Immunospot analyzer

The FFA was performed as previously described (Case et al., 2020 (link)). In brief, Vero E6 cells were plated into 96 well plates at 24,000 cells/well and incubated overnight. Previously propagated SARS-CoV-2 stocks were titrated by plaque forming unit (pfu) assay and diluted to 30 pfu in 15 μL. To the virus, 15 μL of antibody dilutions were added such that the final antibody dilution was 1:10 to 1:1280 in two-fold dilutions and this was incubated at 37°C for 1 hour. All virus and antibody dilutions were prepared in Opti-MEM media with 2% FBS. 30 μL of neutralized virus was then added to the confluent Vero E6 cells and incubated for 1 hour at 37°C. 150 μL of overlay media (Opti-MEM, 2% FBS, 2% Methylcellulose) was then added to each well and incubated for 48 hours at 37°C. Following infection, the plates were fixed using formaldehyde and subsequently blocked for 30 minutes with perm buffer containing 0.1% bovine serum albumin and 0.1% saponin. SARS-CoV-2 RBD and N protein immunized alpaca polyclonal serum was used as primary antibody at 1:5,000 dilution in perm buffer, and anti-Llama-HRP secondary was used at 1:20,000 dilution. Plates were developed with TrueBlue (SeraCare) substrate and imaged with an Immunospot analyzer.