Amicon centrifugal filter columns

The antigen used in this study was the receptor binding domain (RBD) of native (WuHan-Hu-1) SARS-CoV-2 S-protein (anti-RBD). The antigen was produced based on the previously described methodology, with modifications [21] (link). Briefly, the RBD coding sequence, flanked with the signal peptide coding sequence at 5′-end and 6xHis-Tag at 3′-end, was cloned into pCAGGS expression plasmid. Expi293F cell line (ThermoFisher Scientific), a modified HEK293 line, optimized for production of protein exported outside the cell to the cell culture medium, were transfected with expression vector using ExpiFectamine™ reagent (ThermoFisher Scientific) according to the manufacturer’s protocol. After 5 days, the cell suspension was centrifuged at 4000g, 20 min, 4 °C, and the supernatant was collected. The supernatant containing RBD protein was passed through a chromatography column filled with Ni-NTA resin (Therm Fisher Scientific), to immobilize the protein via His-tag. After immobilization, the column was washed with phosphate-buffered saline (PBS), containing 25 mM imidazole and 0.15 M NaCl. The protein was eluted from the resin using PBS containing 230 mM imidazole and 0.2 M NaCl. Subsequently, the protein solution was concentrated and purified with 10 kDa Amicon centrifugal filter columns (Merck, Germany).