Neutralization assays with SARS-CoV-2 WT (plaque reduction neutralization test)

Plaque reduction neutralization tests (PRNTs) with SARS-CoV-2/human/Germany/Heinsberg-01/2020 were performed in Vero E6 cells. 24-well plates were seeded with 1.5·10⁵ Vero E6 cells per well. The following day, nanobodies were subjected to a two-fold dilution series. 120 μl of each nanobody dilution was mixed with 120 μl of OptiPRO SFM cell culture media (Thermo Fisher Scientific) containing 80 pfu of SARS-CoV-2. After 1 hour at 37°C, 200 μl of each mixture was added to 24-well plates. After 1 hour at 37°C, the inoculum was removed, and cells were overlaid with a 1:1 mixture of 1.5% carboxymethylcellulose (Sigma Aldrich) in 2x MEM (Biochrom) with 4% FBS (Thermo Fisher Scientific). After a 3-day incubation at 37°C, cells were fixed with 6% formaldehyde and stained with 1% crystal violet in 20% ethanol. Plaques were counted manually. IC₅₀ values were calculated using the “log(inhibitor) vs. normalized response” equation in GraphPad Prism.