Aquavivid

Using the standard calcium phosphate method, 10 μg of Spike expressor and 2 μg of a green fluorescent protein (GFP) expressor (pIRES-GFP) were transfected into 2 × 10⁶ 293T cells. To determine the Hill coefficients, cells were preincubated with increasing concentrations of soluble ACE2 (0 to 11,500 nM), ACE2-Fc (0 to 500 nM), or the monoclonal antibody CR3022 (0 to 270 nM) 48 h post-transfection. sACE2 binding was detected using a polyclonal goat anti-ACE2 (RND systems). AlexaFluor-647-conjugated goat anti-human IgG (H+L) Ab (Invitrogen) and AlexaFluor-647-conjugated donkey anti-goat IgG (H+L) Ab (Invitrogen) were used as secondary antibodies. The percentage of transfected cells (GFP+ cells) was determined by gating the living cell population based on viability dye staining (Aqua Vivid, Invitrogen). Samples were acquired on an LSRII cytometer (BD Biosciences, Mississauga, ON, Canada) and data analysis was performed using FlowJo vX.0.7 (Tree Star, Ashland, OR, USA). Hill coefficient analyses were done using GraphPad Prism version 8.0.1 (GraphPad, San Diego, CA, USA).

For this, 293T were transfected with full-length SARS-CoV-2 Spikes and a green fluorescent protein (GFP) expressor (pIRES2-eGFP) using the calcium–phosphate method. Two days post-transfection, Spike-expressing 293T cells were stained with the CV3-25 Ab (5 μg/mL) as control or plasma (1:250 dilution) for 45 min at 37 °C. AlexaFluor-647-conjugated goat anti-human IgM, IgG, IgA (1/800 dilution) were used as secondary Abs. The percentage of Spike-expressing cells (GFP + cells) was determined by gating the living cell population based on viability dye staining (Aqua Vivid, Invitrogen, Waltham, MA, USA). Samples were acquired on a LSRFortessa cytometer (BD Biosciences, Franklin Lakes, NJ, USA), and data analysis was performed using FlowJo v10.7.1 (Tree Star). The conformationally independent anti-S2 antibody CV3-25, effective against all Spike variants, was used to normalize Spike expression, as reported [4 (link),26 (link),28 (link),29 (link)]. The Median Fluorescence intensities (MFI) obtained with plasma were normalized to the MFI obtained with CV3-25 and presented as percentage of CV3-25 binding.

293T cells were co-transfected with a GFP expressor (pIRES2-GFP, Clontech, Mountain View, CA, USA) in combination with plasmids encoding the full-length S of SARS-CoV-2 variants; 48 h post transfection, S-expressing cells were stained with the CV3-25 Ab [28 (link)] or plasma (1/500 dilution). AlexaFluor-647-conjugated goat anti-human IgG Abs (1/1000 dilution) was used as a secondary Abs. The percentage of transfected cells (GFP+ cells) was determined by gating the living cell population based on viability dye staining (Aqua Vivid, Invitrogen). Samples were acquired on an LSRII cytometer (BD Biosciences, San Jose, CA, USA) and data analysis was performed using FlowJo v10.7.1 (Tree Star). The seropositivity threshold was established using the following formula: (mean of pre-pandemic SARS-CoV-2 negative plasma + (3 standard deviation of the mean of pre-pandemic SARS-CoV-2 negative plasma). The conformational-independent S2-targeting mAb CV3-25 was used to normalize S expression. CV3-25 was shown to effectively recognize all SARS-CoV-2 S variants [34 (link)].