Potency Validated COVID-19 SARS-CoV-2 neutralizing antibody, Anti-2019-nCoV Spike (Spike RBD domain) monoclonal neutralizing antibody (human IgG1, human IgM, human IgA, mouse IgG1 and Cynomolgus (Non human primate, NHP) IgG1)

Coronavirus disease 2019 (COVID-19) pandemic is caused by SARS-CoV-2 (SARS2, 2019-nCoV) infection, a newly emerged novel coronavirus spreading worldwide. Current efforts are focusing on development of specific antiviral drugs. Therapeutic neutralizing antibodies (NAbs) against SARS-CoV-2(SARS2, 2019-nCoV) will be greatly important therapeutic agents for the treatment of COVID-19. The availability of therapeutic NAbs against SARS-CoV-2 will offer benefits for the control of the current pandemic and the possible re-emergence of the virus in the future, and their development therefore remains a high priority.

GeneMedi’s NAbs has been validated to reduce SARS-CoV-2 lentivirus-based pseudo virus infectivity and thereby blocking the entry of the Coronavirus to its effector/targeting cell: human ACE2-HEK293T cell.

GeneMedi’s NAbs has been validated by
   1. COVID-19 Spike protein and Spike-RBD protein binding affnity. Click here to Spike-RBD(GMP-V-2019nCoV-SRBD001)
   2. 2019nCoV pseudotyped virus (PSV) based neutralization assay in 293T-ACE2 effector cell. Click here to SARS-CoV-2 Pseudotyped virus
   3. Competitively blocking the binding of ACE-2 receptor with SARS-CoV-2 Spike protein. Click here to ACE2-Fc(GMP-H-ACE2002)SRBD001、 S1S2001

GeneMedi’s Coronavirus neutralizing antibodies (Nabs) can either act as positive control in COVID-19 related vaccines and neutralizing antibodies discovery and development, or act as a benchmark in SARS-CoV-2 neutralization potency assay.

GeneMedi’s validated COVID-19 neutralizing antibodies group including:
GMP-V-2019nCoV-SnAb001(human IgG1),
GMP-V-2019nCoV-SnAb002(human IgM),
GMP-V-2019nCoV-SnAb003(human IGA),
GMP-V-2019nCoV-SnAb004(mouse IgG1),
GMP-V-2019nCoV-SnAb005(Cynomolgus (Non human primate, NHP) IgG1)
GMP-V-2019nCoV-SnAb006(human IgG1),
GMP-V-2019nCoV-SnAb007(human IgG1),
GMP-V-2019nCoV-SnAb008(human IgG1).

Potency Validated COVID-19 SARS-CoV-2 neutralizing antibody

SARS-CoV-2 neutralizing antibody validated post download

–Nab discovery and vaccines evaluation through SARS-CoV-2 wildtype/mutant variants pseudovirus based neutralizing assay(PBNA) and Spike-ACE2 competition binding assay

GeneMedi-SARS-CoV-2 WT and Spike Mutation Variants Pseudovirus (PSV) Based Cell Entry

Figure. The Pseudovirus (PSV) Based Cell Entry assay was performed on 293T-hACE2 cells infected with GeneMedi-SARS-CoV-2 WT and Spike Mutation Variants (D614G, S943P, V367F, G476S, V483A, H49Y, Q239K, A831V, P1263L, D839Y/N/E:D839Y, D839N, D839E) Pseudovirus (PSV) Infection rate was determined by RFP+GFP+/GFP+ with FACS validation.

GeneMedi's anti-2019-nCoV Spike Neutralizing antibodies (Nabs) and Spike RBD protein binding validation

Cat No.ProductEC50 (ng/ml)
GMP-V-2019nCoV-SnAb001Anti-2019-nCoV Spike (Spike RBD domain) human monoclonal neutralizing antibody (IgG1)5
GMP-V-2019nCoV-SnAb002Anti-2019-nCoV Spike (Spike RBD domain) human monoclonal neutralizing antibody (IgM)18
GMP-V-2019nCoV-SnAb003Anti-2019-nCoV Spike (Spike RBD domain) human monoclonal neutralizing antibody (IgA)410
GMP-V-2019nCoV-SnAb004Anti-2019-nCoV Spike (Spike RBD domain) mouse monoclonal neutralizing antibody (IgG1)6.8
GMP-V-2019nCoV-SnAb005Anti-2019-nCoV Spike (Spike RBD domain) Cynomolgus monoclonal neutralizing antibody (IgG1)28
Figure. The binding of GeneMedi’s anti-2019-nCoV Spike Neutralizing antibodies (Nabs) to Recombinant 2019-nCoV(SARS-CoV-2) Spike RBD protein (GMP-V-2019nCoV-SRBD001) at 5.0ug/ml (100uL/well) was measured by ELISA.

