Hace 2 fc

For the ACE-2 binding study, the Alhydrogel®-RBD vaccine formulations were blocked overnight with 0.1% BSA. After hACE-2-Fc (LakePharma) was added, the samples were incubated for 2 hours at RT. After incubation, the Alhydrogel® was spun down at 13,000 × g for 5 min. The hACE-2-Fc which did not bind to the RBD on the Alhydrogel® remained in the supernatant. The hACE-2-Fc content in the supernatant was quantified by ELISA using 96-Well MaxiSorp Immuno plates (ThermoFisher) coated overnight with 200 ng/well of RBD219-WT protein. After blocking with 0.1% BSA, 100 µL supernatant samples were added to each well. Plates were washed 4 times with an automated plate washer using PBS with Tween (PBST). A secondary antibody against human Fc was used to detect hACE-2-Fc bound the proteins on the plate. Plates were washed 5 times with an automated plate washer using PBST before 100 µL TMB solution was added. The enzymatic reaction was stopped with HCl and absorption readings were made at 450 nm. The final concentration of the hACE-2 bound on the Alhydrogel® was determined as [hACE-2-Fc on Alhydrogel®] = [Total hACE-2-Fc] – [hACE-2-Fc in supernatant].

384-well Maxisorp plates (Thermo Fisher) were coated overnight at room temperature with 3 μg/mL of SARS-CoV-2 S2P (Pallesen et al., 2017 (link)) in 20mM Tris pH 8 and 150mM NaCl. Plates were slapped dry and blocked with Blocker Casein in TBS (Thermo Fisher) for one hour at 37°C. Plates were slapped dry and NHP sera was serially diluted 1:4 in TBST with an initial dilution of 1:4 for hACE2 competition or 1:2 for antibody competition. Random primary amine biotinylated (Thermo Fisher) hACE2-Fc, CR3022 (Yuan et al., 2020 (link)), or S309 (Pinto et al., 2020 (link)) were added, bringing the concentration of each well to the EC₅₀ values of 0.2nM, 2nm, and 0.01nM, respectively. Plates were left for one hour at 37°C, then washed 4x with TBST using a 405 TS Microplate Washer (BioTek) followed by addition of 1:500 streptavidin-HRP (Thermo Fisher) for one hour at 37°C. Plates were washed 4x and TMB Microwell Peroxidase (Seracare) was added. The reaction was quenched after 1-2 minutes with 1 N HCl and the A450 of each well was read using a BioTek plate reader (BioTek). Data plotted and fit in Prism (GraphPad) using nonlinear regression sigmoidal, 4PL, X is log(concentration) to determine EC₅₀ values from curve fits with upper and lower constraints determined by uncompeted ELISA per antigen.