Hrp conjugated goat anti mouse igg

The SARS-CoV PUMC01 strain ^{46 (link)} was diluted with coating buffer to 4 × 10⁻⁵ TCID₅₀/mL (bicarbonate/carbonate coating buffer, 0.05 M, pH 9.6). The virus was then coated on 96-well plates with 100 μL (4 × 10⁻⁴ TCID₅₀) of virus per well for 12 h at 4 °C. The coating buffer was removed from the wells and 100 μL of blocking buffer was added per well and incubated for 12 h at 4 °C (2% BSA in PBS, pH 7.4. GIBCO, Carlsbad, USA). The plates were washed twice with PBS (pH 7.2). The mouse monoclonal antibodies against SARS-CoV and control mAb against HIV-P27 were diluted to 0.001 μg/μL in the blocking buffer, added to the wells separately and incubated for 1 h at 37 °C. The plates were washed twice with PBS and the secondary antibody (HRP-conjugated goat anti-mouse IgG, (GeneTex, Irvine, USA) diluted 1:1000 in blocking buffer) was added to each well and incubated for 1 h at 37 °C. The secondary antibody was removed and the plate was washed three times with PBS, then 100 μL of TMB substrate solution (Promega, Madison, WI, USA) was added to each well. After incubation for 30 min at room temperature, the absorbance of the test wells was read at 450 nm (A₄₅₀).