Figure 3. Fluorescence‐based assay to assess the neutralization potency of VHH antibodies.

1. Vero E6 cells were infected with SARS‐CoV‐2, pre‐incubated with indicated concentrations of the neutralizing VHH‐72 (Wrapp et al, 2020a). Cells were fixed two days after inoculation, stained with sets of anti‐RBD (green) and anti‐S1ΔRBD (red) nanobodies (see Fig 1B and C), and analyzed by CLSM. We used a cocktail of such fluorophore‐labeled VHH antibodies to ensure that negative fluorescence readings truly indicated neutralization and thus absence of viral infection and not just masking of the IF epitope by the tested nanobody. See Appendix Fig S1 for independent biological replicates.
2. Vero E6 cells were infected as in A. Viral RNA in the culture supernatants was quantified by reverse transcription (RT)–qPCR. RNA signals are normalized to the respective minus‐VHH controls (100%). Note the log10 scale of the y‐axis. "Inoculum" (red dotted line) marks the RNA level detected at the time of infection. See also Appendix Fig S1.