A.GMP-V-2019nCoV-SnAb001:Anti-2019-nCoV Spike (Spike RBD domain) human monoclonal neutralizing antibody (IgG1)
B.GMP-V-2019nCoV-SnAb002:Anti-2019-nCoV Spike (Spike RBD domain) human monoclonal neutralizing antibody (IgM)
C.GMP-V-2019nCoV-SnAb003:Anti-2019-nCoV Spike (Spike RBD domain) human monoclonal neutralizing antibody (IgA)
D.GMP-V-2019nCoV-SnAb004:Anti-2019-nCoV Spike (Spike RBD domain) mouse monoclonal neutralizing antibody (IgG1)
E.GMP-V-2019nCoV-SnAb005:Anti-2019-nCoV Spike (Spike RBD domain) Cynomolgus monoclonal neutralizing antibody (IgG1)

GeneMedi's anti-2019-nCoV Spike Neutralizing antibodies (Nabs) competitive binding assay validation

Cat No. Product IC50 (ng/ml)
GMP-V-2019nCoV-SnAb001 Anti-2019-nCoV Spike (Spike RBD domain) human monoclonal neutralizing antibody (IgG1) 26.3
GMP-V-2019nCoV-SnAb002 Anti-2019-nCoV Spike (Spike RBD domain) human monoclonal neutralizing antibody (IgM) 84.2
GMP-V-2019nCoV-SnAb003 Anti-2019-nCoV Spike (Spike RBD domain) human monoclonal neutralizing antibody (IgA) 20.5
GMP-V-2019nCoV-SnAb004 Anti-2019-nCoV Spike (Spike RBD domain) mouse monoclonal neutralizing antibody (IgG1) 81.9
GMP-V-2019nCoV-SnAb005 Anti-2019-nCoV Spike (Spike RBD domain) Cynomolgus monoclonal neutralizing antibody (IgG1) 243
Figure. GeneMedi’s anti-2019-nCoV Spike Neutralizing antibodies (Nabs) block Recombinant 2019-nCoV(SARS-CoV-2) Spike RBD protein (GMP-V-2019nCoV-SRBD001) and hACE2 (GMP-H-ACE2002) binding.

A.GMP-V-2019nCoV-SnAb001:Anti-2019-nCoV Spike (Spike RBD domain) human monoclonal neutralizing antibody (IgG1)
B.GMP-V-2019nCoV-SnAb002:Anti-2019-nCoV Spike (Spike RBD domain) human monoclonal neutralizing antibody (IgM)
C.GMP-V-2019nCoV-SnAb003:Anti-2019-nCoV Spike (Spike RBD domain) human monoclonal neutralizing antibody (IgA)
D.GMP-V-2019nCoV-SnAb004:Anti-2019-nCoV Spike (Spike RBD domain) mouse monoclonal neutralizing antibody (IgG1)
E.GMP-V-2019nCoV-SnAb005:Anti-2019-nCoV Spike (Spike RBD domain) Cynomolgus monoclonal neutralizing antibody (IgG1)

GeneMedi-SARS-CoV-2 WT and Spike Mutation Variants Pseudovirus (PSV) Based Neutralizing Assay with GeneMedi's anti-2019-nCoV Spike Neutralizing antibodies (Nabs)

Figure. The Pseudovirus (PSV) Based Neutralizing Assay was performed on 293T-hACE2 cells infected with GeneMedi-SARS-CoV-2 WT and Spike Mutation Variants (D614G, S943P, V367F, G476S, V483A, H49Y, Q239K, A831V, P1263L, D839Y/N/E:D839Y, D839N, D839E) Pseudovirus (PSV) under treatment of GeneMedi's anti-2019-nCoV Spike Neutralizing antibodies (Nabs) . Inhibition rate was determined by comparing the relative RFP+GFP+/GFP+ rate.

Pseudotyped virus of SARS-CoV-2 Spike Mutation Variants and Effector cells

The outbreak of COVID-19, caused by SARS-CoV-2 (2019-nCoV), has been a global public health threat and caught the worldwide concern. GeneMedi has developed SARS-CoV-2 wildtype and mutation variants pseudovirus production system, from which the SARS-CoV-2 wildtype and mutation variants pseudotyped virus can be handled in biosafety level 2 (BSL-2).

