Spike RBD Antibody (1049349) [Unconjugated]

HEK293 human embryonic kidney cell line transfected with human ACE-2 and eGFP was incubated with Recombinant SARS-CoV-2 CAL.20C S1 His-Tag protein (10779-CV), then stained with (A) Mouse Anti-SARS-CoV-2 CAL.20C S1 Monoclonal Antibody (Catalog # MAB11055) or (B) Mouse IgG2B Isotype Control Antibody (MAB0041) followed by Allophycocyanin-conjugated Anti-Mouse IgG Secondary Antibody (F0101B). Staining was performed using our Staining Membrane-associated Proteins protocol.

Spike RBD was detected in immersion fixed HEK293 human embryonic kidney cell line transfected with SARS-CoV-2 (positive staining) and HEK293 human embryonic kidney cell line (non-transfected, negative staining) using Mouse Anti-SARS-CoV-2 Spike RBD Monoclonal Antibody (Catalog # MAB11055) at 8 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Mouse IgG Secondary Antibody (red; NL007) and counterstained with DAPI (blue). Specific staining was localized to cytoplasm. Staining was performed using our protocol for Fluorescent ICC Staining of Non-adherent Cells.

• Reactivity: V
• Applications: Flow, ICC/IF
• Clone: 1049349
• Clonality: Monoclonal
• Host: Mouse
• Conjugate: Unconjugated

Spike RBD Antibody (1049349) [Unconjugated] Summary

• Immunogen: Human embryonic kidney cell HEK293-derived SARS-CoV-2 Spike RBD
  Val16-Pro681
  Accession # YP_009724390.1
• Specificity: Detects SARS-CoV-2 Spike RBD in direct ELISAs.
• Source: N/A
• Isotype: IgG2b
• Clonality: Monoclonal
• Host: Mouse
• Purity Statement: Protein A or G purified from hybridoma culture supernatant

Applications/Dilutions

• Dilutions:
  • Flow Cytometry 0.25 ug/10^6 cells
  • Immunocytochemistry 8-25 ug/mL

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 12 months from date of receipt, -20 to -70 °C as supplied.
  • 1 month, 2 to 8 °C under sterile conditions after reconstitution.
  • 6 months, -20 to -70 °C under sterile conditions after reconstitution.
• Buffer: Lyophilized from a 0.2 μm filtered solution in PBS with Trehalose. *Small pack size (SP) is supplied either lyophilized or as a 0.2 µm filtered solution in PBS.
• Reconstitution Instructions: Reconstitute at 0.5 mg/mL in sterile PBS.

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Spike RBD Antibody (1049349) [Unconjugated]

• Spike RBD

Background

SARS-CoV-2, which causes the global pandemic coronavirus disease 2019 (Covid-19), belongs to a family of viruses known as coronaviruses that are commonly comprised of four structural proteins: Spike protein(S), Envelope protein (E), Membrane protein (M), and Nucleocapsid protein (N) (1). SARS-CoV-2 Spike Protein (S Protein) is a homotrimeric glycoprotein that mediates membrane fusion and viral entry. As with most coronaviruses, proteolytic cleavage of the SARS-CoV-2 S protein into two distinct peptides, S1 and S2 subunits, is required for activation. The S1 subunit is focused on attachment of the protein to the host receptor while the S2 subunit is involved with cell fusion (2-5). A SARS-CoV-2 variant (named CAL.20C) carrying the S1 subunit amino acid (aa) change W152C, L452R, and D614G emerged in Southern Califonia (6,7). Based on structural biology studies, the receptor binding domain (RBD), located in the C-terminal region of S1, can be oriented either in the up/standing or down/lying state (8). The standing state is associated with higher pathogenicity and both SARS-CoV-1 and MERS can access this state due to the flexibility in their respective RBDs. A similar two-state structure and flexibility is found in the SARS-CoV-2 RBD (9). Based on amino acid (aa) sequence homology, the SARS-CoV-2 S1 subunit has 65% identity with SARS-CoV-1 S1 subunit, but only 22% homology with the MERS S1 subunit. The low aa sequence homology is consistent with the finding that SARS and MERS bind different cellular receptors (10). The S Protein of the SARS-CoV-2 virus, like the SARS-CoV-1 counterpart, binds Angiotensin-Converting Enzyme 2 (ACE-2), but with much higher affinity and faster binding kinetics (11). Before binding to the ACE-2 receptor, structural analysis of the S1 trimer shows that only one of the three RBD domains in the trimeric structure is in the "up" conformation. This is an unstable and transient state that passes between trimeric subunits but is nevertheless an exposed state to be targeted for neutralizing antibody therapy (12). Polyclonal antibodies to the RBD of the SARS-CoV-2 S1 subunit have been shown to inhibit interaction with the ACE-2 receptor, confirming S1 subunit especially the RBD as an attractive target for vaccinations or antiviral therapy (13). There is also promising work showing that the RBD may be used to detect presence of neutralizing antibodies present in a patient's bloodstream, consistent with developed immunity after exposure to the SARS-CoV-2 virus (14). Lastly, it has been demonstrated the S Protein can invade host cells through the CD147/EMMPRIN receptor and mediate membrane fusion (15, 16).

1. Wu, F. et al. (2020) Nature 579:265.
2. Tortorici, M.A. and D. Veesler (2019) Adv. Virus Res. 105:93.
3. Bosch, B.J. et al. (2003) J. Virol. 77:8801.
4. Belouzard, S. et al. (2009) Proc. Natl. Acad. Sci. 106:5871.
5. Millet, J.K. and G.R. Whittaker (2015) Virus Res. 202:120.
6. Zhang, W. et al. (2021) JAMA https://doi.org/10.1001/jama.2021.1612.
7. Zhang, W. et al. (2021). MedRxiv https://doi.org/10.1101/2021.01.18.21249786.
8. Yuan, Y. et al. (2017) Nat. Commun. 8:15092.
9. Walls, A.C. et al. (2010) Cell 180:281.
10. Jiang, S. et al. (2020) Trends. Immunol. https://doi.org/10.1016/j.it.2020.03.007.
11. Ortega, J. T. et al. (2020) EXCLI J. 19:410.
12. Wrapp, D. et al. (2020) Science 367:1260.
13. Tai, W. et al. (2020) Cell. Mol. Immunol. https://doi.org/10.1016/j.it.2020.03.007.
14. Okba, N.M.A. et al. (2020). Emerg. Infect. Dis. https://doi.org/10.3201/eid2607.200841.
15. Wang, X. et al. (2020) https://doi.org/10.1038/s41423-020-0424-9.
16. Wang, K. et al. (2020) ioRxiv https://www.biorxiv.org/content/10.1101/2020.03.14.988345v1.

Limitations

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

Bioinformatics

• Uniprot:
  • YP_009724390.1(Rabbit)