CR3022-derived Anti-SARS-CoV2 RBD mAbs, human & mouse isotypes

Simplified schematic of CR3022- and
ACE2-binding sites in Spike RBD

Anti-SARS-CoV-2 & SARS-CoV Spike recombinant antibody

Antibody description

InvivoGen provides a family of Anti-Spike RBD CR3022-derived monoclonal antibodies (mAbs) in multiple human and mouse isotypes. These mAbs feature:

• the variable region of the CR3022 human mAb that specifically targets the Spike receptor-binding domain (RBD) [1]
• a constant region that mediates different effector functions depending on the isotype (human IgG1, hIgG1NQ, hIgA1, or hIgM, and mouse IgG2a or mIgG1e3 ) (See Table)

The Anti-Spike RBD clone CR3022 is a SARS-CoV neutralizing antibody that was obtained from the screening of an antibody-phage library and converted to a human IgG1 format [1]. SARS-CoV and SARS-CoV-2 (2019-nCoV) RBD share a highly conserved epitope (86% shared identity) that is recognized by CR3022 [2, 3]. To date, the neutralization potency of CR3022 for SARS-CoV-2 is still unclear [3, 4]. Indeed, CR3022 does not interfere with the ACE2 binding motif [2-4].

 More details

Applications

• Detection of the presence of viral particles in culture supernatant or in serum (ELISA)
• Control of antibody-mediated effector functions  (i.e. absence, increase or decrease in ADCC, ADCP, or CDC) (See Table)

Quality control

• Isotype confirmed by ELISA
• Functionality validated by ELISA using a coated Spike-RBD-His fusion peptide

Our CR3022 variants have been generated by recombinant DNA technology, produced in CHO cells, and purified by affinity chromatography.

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References

1. Ter Muelen J. et al., 2006. Human monoclonal antibody combination against SARS coronavirus: synergy and coverage of escape mutants. PLos Med. 3(7):e237.
2. Yuan M. et al., 2020. A highly conserved cryptic epitope in the receptor-binding domains of SARS-CoV-2 and SARS-CoV. Science. DOI: 10.1126/science.abb7269.
3. Tian X. et al., 2020. Potent binding of 2019 novel coronavirus spike protein by a SARS coronavirus-specific human monoclonal antibody. Emerging Microbes & Infections. 9(1):382-385.
4. Huo J. et al., 2020. Neutralization of SARS-CoV-2 by destruction of the prefusion Spike. bioRxiv. DOI:10.1101/2020.05.05.079202.

Figures

SARS-CoV-2 Spike-RBD-His fusion peptide (10 μg/ml) was coated on ELISA plates overnight. A 3-fold serial dilution of the Anti-Spike-RBD hIgG1 (red curve) or of the Anti-βGal hIgG1 control antibody (grey curve) was performed for the capture step. A HRP-labelled anti-hIgG1 antibody (1/1000 dilution) and the HRP substrate OPD (o-phenylenediamine dihydrochloride) were used for the detection step. Absorbance was read at 490 nm

SARS-CoV-2 Spike-RBD-His fusion peptide (5 μg/ml) was coated on ELISA plates overnight. A 3-fold serial dilution of the Anti-Spike-RBD hIgG1NQ (red curve) or of the Anti-βGal hIgG1 control antibody (grey curve) was performed for the capture step. A HRP-labelled anti-hIgG1 antibody (1/1000 dilution) and the HRP substrate OPD (o-phenylenediamine dihydrochloride) were used for the detection step. Absorbance was read at 490 nm.

SARS-CoV-2 Spike-RBD-His fusion peptide (10 μg/ml) was coated on ELISA plates overnight. A 3-fold serial dilution of the Anti-Spike-RBD hIgM (red curve) or of the Anti-βGal hIgM control antibody (grey curve) was performed for the capture step. A HRP-labelled anti-hIgM antibody (1/1000 dilution) and the HRP substrate OPD (o-phenylenediamine dihydrochloride) were used for the detection step. Absorbance was read at 490 nm.

SARS-CoV-2 Spike-RBD-His fusion peptide (10 μg/ml) was coated on ELISA plates overnight. A 3-fold serial dilution of the Anti-Spike-RBD hIgA1 (red curve) or of the Anti-βGal hIgA1 control antibody (grey curve) was performed for the capture step. A HRP-labelled anti-hIgA antibody (1/1000 dilution) and the HRP substrate OPD (o-phenylenediamine dihydrochloride) were used for the detection step. Absorbance was read at 490 nm.