GeneMedi develop COVID-19 related SARS-CoV-2 (2019-nCoV) Pseudotyped virus of SARS-CoV-2 Spike Mutation Variants including D614G, N501Y, E484K, E484Q, L452R, K417N, K417T, S477N, S477G, D253G, and so on.

GeneMedi’s Pseudovirus Based Neutralization Assay (PBNA) is a conventional assay method that is suitable for High-Throughout Screening (HTS) without live virus engaged. The Pseudovirus Based Neutralization Assay can be used for evaluating:

1) Neutralizing antibodies
2) Peptides blockers (peptide inhibitors)
3) Types of Vaccines3
4) Compounds targeting Spike induced cell-fusion.

GeneMedi offers:

1.SARS-CoV-2 Pseudotyped virus packaging and production
2.Effector cells: human ACE2 overexpression stable HEK293T cell lines
3.SARS-CoV-2(2019nCoV) Pseudotyped Virus Based Neutralization Assay service.

Protocol of SARS-CoV-2 Pseudovirus (PSV)-Based Neutralization Assay For Vaccines, therapeutic antibodies, peptides and compounds against COVID-19

SARS-CoV-2 (2019nCoV) pseudotype virus (pseudovirus, PSV) for COVID-19 related vaccines and neutralizing antibodies evaluation.

The outbreak of COVID-19, caused by SARS-CoV-2 (2019-nCoV), has been a global public health threat and caught the worldwide concern. Due to its high pathogenicity and infectivity1, live SARS-CoV-2 should be handled under biosafety level 3 (BSL-3) conditions. GeneMedi has developed SARS-CoV-2 pseudovirus production system, from which the SARS-CoV-2 pseudotyped virus can be handled in biosafety level 2 (BSL-2)2.

GeneMedi’s SARS-CoV-2 (2019nCoV) pseudotype virus (pseudovirus, PSV) based neutralization assay is a standard evaluation procedure for COVID-19 related vaccines and neutralizing antibodies potency evaluation. GeneMedi’s SARS-CoV-2 PSV is the core ingredient of diagnostics for neutralization serology after vaccinotherapy.

GeneMedi’s SARS-CoV-2 pseudotyped virus includes wildtype and the spike mutation variants (D614G, S943P, V367F, G476S, V483A, H49Y, Q239K, A831V, P1263L, D839Y/N/E: D839Y, D839N, D839E). The GeneMedi’s SARS-CoV2 PSV panel help for all-in-one vaccinotherapy evaluation.

Application-SARS-CoV-2(2019nCoV) Pseudotyped Virus Based Neutralization Assay3

In the Pseudovirus-Based Neutralization Assay (PBNA), the inhibition of viral entry into cells by neutralizing antibodies is correlated to the decreased levels of firefly luciferase signals in the ACE2 expression cells (hACE2-HEK293T, Cat. GM-SC-293T-hACE201) . GeneMedi’s Pseudovirus Based Neutralization Assay (PBNA) is a conventional assay method that is suitable for High-Throughout Screening (HTS) without live virus engaged. The Pseudovirus Based Neutralization Assay can be used for evaluating: 1) Neutralizing antibodies (NAbs)3,4 2) Peptides blockers5,6 (peptide inhibitors) or protein7,8 3) Types of Vaccines (Immunized serum)9 4) Compounds targeting Spike induced cell-fusion10.

Protocol of SARS-CoV-2 Pseudovirus (PSV)-Based Neutralization Assay

Materials 1. SARS-CoV-2 Pseudovirus-RFP-fLuciferase (GM-2019nCoV-PSV01) 2. Efforter cell: Alternative A. hACE2-HEK293T stable cell line (GM-SC-293T-hACE2-01) B. Wildtype HEK293T cell line, hACE2 vector for transfection (GMV-V-2019nCoV-041) Protocol If your efforter cell is hACE2-HEK293T stable cell line, please begin in Step 2. 1.Transfect HEK293T with hACE2-GFP vector (GMV-V-2019nCoV-041) 24hrs before planting the cell into 96-well. 2.Plant the hACE2-HEK293T into 96-well (5,000~10,000 per well) overnight before SARS-CoV-2 PSV infection. 3.Generation of 100ul PSV-Sample mixture:
100ul PSV-Sample mixture Volume
GM-2019nCoV-PSV01* 50ul or 5ul
Sample(NAbs, peptides, serum, etc) flexible (According to your own products)
Total add culture medium to 100ul
* For GM-2019nCoV-PSV01-1, add 50ul in recommendation (range from20ul~100ul). For GM-2019nCoV-PSV01-2, add 5ul in recommendation. (range from2ul~10ul).

