Ctl immunospot s6

Focus forming reduction neutralization assays (FRNT) were performed in a similar manner to those described (61 (link)) and were used to determine the 50% and 90% effective neutralizing concentration of immunoglobulins against Zika virus and DENV. Briefly, four-fold serial dilutions of antibody (2.5 mg/mL to 19 ng/mL) were mixed with ~80 focus forming units (FFU) of virus, incubated at 37°C for 1 hour, then added to Vero cell monolayers in 96 well plates for 1 hour at 37°C to allow virus adsorption. Cells were overlaid with 1% methylcellulose and incubated for two days. Monolayers were fixed for 1 hour at room temperature, washed and permeabilized. Infected cell foci were stained by incubating cells with 500 ng/mL of flavivirus cross-reactive mAb 4G2 for 1 hour at 4°C, washed and detected by incubating cells with a 1:5000 dilution of horseradish peroxidase conjugated goat anti-mouse IgG for 1 hour. After washing, staining was visualized by addition of TrueBlue™ detection reagent. Infected foci were enumerated using a CTL-Immunospot S6 (Cellular Technology Limited).

IFN-γ ELISPOT was performed using IFN-γ ELISPOT Kit as manufacturer instructions (eBiosciences, San Diego, CA, USA) in PVDF plates (Millipore, Billerica, MA, USA). In brief, plates were covered with 100 µL of capture antibody and after overnight incubation at 4°C washed and blocked with RPMI-1640 +10% FCS. Subsequently, 10⁶ splenocytes in 100 µL of RPMI supplemented with 10% FCS were seeded per well and peptides (T*-1, QNT-5, QNT-Y, HA) at a final concentration of 10 µg/mL added. After 48 hours incubation at 37°C, 5% CO₂ plates were washed 3 times with 1× PBS, 0.05% Tween-20 and cytokine revealed by successive incubation with detection antibody, wash, peroxidase-HRP incubation, wash and final visualization with AEC substrate. Plates were scanned and spots counted with the help of an immunospot Analyzer (CTL-immunospot S6, Cellular Technology Limited, USA).