Maxsorp plates

To evaluate antibody responses in the serum, MaxSorp plates (Nunc) were coated overnight with 0.2 μg/well of either HA, gp120, or ovalbumin. The plates were blocked by 3% skimmed milk for (1h) at room temperature. After blocking, the plates were incubated for 1h with serial dilutions of pre- and post-immune sera. The samples were initially diluted at 1:20 in PBS followed by a 1:5 serial dilution in the same buffer. The wells were then washed 3x with PBST, incubated for 1h with a 1:5000 dilution of either: goat anti-murine IgM (IgG-HRP, Southern Biotech) or sheep anti-murine IgG (HRP-IgG, GE Healthcare). The plates were washed (3x PBST) and then developed using TMB substrate (Dako). The developer reaction was quenched by the addition of 1N sulphuric acid and the plates were then read at 450nm using a Spectomax Plus (Molecular Devices). Antibody endpoint dilutions were interpolated using Graphpad Prism version 5.0 (GraphPad Software Inc.), as previously described (Kanekiyo et al., 2013 (link); Yassine et al., 2015 (link)). The concentrations of IL-6 and sIL6R in mouse sera were quantified according to the instructions provided in commercial sandwich ELISA kits from R&D Systems (Quantikine-M6000B) and Abcam (ab203360), respectively.

Serum from HLA-A2 mice was collected by aspiration from whole blood prior to vaccination or infection and at 2-weeks and 8-weeks after viral exposure by centrifugation at 11,000 g for 5min. Samples were tested for vaccinia-specific serum antibody by neutralizing antibody ELISA(Frey et al., 2002 (link)). 96-well maxSorp plates (ThermoFisher 44–2404-21) were coated with crude extract from lysed BSC-1 cells infected with live ACAM2000 smallpox vaccine. Plates were washed 3x with PBS and wells were coated with ~5x10⁴ PFU live ACAM2000 diluted in carbonate coating buffer (0.1M Na₂CO₃ 0.1M NaHCO₃ pH 9.3) in 100ul overnight at 4^○C. Wells were washed and blocked with blocking buffer containing 5% BSA in PBS for 30min at RT. Plates were washed 3x with PBS and serum samples diluted 1:20 in blocking buffer were added (in duplicate) to wells and incubated for 2 hours at RT. Plate was washed 3x with PBS, then 100uL anti-mouse HRP-conjugated antibody (Sigma A8924) was diluted 1:2500 in blocking buffer was added and allowed to incubate for 1h. Plates were washed 3x with PBS and 75ul of True Blue peroxidase substrate (KPL, 71–00-65) was added to each well and incubated for 15min in the dark at RT. 75ul of 1N HCl was added to each well and OD measured at 450nm.