Incubate PSV-Sample mixture for 1h at room temperature.

4.Remove the medium of efforter cell in 96-well, add 100ul PSV-Sample mixture to 96well cells for infection. 3wells for a group.

5.Fluorescence imaging (RFP) 72hrs after SARS-CoV-2 PSV infection. The firefly luciferase reporter is measured by following the Promega Luciferase Assay Reagent manual.

Tips

 

If your samples are serum
A standard curve should be generated using serially diluted Nabs (neutralizing antibodies) as a positive control.

If your samples are therapeutic antibodies or peptides candidates
Dilute the samples into concentration gradient for IC50 value evaluation.

GeneMedi SARS-CoV-2 Pseudovirus (PSV) Based Cell Entry

Figure. GeneMedi-SARS-CoV-2 Pseudovirus (PSV) Based Cell Entry validation with fluorescence(A), Luciferase activity (B) and FACS (C) after 72 hours of HEK293T infection. hACE2 significantly enhanced the infection efficiency of the SARS-CoV-2 PSV. GeneMedi SARS-CoV-2 PSV-Luciferase (Cat.GM-2019nCoV-PSV01) is recombinant pseudotyped lentiviral particles containing SARS-CoV-2 spike protein to mimic SARS-CoV-2 (2019nCoV) cell infection. GM-2019nCoV-PSV01 is a powerful tool for SARS-CoV-2 related vaccine efficacy evaluation, neutralizing antibodies, peptides blockers competitors neutralization assay, and tissue-specific infection determination.

References

1 XiaolongCai. An Insight of comparison between COVID-19 (2019-nCoV) and SARS-CoV in pathology and pathogenesis. Preprint, doi:10.31219/osf.io/hw34x (2020, paper of GeneMedi). 2 Jean K. Millet1, Tiffany Tang3, Lakshmi Nathan3, Javier A. Jaimes4, Hung-Lun Hsu3,5, & Susan Daniel3, G. R. W. Production of Pseudotyped Particles to Study Highly Pathogenic Coronaviruses in a Biosafety Level 2 Setting. J Vis Exp, doi:10.3791/59010 (2019). 3 Nie, J. et al. Establishment and validation of a pseudovirus neutralization assay for SARS-CoV-2. Emerg Microbes Infect 9, 680-686, doi:10.1080/22221751.2020.1743767 (2020). 4 Jiang, S., Hillyer, C. & Du, L. Neutralizing Antibodies against SARS-CoV-2 and Other Human Coronaviruses. Trends Immunol, doi:10.1016/j.it.2020.03.007 (2020). 5 Zhang, G., Pomplun, S., Loftis, A. R., Loas, A. & Pentelute, B. L. The first-in-class peptide binder to the SARS-CoV-2 spike protein. doi:10.1101/2020.03.19.999318 (2020). 6 Xia, S. et al. Inhibition of SARS-CoV-2 (previously 2019-nCoV) infection by a highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate membrane fusion. Cell Res 30, 343-355, doi:10.1038/s41422-020-0305-x (2020). 7 Monteil, V. et al. Inhibition of SARS-CoV-2 Infections in Engineered Human Tissues Using Clinical-Grade Soluble Human ACE2. Cell, doi:10.1016/j.cell.2020.04.004 (2020). 8 Lei, C. et al. Neutralization of SARS-CoV-2 spike pseudotyped virus by recombinant ACE2-Ig. Nat Commun 11, 2070, doi:10.1038/s41467-020-16048-4 (2020). 9 HuajunBai, X., XiaolongCai. Vaccines And Advanced Vaccines: -A landscape for advanced vaccine technology against infectious disease ,COVID-19 and tumor. Preprint, doi:10.31219/osf.io/ypgx4 (2020). 10 Hoffmann, M. et al. The novel coronavirus 2019 (2019-nCoV) uses the SARS-coronavirus receptor ACE2 and the cellular protease TMPRSS2 for entry into target cells. bioRxiv, doi:10.1101/2020.01.31.929042 (2020).
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