2.18. Plaque reduction neutralization test

Live virus experiments were performed in approved biosafety level 3 (BSL-3) facilities at Georgia State University and strictly followed the approved standard operation procedures. Neutralizing Abs were measured by plaque reduction neutralization assay (PRNT) using ancestral B.1 Wuhan virus (BEI# NR-52281). For PRNT assay Vero E6-TMPRSS2-T2A-ACE2 cells (BEI NR-54970) were seeded in 6-well plates at 200,000 cells/well in M199 medium with Earle’s salts (10X) supplemented with 5% inactivated fetal bovine serum, buffered with 3% sodium bicarbonate and 1% Penicillin-Streptomycin for 3 days (preformed monolayers). Serum samples were heated at 56oC for 1 hour to inactivate complement. Heat-inactivated serum samples were diluted at 1:10 and were further serially diluted 4-fold from 1:10 to 1:5120. 60μl of serially diluted serum was mixed in 96-well plates with an equal volume of 100 PFU of SARS-CoV-2. Serum/virus mixtures were incubated for 30 mins at 37°C. After incubation, 100 ul of serum/virus mixture was transferred to monolayered cells and incubated for 1 h at 37°C with gentle rocking every 15 minutes. After incubation, 2 ml of 1% low melting agarose media was added to each well and plates were incubated at 37°C for 2 days. After two days, plates were overlaid with 2% neutral red in 1% low-melting agarose for visualizing plaque formation. The number of plaques were counted and recorded in each well. The neutralization titer for each dilution was calculated as follows: NT = [1-(plaque count with sera/plaque count without sera)]x100.