Spectomax plus

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.