Sars cov 2 spike protein s1 s2 ecd his tag

Assay specificity was tested using a depletion method. Magnetic beads (Dynabeads^® Antibody Coupling Kit, Thermo Fisher Scientific, Waltham, Massachusetts, USA) were coupled with homologous or one of two heterologous competitor proteins by overnight incubation with 20 μg protein per mg of beads followed by washing in kit storage buffer. The homologous competitor was the SARS-CoV-2 spike protein (S1 + S2 ECD His tag; Sino Biological). The heterologous competitor proteins were influenza A/Hong Kong/45/2019 (H3N2) neuraminidase His-Tag and human coronavirus OC43 spike glycoprotein (full-length) sheep Fc-Tag (HEK293) (both heterologous antigens from LGC Ltd, Teddington, UK). Mock samples, with beads incubated with water and phosphate-buffered saline, were used as the comparator. For antibody depletion, four serum samples were incubated separately with each type of antigen-coupled beads and mock beads for 2 hours. Following removal of the coupled beads, depleted and non-depleted serum samples were tested together on the same plate at the primary sample dilution series.

Ficoll-Paque was used to isolate human peripheral blood mononuclear cells (PBMCs) from the peripheral blood of 3 healthy control donors. PBMCs from each donor were resuspended in complete RPMI-1640 media and plated into non-adherent 12-well plates at a density of 1 ×10⁶ cells per well overnight. On the day of the treatment, cells were primed with 150ng/ml lipopolysaccharide (LPS) (O111:B4) from Escherichia coli (Sigma-Aldrich, Merck, Darmstadt, Germany) for 3 hours. Then, 10nM SARS-CoV-2 spike protein (S1+S2 ECD, His Tag) (Sino Biological, 40589-V08B1) was added for 16 h after which the cells were harvested for lysate preparation, and the supernatants were collected for cytokine/chemokine release by ELISA. For VitD3 treatment conditions, 50nM calcitriol (Sigma-Aldrich, USA) was added during the priming step along with LPS prior to inflammasome activation by SARS-CoV-2 spike protein. MCC950 (Tocris Bioscience, CAS-256373-96-3), a selective and highly potent NLRP3 inhibitor was used at 10uM as a positive control.