Rprotein a sepharose

To make human ACE2 protein, pcDNA3-sACE2-WT(732)-IgG1⁴³ (link) (Addgene plasmid #154104, gift of Erik Procko) plasmid was transfected into Expi293 cells using PEI at a ratio of 1:3, and the supernatants were collected after five days. hACE2 was purified from the cell supernatant by using rProtein A Sepharose (GE) followed by running through a Superdex 200 Increase 10/300 GL column. For the spike trimer proteins, paH-spike was transfected into Expi293 cells using PEI at a ratio of 1:3, and the supernatants were collected five days later. The spike proteins were purified using Excel resin (Cytiva) according to the manufacturer’s instructions. The molecular weight and purity were checked by running the proteins on SDS-PAGE.

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)}.

All animal care and experimental protocols were performed in accordance with the guidelines for the care and use of laboratory animals at Chugai Pharmaceutical Co., Ltd. The protocol was approved by the Institutional Animal Care and Use Committee at Chugai Pharmaceutical Co., Ltd. Five New Zealand White rabbits (Kitayama Labes) were immunized four times with purified FLAG-tagged mouse PlxnA1 ectodomain. Then, the rabbits were humanely sacrificed by exsanguination under anesthesia, and their peripheral blood mononuclear cells and splenocytes were isolated. B cells positive for the production of anti-PlxnA1 were cultivated using the method reported by Seeber et al. [19 (link)] and were subjected to sequencing of the genes encoding the antibody variable regions. The DNA segment encoding each rabbit’s Ig variable region was inserted into an expression vector containing the constant region of various IgG species, including rabbit IgG/kappa, mouse IgG1/kappa, mouse IgG2a/kappa, or human IgG4/kappa, to express as recombinant IgG in FreeStyle293 cells. Recombinant IgGs were purified from the culture supernatants using rProtein A-Sepharose (GE Healthcare). Fab fragments were prepared and purified as previously described [20 (link)].

Co‐IP assays were performed as described previously (Zhang et al., 2017 (link)). The OsHDAC1‐HA and OsGRAS30‐MYC constructs or HA and OsGRAS30‐MYC constructs were transformed into rice protoplasts using a plant protoplast preparation and transformation kit (Real‐Times, Cat. # RTU4052, Beijing, China) in accordance with the manufacturer's instructions. Then, total proteins were extracted from rice protoplasts and incubated with rProtein A sepharose (GE Healthcare, USA) and an anti‐HA antibody. Proteins bound to sepharose were detected using an anti‐MYC antibody.

For each antibody, variable genes were optimized for human cell expression and synthesized by GenScript. VH and VL were inserted separately into pcDNA3.4 plasmids encoding the constant region for heavy chain and light chain, respectively. Monoclonal antibodies were expressed in Expi293F cells (Thermo Fisher Scientific, Catalog # A14527, RRID: CVCL_D615) by co-transfection of heavy chain and light chain expressing plasmids using PEI (Polysciences) and cultured in a 37°C shaker at 125 RPM under 8% CO₂. Supernatants were collected five days post-transfection, and then antibodies were purified by affinity chromatography using rProtein A Sepharose (GE).

Antibodies were expressed as previously described^{18 (link)}. Briefly, Vh and Vl genes for each antibody were codon optimized and synthesized (GenScript), and then inserted into mammalian expression vectors. These plasmids were transiently transfected into Expi293 cells (Thermo Fisher) using polyethylenimine and cultured for 5 days, and then the antibody was purified by affinity chromatography using rProtein A Sepharose (GE). REGN10933, REGN10987, COV2-2130 and COV2-2196 were provided by Regeneron Pharmaceuticals, Brii-196 and Brii-198 were provided by Brii Biosciences, and CB6 was provided by B. Zhang and P. Kwong (NIAID).