Sars cov 2 rbd mfc protein

HEK293 cells transfected with ACE2-EGFP (Vectorbuilder) were seeded overnight at a density of 5000 cells/well on a sterile coverslip inside a 24-well plate. To begin, corilagin and SARS-CoV-2-RBD-mFc protein (Sino Biological) were co-incubated for 30 min before being added into the cells for a further incubation of 40 mins. After treatments, cells were fixed with 4% PFA for 10 min and blocked with 3% BSA for another 30 min. Cells were then incubated with anti-mouse IgG Fc TRITC antibody purchased from Invitrogen (Carlsbad, CA, USA) for 2 h. The coverslips with cells were mounted with FluorSave reagent (Calbiochem) and visualized by confocal microscope with Leica SP8 (Wetzlar, Germany). For semi-quantitative determination, fluorescence images were analysed by the Image J. The optical density of the TRITC-labelled SARS-CoV-2-RBD was measured. The reduction (%) of TRITC optical density compared to the un-treated control wells were calculated as the neutralizing potency. Quantification bar chart represented the data from 3 independent experiments.

Anti-SARS-CoV-2 Spike RBD antibodies were isolated from an HX02 human Fab phage display library (Humanyx Pte Ltd) via in vitro selection. Briefly, biopanning was performed using SARS-CoV-2 RBD (YP_009724390.1) (Arg319-Phe541) with a mouse Fc tag (Sino Biological, Cat#40592-V05H) biotinylated using the EZ-Link NHS-PEG4-Biotin labeling kit (Thermo Fisher Scientific, Cat#A39259). In both rounds of biopanning, biotinylated SARS-CoV-2 RBD-mFc protein (Sino Biological, Cat#40592-V05H) was immobilized on M280 streptavidin-coated magnetic beads (Life Technologies, Cat#11205D); 3.5 × 10¹² cfu phage in 1ml 1% casein-PBS blocking buffer was used in the first round, and 1.64 × 10¹¹ cfu phage were used in the second round. During the biopanning process, binders to mouse Fc were removed by pre-incubation of phage with 2 μM mouse IgG before mixing with the RBD-mFc antigen. After two rounds of biopanning, the Fabs of selected clones were expressed in E. coli HB2151 cells (Stratagene) to screen for RBD binders by ELISA. Unique clones were identified by DNA sequencing.