MAbs were screened for binding to MDCK-SIAT1 cells expressing the N-terminal domain (NTD) of SARS-CoV-2 spike glycoprotein (MDCK-NTD). MDCK-NTD was created by stably transfecting MDCK-SIAT1 cells (ECACC 05071502) (Matrosovich et al., 2003) with cDNA encoding the SARS-CoV-2 NTD (amino acids VNLT…TLKS) fused to the transmembrane domain of haemagglutinin H7 (A/HongKong/125/2017) (EPI977395) at the C terminus for surface expression using a second-generation lentiviral vector system. NTD expressing cells were FACS sorted using the FD7C mAb (Huang et al., 2020). In brief, MDCK-NTD cells were seeded at 3 × 104 per well in flat-bottomed 96-well plates (TPP) in high glucose DMEM containing 10% fetal bovine serum (FBS) at 37°C overnight. The medium was then removed and washed with 2% FBS in PBS (PBS/2% FBS) twice. 10 μg/ml of mAbs supernatants from transfected 293T cells were added (50 μl per well) and incubated at room temperature for 1 h. A second antibody Goat anti-human IgG Fc specific-FITC (F9512, Sigma-Aldrich) diluted 1:300 in PBS/2% FBS was then added (50 μl per well) and incubated for another 1 h at room temperature. After washing twice with PBS, the wells were fixed with 1% formaldehyde in PBS. The binding antibodies were detected by fluorescence intensities using a Clariostar plate reader (BMG, Labtech).

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