SARS-CoV-2 Spike-RBD-His fusion peptide (5 μg/ml) was coated on ELISA plates overnight. A 3-fold serial dilution of the Anti-Spike-RBD mIgG2a (red curve) or of the Anti-βGal mIgG2a control antibody (grey curve) was performedfor the capture step. A HRP-labelled anti-mIgG2a antibody (1/1000 dilution) and the HRP substrate OPD (o-phenylenediamine dihydrochloride) were used for the detection step. Absorbance was read at 490 nm.

SARS-CoV-2 Spike-RBD-His fusion peptide (5 μg/ml) was coated on ELISA plates overnight. A 3-fold serial dilution of the Anti-Spike-RBD mIgG1e3 (red curve) or of the Anti-βGal mIgG1e3 control antibody (grey curve) was performed for the capture step. A HRP-labelled anti-mIgG1 antibody (1/1000 dilution) and the HRP substrate OPD (o-phenylenediamine dihydrochloride) were used for the detection step. Absorbance was read at 490 nm.

Specifications

Specificity:  Target SARS-CoV and SARS-CoV-2 Spike RBD
Clonality: Monoclonal antibodies
Source: CHO cells
Formulation: 0.2 μm filtered solution in a sodium phosphate buffer with glycine, saccharose, and stabilizing agents.
Purification: 

• hIgG1 and hIgG1NQ: purified by affinity chromatography with protein G
• hIgA1: purified by affinity chromatography with peptide M
• hIgM: purified by affinity chromatography with protein L
• mIgG1e3 and mIgG2a: purified by affinity chromatography with protein A

Quality control:

• The complete sequence of each antibody construct has been verified.
• Each antibody isotype has been confirmed by ELISA.
• The functionality of each antibody has been validated by ELISA using a coated Spike-RBD-His fusion peptide.
• The absence of bacterial contamination (e.g. lipoproteins and endotoxins) has been confirmed using HEK-Blue™ TLR2 and HEK-Blue™ TLR4 cellular assays.

Contents

• 3 x 100 µg purified antibody, azide-free, and lyophilized

 The product is shipped at room temperature.

 Store lyophilized antibody at -20°C.

Details

• the CR3022 epitope in RBD does not overlap with the ACE2 receptor binding motif [2-5]
• the CR3022 epitopes in SARS-CoV and SARS-CoV-2 RBD share 24 residues out of 28 (86% identity in amino acids) [4]
• Spike trimers stochastically exhibit their RBD in a “down” or “up” state [7, 8]
• the CR3022 antibody only accesses its epitope when at least two RBDs are in the “up” conformation and slightly-rotated [4, 5]
• CR3022 does not sterically prevent ACE2 binding to RBD [4, 5]

References

1. Jiang S. et al. 2020. Neutralizing antibodies against SARS-CoV-2 and other human coronaviruses. Trends Immunol. 41 (5):355-359.
2. Ter Muelen J.et al., 2006. Human monoclonal antibody combination against SARS coronavirus: synergy and coverage of escape mutants. PLos Med. 3(7):e237.
3. Tian X. et al., 2020. Potent binding of 2019 novel coronavirus spike protein by a SARS coronavirus-specific human monoclonal antibody. Emerging Microbes & Infections. 9(1):382-385.
4. Yuan M. et al., 2020. A highly conserved cryptic epitope in the receptor-binding domains of SARS-CoV-2 and SARS-CoV. Science. DOI: 10.1126/science.abb7269.
5. Huo J. et al., 2020. Neutralization of SARS-CoV-2 by destruction of the prefusion Spike. bioRxiv. DOI:10.1101/2020.05.05.079202.
6. Korber B. et al., 2020. Spike mutation pipeline reveals the emergence of a more transmissible form of SARS-CoV-2. bioRxiv. DOI:10.1101/2020.04.29.069054.
7. Walls A.C. et al., 2020. Structure, function, and antigenicity of the SARS-CoV-2 Spike glycoprotein. Cell. 181(2):281-292.e6.
8. Wrapp D. et al., 2020. Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science. 367:1260-1263.