Sars cov 2 rbd protein

RBD binding antibody titers in serum and BAL samples were measured by ELISA. In brief, sera and BAL samples were heat-inactivated at 56°C for 30 min before use. Next, 96-well ELISA plates were coated with 100 μL of 1 mg/mL recombinant SARS-CoV-2 RBD protein (Z03483-1, Genscript, Piscataway, NJ, USA) at 4°C overnight. After washing the plates with 300 μL PBS containing 0.5% Tween-20 (PBS-T) and blocking with 200 μL 5% non-fat milk in PBST (PBST/5% milk) for 2 h at room temperature (RT), a 2-fold dilution series (generally starting from 1:100) of mouse serum and BAL samples were added, followed by 3-h incubation at RT. For immunoglobulin G (IgG) measurement, a 1:5,000 dilution of horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (ZB-5305, Zsbio, Beijing, China) was applied in 100 μL PBST/5% milk. After 1 h incubation at RT, the plates were extensively washed with 300 μL PBS-T before addition of the substrate OPD (one SIGMAFAST OPD tablet [SLCC0308, Sigma, St. Louis, MO, USA] in 20 mL of deionized water). The reactions lasted for 5 min at RT and were terminated by adding 1M H₂SO₄, followed by reading at OD492 with a Synergy Microplate Reader (Bio-Tek, Winooski, VT, USA). ELISA endpoint titers were defined as the highest dilution that yielded an absorbance that was 2-fold greater than the background value.

To perform the assay, a fixative solution containing 1 µg/mL of SARS-CoV-2 RBD protein (GenScript) was prepared in carbonate-bicarbonate buffer (pH 9.6) and then a Nunc MaxiSorp flat bottom plate (Sigma) was coated with 100 µL of the fixative solution and incubated at 4°C overnight. The next day, the plate was washed five times with DPBS 0.05% (v/v) Tween-20 buffer (0.05% DPBS-T) and blocked with 3% (w/v) of skim milk (BD Biosciences) in 0.05% DPBS-T for 2 hours at room temperature, the plate was then washed five times with 0.05% DPBS-T. 100 µL of sera diluted 1/2000 (week 1 post-vaccination to week 7 post-vaccination) and 100 µL of purified total IgY antibodies (0.3 mg/mL) diluted 1/800 (week 1 post-vaccination to week 10 post-vaccination) both with 1% (w/v) of skim milk were added to the plate and incubated for 1 hour at 37°C. Later, wells were washed five times with 0.05% DPBS-T and immediately incubated with 100 µL of Goat anti-Chicken IgY secondary antibody conjugated with HRP (Genscript) diluted 1/2000 in 1% non-fat milk in 0.05% DPBS-T for 1 hour at 37°C. The plate was then washed five times with 0.05% DPBS-T and incubated with 100 µL of 3,3’,5,5’-tetramethylbenzidine (TMB) for 15 min at room temperature. The reaction was stopped by the addition of 50 µL of 2N H₂SO₄ per well, and the plate was read at 450 nm using an Epoch 2 microplate reader (Bioteck).

The titers of SARS-CoV-2 RBD-specific antibodies were investigated by ELISA following our previously optimized procedures [11 (link)]. Briefly, 96-well plates were coated by Sf9-produced SARS-CoV-2 RBD protein (GenScript, USA) and incubated overnight at 4 °C. The plates were washed by 1xPBST (1xPBS plus 0.05% Tween-20) three times and blocked by 5% skim milk in 1xPBS for 2 h at 37 °C. After three washes with 1xPBST, two-fold serial dilutions of immunized mouse sera, starting at a 1:100 dilution, were added into the wells for 2 h at 37 °C. Subsequently, mouse-specific IgG-detection antibodies, such as goat anti-mouse IgG (HRP) (Jackson ImmunoResearch, USA), goat anti-mouse IgG1 (HRP), and goat anti-mouse IgG2a (HRP) (Abcam, UK) at a dilution of 1:2000 in 1xPBS were incubated at 37 °C for 1 h for the detection of anti-RBD specific total IgG, IgG1, and IgG2a, respectively. The colorimetric reactions were visualized by the addition of a 3,3′,5,5′-tetramethylbenzidine (TMB) solution (Promega, USA) and terminated with 1M H₂SO₄.The absorbance at 450 nm (A₄₅₀) was read by an ELISA microplate reader (BMG Labtech, Germany). The titers of mouse antibodies were expressed as the reciprocal of the highest dilution of serum that has A₄₅₀ more than the cut-off [52 (link)].