Monoclonal Antibody Generation from SARS-CoV-2 Convalescent Patients

SARS-CoV-2 convalescent patients and immunized healthy adults were enrolled and peripheral blood was collected. Peripheral blood mononuclear cells (PBMCs) were prepared from peripheral blood using Ficoll-Paque (Sigma-Aldrich, USA). Single B cells were isolated with or without using biotinylated RBD (Beta variant) into the 96-well PCR plate containing lysis buffer as previously described^{6 (link)}. After performing reverse transcription to obtain cellular Ig cDNA, variable domain-encoding genes for heavy, kappa and lambda chains were amplified and inserted into human IgG1 expression vectors. For antibody expression, heavy and light chain expression vectors were transiently transfected into ExpiCHO cells (Thermo Fisher Scientific, A29133) using the ExpiCHO expression system kit. Human IgG1 monoclonal antibody-containing supernatant was harvested and purified by rProtein A Sepharose (GE healthcare), with the resulting monoclonal antibodies collected for further analysis.
To determine the individual gene segments employed by VDJ and VJ rearrangements and the number of nucleotide mutations and amino acid replacements, the variable domain sequences were aligned with germline gene segments using the international ImMunoGeneTics (IMGT) alignment tool (http://www.imgt.org/IMGT_vquest/input)^{34 (link)}.