Sea block blocking buffer

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)].

Primary human B cells were treated with various concentrations of GML or 0.009% EtOH as a vehicle control in serum free RPMI 1640 media. Cells were then stimulated for 10 min with anti-IgM (Jackson ImmunoReserach 109-006-129) and recombinant IL-4 (BioLegend 574004). Cells were lysed with 2 × sample buffer, heated to 95 °C, and sonicated. Cell lysates were separated across a 4–15% polyacrylamide gel via gel electrophoresis and transferred to PVDF. The membranes were blocked with SEA BLOCK blocking buffer (ThermoFisher) diluted in PBS and incubated overnight at 4 °C with primary antibody. After washing, samples were incubated with secondary antibodies for 30 min at room temperature and then imaged and quantified using a Licor Odyssey. Samples were normalized to GAPDH and analyzed as a fold change with stimulated EtOH control set to 1. Data presented is 3 experimental replicates and analyzed using One-Sample T-test with theoretical value 1.

Cells were washed in PBS and incubated for 15 minutes at 4°C and in NADOC (0.5% Tritonx-100, 0.5% sodium deoxycholate, 50 mM Tris, and 150 mM NaCl) lysis buffer (Thermo Fisher Scientific) with protease (HaltTM Protease Inhibitor Cocktail 100X, Thermo Fisher Scientific) and phosphatase inhibitors (HaltTM Phosphatase Inhibitor Cocktail 100X, Thermo Fisher Scientific). The extracts were centrifuged at 14,000g for 15 minutes. Protein concentrations in the supernatant were measured using a Pierce BCA protein assay kit (Thermo Fisher Scientific) and a Tecan Safire® plate reader. Protein extracts (25 μg) were resolved by 4-12% SDS-PAGE (Invitrogen) and transferred to nitrocellulose membranes (iBlot, Invitrogen). Membranes were blocked with SEABLOCK blocking buffer (Thermo-Scientific) for 1 hour at room temperature and then incubated overnight at 4°C with primary antibodies diluted in blocking buffer. After washing in PBS-Tween buffer, the membranes were incubated with secondary antibodies conjugated to IRDye fluorophores. The infrared signal of the membranes was detected using an Odyssey detection system (Li-Cor biosciences).