SARS-CoV-2 Spike Protein Binding Assay

The SARS-CoV-2 spike (S) ectodomain containing the prefusion stabilizing hexapro mutations [74 (link)] and a mutated furin cleavage site was produced and purified from Expi293 cells as previously described [75 (link)]. The SARS-CoV-2 S protein was diluted to 0.003 mg/mL in PBS and used to coat 384-well Nunc Maxisorp plates (Thermo Fisher) overnight at room temperature. The plates were slapped dry and blocked for 1 hour at 37°C using Casein in PBS (Thermo Fisher). Following blocking, the plates were again slapped dry and sera from infected bats was added to the plates beginning at a 1:30 dilution in TBST followed by a 1:3 serial dilution thereafter. Plates were incubated for 1 hour at 37°C, slapped dry, and washed four times with TBST. Recombinant protein A/G conjugated to HRP (Thermo Fisher) diluted 1:500 in TBST was added each well and the plates were incubated for 1 hour at 37°C then washed four times with TBST. To measure binding titers, TMB Microwell Peroxidase (Seracare) was added to each well and, after 2 minutes, the reaction was quenched with 1 N HCl. The absorbance at 450 nm was measured using a BioTek Neo2 plate reader and analyzed in GraphPad Prism 9 with ED₅₀ values being determined using a four-parameter logistic regression model. Two technical replicates were performed for each sample.