The animal studies were approved by the Institutional Animal Care and Use Committee at the Institut Pasteur of Shanghai. The mice were kept in the SPF (specific pathogen free) animal facility with controlled temperature (20–26 °C), humidity (40–70%), and lighting conditions (12 h light/12 h dark cycle).
To generate MAbs, female BALB/c mice aged 6–8 weeks were each primed with 100 μg of RBD-mFc protein (Sino Biological) formulated with 0.5 mg of aluminum hydroxide adjuvant (Invivogen, USA) and 25 μg of CpG oligonucleotides (Sangon Biotech, China) via the i.p. route on day 0. The mice were boosted via the subcutaneous route on day 8 with RBD-mFc (50 μg/mouse) emulsified with Freund’s complete adjuvant (Sigma), and on day 13 with RBD-mFc (50 μg/mouse) emulsified with Titermax adjuvant (Sigma). On day 22, one mouse was injected with 75 μg of HEK 293F-expressed RBD protein in PBS in a tail vein. On day 26, splenocytes were isolated and fused with SP2/0 cells using polyethylene glycol 1450 (Sigma). Fused cells were then selected in a hypoxanthine, aminopterine, and thymidine (HAT; Sigma) medium. Eight days later, hybridoma supernatants were screened for their ability to bind to RBD protein and to block the ACE2-hFc/SARS-CoV-2 RBD binding by ELISA, as described below. ELISA-positive hybridoma cells were cloned by limiting dilution method and the resulting monoclonal cell lines were expanded. Purified MAbs were prepared from ascitic fluids using HiTrap™ Protein G HP column (GE Healthcare, USA).
To generate MAbs, female BALB/c mice aged 6–8 weeks were each primed with 100 μg of RBD-mFc protein (Sino Biological) formulated with 0.5 mg of aluminum hydroxide adjuvant (Invivogen, USA) and 25 μg of CpG oligonucleotides (Sangon Biotech, China) via the i.p. route on day 0. The mice were boosted via the subcutaneous route on day 8 with RBD-mFc (50 μg/mouse) emulsified with Freund’s complete adjuvant (Sigma), and on day 13 with RBD-mFc (50 μg/mouse) emulsified with Titermax adjuvant (Sigma). On day 22, one mouse was injected with 75 μg of HEK 293F-expressed RBD protein in PBS in a tail vein. On day 26, splenocytes were isolated and fused with SP2/0 cells using polyethylene glycol 1450 (Sigma). Fused cells were then selected in a hypoxanthine, aminopterine, and thymidine (HAT; Sigma) medium. Eight days later, hybridoma supernatants were screened for their ability to bind to RBD protein and to block the ACE2-hFc/SARS-CoV-2 RBD binding by ELISA, as described below. ELISA-positive hybridoma cells were cloned by limiting dilution method and the resulting monoclonal cell lines were expanded. Purified MAbs were prepared from ascitic fluids using HiTrap™ Protein G HP column (GE Healthcare, USA).
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