Anti sars cov 2 spike receptor binding domain polyclonal ab

Vero E6 cells were seeded in 96-well plates with 2 × 10⁴ cells/well in 5% DMEM 24 h prior to infection and checked to verify ≥80% confluency. Dilutions of virus and/or virus treated with inactivation reagents in Opti-MEM (50 μl) were incubated on Vero E6 monolayers for 2 h absorption at 37°C without rocking. After absorption, cells were overlaid with 2% methylcellulose in Opti-MEM (Life Technologies) supplemented with 2% FBS, 2.5 μg of amphotericin B (MilliporeSigma), and 20 μg/ml ciprofloxacin (MilliporeSigma) for 72 h at 37°C in a 5% CO₂, humidified incubator. At 72 hpi, methylcellulose was carefully removed and the cells were fixed with a 1:1 methanol/acetone mixture for 30 min at RT, then blocked with 200 μl of 5% milk in 1× PBS for 20 min.Cells were incubated with an anti–SARS-CoV-2 spike receptor-binding domain polyclonal Ab (Gentaur) at 1:3000 overnight at 37°C. Cells were washed to remove excess Ab, then incubated with a secondary HRP-conjugated anti-human IgG for 1 h at 37°C. Cells were washed to remove excess Ab and foci were visualized using the TrueBlue peroxidase substrate (SeraCare Life Sciences) incubated for 1 h at RT with rocking prior to imaging with an ELISPOT reader for foci quantification. Data are reported as focus forming units per milliliter.