Quantifying HSV-2-Specific Antibodies in Mice

CSF and serum were collected from mice as previously described^{9 (link)}. The serum and CSF were then diluted with PBS containing 0.1% BSA in a 1:1 ratio. Plates (96-well) were coated with 100 μl of heat-inactivated or PFA-inactivated purified HSV-2 (10⁴ to 10⁵ plaque-forming units equivalent per 100 μl) for virus-specific immunoglobulin measurement or goat anti-mouse immunoglobulin (SouthernBiotech, 1010-01, 1:1,000) and then incubated overnight at 4 °C. These plates were then washed with PBS–Tween 20 and blocked for 2 h with 5% FBS in PBS. Samples were then plated in the wells and incubated for at least 4 h at room temperature. After being washed in PBS–Tween 20, HRP-conjugated anti-mouse immunoglobulin antibodies (SouthernBiotech, 1010-05, 1:5,000) were added in the wells for 1 h, followed by washing and addition of TMB solution (eBioscience). Reactions were stopped with 1 N H₂SO₄, and absorbance was measured at 450 nm. The total antibody titres were defined by using an immunoglobulin standard (C57BL/6 mouse immunoglobulin panel; SouthernBiotech).