Hek293f

N3a peptide binding scAb, N3aB02, was reformatted into a mouse monoclonal antibody by cloning its variable heavy (VH) and variable light (Vk) genes into a dual plasmid eukaryotic vector system encoding the constant domain genes of mouse IgG2a isotype and murine kappa chain respectively. Human Embryonic Kidney cells (HEK293-F) (Life Technologies, Carlsbad, CA, USA) were transfected with plasmids harbouring reformatted antibody heavy and light chain genes using polyethyleneimine (PEI). Transfection was carried out using 1 mg of total DNA (500 μg each of VH and Vκ plasmid DNA) and 1000 mL of cultured HEK293-F cell suspension maintained in sterile Freestyle 293 expression medium (Invitrogen) without antibiotics at 37 °C, with 8% CO₂, 125 rpm shaking. The transfected cells were allowed to express N3aB02 mAb for a week before the supernatant was collected and purified using protein A beads (ProSep^® Ultra Plus, Merck Millipore, Burlington, MA, USA). N3aB02 mAb was eluted using 100 mM glycine (pH 3.0), dialysed against 1× PBS and quantified as above.

For laboratory-scale expression of mAbs, plasmids bearing chimeric antibody heavy and light chain genes were prepared (EndoFree plasmid mega prep kit; Qiagen) and transfected into human embryonic kidney cells (HEK293-F) (Life Technologies) using polyethylenimine (PEI). Transfections were carried out using 1 mg of total DNA (500 μg each of VH and Vκ plasmid DNAs) and 1 L of the cultured HEK293-F cell suspension maintained in sterile Freestyle 293 expression medium (Invitrogen) without antibiotics at 37°C with 8% CO₂, with shaking at 125 rpm. The transfected cells were grown for 8 days and purified using ProSep A beads (Millipore) and Econo-Pac chromatography columns (Bio-Rad). Recombinant mAbs were eluted in 100 mM glycine (pH 3.0) before neutralization with 1 M Tris-HCl (pH 8.0). Purified mAbs were quantified by SDS-PAGE and A₂₈₀ measurements.

All experimental models were described before^{29 (link)}. The HEK293F (Life Technologies) and HEK293T (ATCC CRL-11268) cell lines are human embryonic kidney cells transformed to have increased recombinant protein production and increased retrovirus production. HEK293T cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), streptomycin (100 μg/mL) and penicillin (100 U/mL) at 37 °C with 5% CO₂ in flasks. HEK293F cells were cultured in 293FreeStyle expression medium (Life Technologies) at 37 °C with 8% CO₂ and shaking at 125 rpm. HEK293T/ACE2 cells are transformed to express human angiotensin-converting enzyme 2. The HEK293T/ACE2 cells were cultured in DMEM supplemented with 10% FBS, streptomycin (100 μg/mL) and penicillin (100 U/mL) at 37 °C with 5% CO₂ in flasks.

The HEK293T (ATCC CRL-11268) and HEK293F (Life Technologies) cell lines are human female embryonic kidney cells engineered to enhance the production of recombinant protein or retrovirus. HEK293F cells are designed for suspension growth. HEK293T cells were maintained in flasks with DMEM + 10% FBS + 1% penicillin-streptomycin at 37°C with 5% CO₂. HEK293F cells were cultivated in 293FreeStyle expression medium (Life Technologies) at 37°C with 8% CO₂ and agitation at 125 rpm. HEK293T/ACE2 represents a human embryonic kidney cell line expressing Human Angiotensin-Converting Enzyme 2. HEK293T/ACE2 cells were cultured in flasks with DMEM + 10% FBS + 1% penicillin-streptomycin + 5 μg/ml blasticidin at 37°C with 5% CO₂. Huh7 cells were maintained in DMEM + 10% FBS + 1% penicillin-streptomycin + 1x MEM NEAA + 1mM HEPES at 37°C with 5% CO₂.

HEK293F (Life Technologies) and HEK293T (ATCC CRL-11268) are human embryonic kidney cell lines transformed for increased production of recombinant protein or retrovirus. HEK293F cells are adapted to grow in suspension. HEK293F cells were cultured at 37°C with 8% CO₂ and shaking at 125 rpm in 293FreeStyle expression medium (Life Technologies). HEK293T cells were cultured at 37°C with 5% CO₂ in flasks with DMEM supplemented with 10% fetal bovine serum (FBS), streptomycin (100 μg/mL) and penicillin (100 U/mL). VeroE6 (ATCC CRL-1586) is a kidney epithelial cell from African green monkeys. VeroE6 cells were cultured at 37°C with 5% CO₂ in DMEM supplemented with or without streptomycin (100 μg/mL) and penicillin (100 U/mL) and with or without 5 or 10% FBS, and with or without TPCK-trypsin. HEK293T/ACE2 cells (Schmidt et al., 2020 (link)) are a human embryonic kidney cell line expressing human angiotensin-converting enzyme 2. HEK293T/ACE2 cells were cultured at 37°C with 5% CO₂ in flasks with DMEM supplemented with 10% FBS, streptomycin (100 μg/mL) and penicillin (100 U/mL). Ramos is a human Burkitt’s lymphoma cell line that has B lymphocyte characteristics. Ramos B cells were cultured at 37°C with 5% CO₂ in RPMI supplemented with 10% FBS, streptomycin (100 μg/mL) and penicillin (100 U/mL).