Pe conjugated goat anti human igg polyclonal antibodies

Fabs were titrated on yeast surface displayed RBD variants to determine EC₅₀ concentrations. Briefly, induced cells were aliquoted (0.2 OD600 / well) into 96-well plates and washed with PBSF. Cells were then resuspended in 50 μL of Fab solution diluted in PBSF and incubated on ice for 2 hours. Cells were subsequently washed twice with PBSF and labeled with 50 μL of APC-conjugated monoclonal mouse anti-hemagglutinin tag (HA).11 antibody (BioLegend, Cat # 901524), PE-conjugated goat anti-human IgG polyclonal antibodies (Southern Biotech, Cat # 2040-09), and propidium iodide (Invitrogen, Cat #P1304MP) for 15 min on ice. Cells were washed twice with PBSF before analyzing via flow cytometry on a BD FACS Canto II (BD Biosciences). Mean anti-human IgG PE fluorescence signal values were fitted as nonlinear regression curves in GraphPad Prism using the following equation: Y=Y_minimum+ (X^Slope) * (Y_maximum-Y_minimum)/(X^Slope + EC₅₀^Slope)), where X is the Fab concentration and Y is the PE MFI. Concentrations displaying hook effects, defined as concentrations higher than those generating the maximum PE MFI signal, were excluded from analysis. The percent reduction in binding activity to the mutant RBD relative to the WT SARS-CoV-2 RBD was calculated using the following equation: (WT EC₅₀/Variant EC₅₀) * 100.

To assess binding breadth, IgGs and recombinant human Fc-conjugated hACE2 (Sino Biological, 10108-H02H) were tested against the panel of 17 sarbecovirus RBDs expressed by yeast display as previously used^{17 (link)}. EBY100 yeast were transformed with a plasmid encoding the yeast mating protein Aga2p linked to sarbecovirus RBD on the C terminus. To induce RBD expression, 0.5 OD₆₀₀/ml of yeast were transferred to SGCAA media and cultured at 20 °C for 16–20 h with 180 rpm shaking. Next, IgGs and hACE2 were titrated via 3-fold serial dilutions from 100 nM to 0.5 pM. RBD-expressing cells were aliquoted into 96-well plates and incubated with 100 µl of 100 nM IgG for 30 min on ice. Next, cells were washed twice with PBSF (1× PBS, 0.1% BSA) before secondary detection with 1:100 dilutions of APC-conjugated mouse anti-hemagglutinin tag (HA).11 antibody (BioLegend, 901524), PE-conjugated goat anti-human IgG polyclonal antibodies (Southern Biotech, 2040-09), and propidium iodide (Invitrogen, P1304MP) for 20 min on ice. Cells were washed twice with PBSF before analyzing via flow cytometry on a BD FACS Canto II (BD Biosciences). Binding curves were fitted with four-parameter non-linear regression analysis to calculate the apparent equilibrium binding constant (K_D^App) in GraphPad Prism 9. Points exhibiting hook effects at higher concentrations were excluded from analysis.