 h. The virus inocula were aspirated and replaced with 100 µL of DMEM plus 5% FBS and 20 mM NH₄Cl or 1% carboxymethylcellulose in modified Eagle’s Medium supplemented with 2% heat-inactivated FBS and 10 mM Hepes pH 7.4. For ICA, 48 h post-infection, cells were fixed with 4% paraformaldehyde (PFA, Electron Microscopy Science) and stained with RuV pAb and fluorescently labeled secondary antibody, and infection was quantitated by fluorescence microscopy. For FFA, cells were fixed by adding 100 µL of prewarmed 1% PFA in PBS to the overlay and incubating for 1 h. Cells were washed with PBS, permeabilized with 0.1% saponin in PBS containing 0.1% bovine serum albumin (BSA), incubated with RuV pAb followed by incubation with horseradish peroxidase-conjugated rabbit anti-goat IgG (Seracare, Milford, MA). Foci were developed using TrueBlue Peroxidase substrate (Seracare) and quantified using an ImmunoSpot S6 Macroanalyzer with Biospot 7.0.9.10 software (Cellular Technologies, Shaker Heights, OH). The titer for psVSV-RuV was determined by ICA, with initial infection for 2 h and scoring of eGFP-positive cells 24 h post-infection.

Serial dilution of mAbs were incubated with 100–150 FFU of CHIKV 181–25 vaccine strain for 1 h at 37°C. Antibody-virus complexes were then added to Vero cell monolayers in 96-well plates. Infection proceeded for 90 min at 37°C and cells were then overlaid with 0.5% carboxylmethylcellulose in Modified Eagle Media (MEM), supplemented with heat inactivated 2% FBS and 10 mM Hepes pH 7.4. Plates were fixed 16 h post-infection with 1% PFA diluted in PBS. After fixation, plates were incubated with 250 ng/mL of 5G11 (USAMRIID) and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG in PBS supplemented with 0.1% Saponin and 0.1% BSA. Foci were then visualized using TrueBlue Peroxidase substrate (KPL). Developed foci where quantified on ImmunoSpot S6 Macroanalyzer (Cellular Technologies Ltd.). Infection in wells containing mAb was calculated relative to wells containing CHIKV 181/25 alone. Non-linear regression analysis was performed using Prism 7 software (GraphPad Software, La Jolla CA).