Goat anti human igg fc specific fitc antibody

293 T cells were transfected with a plasmid encoding the full-length S protein (kindly provided by O. Schwartz) [39 ] or a control plasmid using Lipofectamine 2000 (Life technologies). Twenty-four hours post-transfection, the cells were detached using PBS-EDTA and transferred into U-bottom 96-well culture plates (200.000 cells/well). Cells were saturated with 10% fetal bovine serum at 4 C for 10 min and incubated with the patients’ sera (1:300 dilution) in PBS containing 0.5% BSA for 30 minutes at 4 °C. Cells were then washed and stained for 30 min at 4 °C using a goat anti-Human IgG (Fc specific)-FITC antibody that specifically recognizes the Fc fragment and not the light chains of the Ig (Sigma). After washing, cells were fixed with 2% PFA and analyzed on an Attune™ Nxt flow cytometer using FlowJo software (Tree Star, Inc.).

A fluorescence immunoassay was performed to determine the presence of anti-SARS-CoV-2 antibodies in the serum of enrolled participants. For this purpose, 20 µL of serum at 1:20 dilution with PBS were added into slides containing Vero E6 cells infected with B.1 lineage of SARS-CoV-2 and then were incubated for 30 minutes at 37°C. Serum from a convalescence patient pre-analyzed was used as a positive control, and non-infected cells were used as mock. Later, the slides were washed twice with PBS, considering that the whole slide had to be submerged and was allowed to air dry. Then, 20 µL of goat anti-human IgG (Fc specific)- FITC antibody (Sigma-Aldrich) at 1:40 dilution with PBS was added and incubated for 30 minutes using a Humidifying Chamber. Finally, immunofluorescence was assessed by microscopy in an Axio Vert.A1™ (Zeiss, Oberkochen, Germany)