Tmb microwell peroxidase

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.

For ELISA experiments with NTD-targeted mAbs, 384-well Maxisorp plates (Thermo Fisher Scientific) were coated overnight at 4 °C with 2 μg/mL of S glycoprotein in 20 mM HEPES pH 8 and 150 mM NaCl. For ELISA experiments with RBD-targeted mAbs, 384-well Maxisorp plates (Thermo Fisher Scientific) were coated overnight at 4 °C with 4 µg/mL of hACE2-His in 20 mM Sodium Phosphate pH 8 and 100 mM NaCl. The antibodies that were used were purified previously^{13 (link),36 (link)}. Plates were slapped dry and blocked with Blocker Casein in TBS (Thermo Fisher Scientific 37532) for one hour at 37 °C. Plates were slapped dry and mAbs were serially diluted in TBST with an initial concentration of 50 µg/ml. Plates were left for one hour at 37 °C and washed 4X with TBST, then 1:5000 Goat anti-Human (Thermo Fisher Scientific A18817) was added. Plates were left for 1 h at 37 °C and washed 4x with TBST, and then TMB Microwell Peroxidase (Seracare 5120-0083) was added. The reaction was quenched after 4 min with 1 N HCl and the A450 of each well was read using a BioTek plate reader.

96-well Maxisorp plates (Thermo Fisher) were coated overnight at 4°C with 2 μg/mL of NiV G or LayV G central stalk stabilized ectodomain in 50mM Tris and 150mM NaCl at pH 8. Plates were slapped dry, washed 3X in Tris Buffered Saline Tween (TBST) and blocked with Blocker Casein (ThermoFisher) for 1 h at 37°C. Plates were slapped dry and washed 4X in TBST. 1:4 serial dilutions of NHP sera were made in 50 μL TBST and incubated at 37°C for 1 h. Plates were slapped dry and washed 4X in TBST followed by addition of 50 μL 1:5000 Goat anti-Human IgG Fc Secondary Antibody with HRP (Invitrogen) for one hour at 37°C. Plates were slapped dry and washed 4X in TBST followed by addition of 50 μL TMB Microwell Peroxidase (Seracare). The reaction was quenched after 4 minutes with 1 N HCl and the A450 of each well was read using a BioTek Synergy Neo2 plate reader. Data was plotted and fit in Prism (GraphPad) using nonlinear regression sigmoidal, 4PL, X is log(concentration).