Igg elution buffer

Plasmids of c13G8 and CC5-17 were standardized to a human IgG1 Fc backbone and were then expressed by transient transfection of expiCHO-S cells. Briefly, expiCHO-S cells were transfected with plasmids of the heavy and light chains of antibodies and expressed according to the manufacturer’s instructions for 10 days at 32 °C. Culture supernatants were collected and clarified, and antibodies were purified with HiTrap MabSelect Sure pcc (Cytiva) protein A affinity chromatography column on AKTA start fast protein liquid chromatography system. The CC5-17 antibody was eluted with pH2.2 glycine and c13G8 was eluted using IgG Elution Buffer (Thermo Scientific). Eluted fractions were neutralized with 2 M Tris. Following neutralization fractions containing antibodies were applied to HiLoad 26/600 Superdex 200 pg column to both improve the purity and buffer exchange to PBS. Pure fractions of antibodies were pooled, quantified by Nanodrop 2000c (Thermo Scientific), and stored at −20 °C. For the animal experiments, aliquots of mAbs were diluted in sterile PBS pH 7.4 at 0.25 mg/ml and 1 mg/ml for c13G8 and CC5-17 and 1 mg/ml for the isotype control. Diluted antibodies were further quantified using BLI in Octet R8 (Sartorius) with Protein A biosensors to confirm accuracy of the doses.

Production of antibodies was performed as previously described.⁵² (link) Briefly, HEK293T cells in 175-mm plates were transfected with 80 μg total DNA/plate at 50% confluency with a calcium phosphate transfection kit (Takara, Mountain View, CA). For human antibody production, cells were co-transfected with both heavy-chain vector and light-chain vector at a 1:1 ratio. For rhesus antibody production, cells were co-transfected with AAV transfer plasmid and a plasmid encoding furin at a 4:1 ratio. At 12–16 hr post-transfection, 10% fetal bovine serum (FBS)-DMEM was replaced with serum-free 293 Freestyle media (Invitrogen, Carlsbad, CA). Media were collected after 48 h, and debris was cleared by centrifugation for 10 min at 1,500 × g and filtered using 0.45-μm filter flasks (Millipore Sigma, Billerica, MA). Proteins were isolated with HiTrap columns (GE Healthcare, Pittsburgh, PA) and eluted with IgG Elution Buffer (Thermo Scientific, Waltham, MA) into 1 M Tris-HCl Buffer (pH 9.0) (G Biosciences, St. Louis, MO). Buffer was exchanged with PBS and protein concentrated to 1 mg/mL with Amicon Ultra Centrifugation Filters (Millipore Sigma, Billerica, MA). Antibodies were stored at 4°C.

The Expi293 expression system (Thermo Fisher Scientific) was used for mammalian recombinant ORF8 protein production. Expi293F cells were transfected with pCAGEN-ORF8-His using Expifectamine 293 (Thermo Fisher Scientific) according to the manufacturer’s instructions. After 5–6 days’ post-transfection, serum-free medium, carrying secreted ORF8 protein, was harvested and purified by anti-his affinity resin (GenScript, China). Recombinant His-ORF8 protein was eluted using low pH amine-based elution buffer (IgG Elution Buffer, ThermoFisher), which is immediately neutralized with 1M Tris-Cl, pH 8.8.

Simple cloning was performed to produce the ICAM‐1 protein. Mouse or human ICAM‐1 extracellular domain was cloned into a mouse (Invivogen, pfuse‐mchg1) or human IgG expression vector (Invivogen, pfuse‐hg40fc1). The plasmid was transfected into Expi293F cells using the ExpiFectamine 293 Transfection kit (Gibco, A14524). The protein was then purified from the supernatant using protein A beads (REPLIGEN, 10‐2500‐03) and an IgG Elution Buffer (Thermo Fisher Scientific, 21004).