Sars cov 2 spike rbd his recombinant protein

The competitive ELISA was achieved by the competition of neutralizing antibodies against the RBD in vaccinated plasma and the spike neutralizing mouse mAb. The wells were coated with 50 µL of 1.0 µg/mL SARS-CoV-2 spike RBD-His recombinant protein (Sino Biological, Beijing, China) (Cat. 40592-V08H31) (diluted in coating buffer 1M NaHCO₃ (pH 9.6)) and kept overnight at 4 °C in the moisture chamber. The coated wells were washed four times with 0.05% Tween 20 in PBS (pH 7.4) (PBST) and non-specific binding was blocked with 200 µL of 2% skimmed milk in PBS at room temperature for 1 h. After washing, plasma samples at dilution 1:5 were combined with 2.5 µg/mL spike neutralizing antibody mAb (40591-MM43) (Sino Biological, Beijing, China) (Cat. 40591-MM43), then added to ELISA wells and incubated for 1 h. The wells were washed and HRP-conjugated goat anti-mouse Igs was added at dilution 1:3,000 (KPL, Gaithersburg, MD, USA) (Cat. 074-1807). After incubation for 1 h, the wells were washed and 50 µL of TMB substrate was added (Seracare, Milford, MA, USA) (Cat. 5120-0076). The reaction was stopped with 1 N HCl, and the OD at 450 nm was measured.

Binding ELISAs were used to confirm the epitope specificities of DMAbs 2130, 2196 and 2381. NUNC 96-well MaxiSorp plates were coated with recombinant RBD proteins (3 µg ml⁻¹ in 1× PBS) containing mutations at residues F444A (RBD-F444A) or F486 (RBD-F486A) (AstraZeneca), which are key residues required for the binding of clones 2130 and 2196/2381, respectively. To evaluate the relative binding of each construct to different VoC, the following coating antigens were used (0.5–1 µg ml⁻¹ in 1× PBS): SARS-CoV-2 Spike RBD-His Recombinant Protein (Sino Biologicals; 40592-V08B), Spike S1(D614G)-His Recombinant Protein (Sino Biologicals; 40591-V08H3), RBD-His K417N Recombinant Protein (Sino Biologicals; 40592-V08H59), RBD-His E484K Recombinant Protein (Sino Biologicals; 40592-V08H84), RBD-His N501Y (Sino Biologicals; 40592-V08H82), Spike S1-K417N/E484K/N501Y/D614G Recombinant Protein (Sino Biologicals; 40591-V08H10), B.1.1.529(BA.1) S1 + S2 Trimer-His Recombinant Protein (Sino Biologicals; 40589-V08H26). ELISA procedure was completed as described above.

Balb/c mice (female, 8–12 weeks) were used to produce the neutralizing antibody targeting SARS-CoV-2 RBD. The antigen (SARS-CoV-2 Spike RBD-His Recombinant Protein, 40592-V08H, Sinobiological, 70 μg) was injected subcutaneously into the neck and back of mice after mixing with equal volume of Freund’s complete adjuvant. Two weeks later, repeat the antigen injection as above. This step was repeated once. Subsequently, 70 μg of antigen was injected by intraperitoneal injection and antibody titers were measured by ELISA assay. The last booster was given 3 days prior to cells fusion with 100 μg of antigen by intraperitoneal injection. Sp2/0 cells were used for fusion with spleen cells using PEG1500. After screening by HAT and HT medium, stable expression cell lines were finally obtained by multiple cloning culture and ELISA detection. For purification, the supernatant of monoclonal cells was collected for centrifugation at 8000 rpm for 30 mins and filtration using 0.2 µm strainer, and then the AKTA Purifier system was used for chromatography (Mabselect Xtra 25 ml, 17526907, Cytiva), the binding buffer and elution buffer were 0.02 M PBS buffer (pH7.3) and 0.05 M Glycine-hydrochloric acid buffer (pH2.45), respectively. Finally, the eluent was concentrated using 30 kD centrifugation ultrafiltration tube and stored at −80 °C.