Nunc maxisorb plates

For the tests of VLP antigenicity, 96-well Nunc Maxisorb plates (Thermo Fisher Scientific) were coated with 0.5 μg of purified VLPs per well diluted in 50 μL of carbonate coating buffer (15 mM Na₂CO₃, 36 mM NaHCO₃, pH 9.6) and incubated overnight at 4 °C. The plates were washed with PBS containing 0.05% Tween-20 (PBST) and blocked with Sea Block blocking buffer (Thermo Fisher Scientific) for 1 h at room temperature. 2-fold serial diluted human sera in blocking buffer were added to the plates and incubated for 1 h at room temperature. After three washes, the bound antibody was detected with goat anti-human Ig (γ chain specific)-HRP conjugated secondary antibody (Thermo Fisher Scientific) diluted 1:10,000 in blocking buffer. The assay reaction was developed with the 1-step Ultra TMB-ELISA substrate (Thermo Fisher Scientific). The reaction was left to develop for 5 min and then stopped with 2 M H₂SO₄. The optical density was determined at 440 nm using a plate reader. The neutralizing antibody response following immunization was measured using the “cPass™ SARS-CoV-2 Neutralization Antibody Detection Kit” (Genscript, L00847, Piscataway, NJ, USA). Used as directed, the assay detects functional neutralizing antibodies antibodies that prevent the receptor binding domain (RBD) of SARS-CoV-2 binding to ACE-2 expressed as % of blocking activity [18 (link)].

The concentrations of CXCL8 and IL-1β in the cell culture supernatant were measured using Cytoset (Invitrogen by Thermo Fischer Scientific, Waltham, MA, USA and Novex by Life Technologies Waltham, MA, USA) or Duoset (R&D systems, Minneapolis, MN, USA) enzyme-linked immunosorbent assay (ELISA) kits, respectively, both applied according to the manufacturer’s instructions. Samples were diluted in the recommended assay buffer to stay within the appropriate range of the assay. ELISA was performed in Nunc Maxisorb plates (Thermo Scientific, Waltham, MA, USA). Absorbance was measured using a Sunrise plate reader (Tecan, Männerdorf, Switzerland).

The concentrations of cytokines in the cell culture supernatants were measured using Cytoset (TNFα and CXCL8, Invitrogen by Thermo Fischer Scientific and Novex by Life Technologies) or Duoset (IL-1α and IL-1β, R&D systems, Minneapolis, MN, USA) enzyme-linked immunosorbent assay (ELISA) kits, both applied according to the manufacturer’s instructions. ELISA was performed in Nunc Maxisorb plates (Thermo Scientific, Waltham, MA, USA). Absorbance was measured using a Tecan Sunrise plate reader (Tecan, Männedorf, Switzerland).