H4-derived Anti-SARS-CoV2 RBD mAbs, human & mouse isotypes

SARS-CoV-2 spike-specific neutralizing mAb (clone H4)

Specific SARS-CoV-2 Spike-RBD neutralizing recombinant antibody 

Antibody description

InvivoGen provides Anti-CoV2RBD-c1-hIgG1 and Anti-CoV2RBD-c1-mIgG2a isotypes, derived from the specific SARS-CoV-2 neutralizing recombinant monoclonal antibody (mAb) originally described as 'clone H4' [1]. They feature:

• The variable region from 'clone H4' that specifically targets the Spike receptor-binding domain (S-RBD) of SARS-CoV-2 [1]
• The human IgG1 or mouse IgG2a constant region 

Clone H4 was isolated from a convalescent COVID-19 patient and subsequently, confirmed to specifically neutralize SARS-CoV-2, in vitro, by blocking the interaction between the SARS-CoV-2 S-RBD and the host receptor ACE2 [1].  Early in the pandemic, a previously characterized SARS-CoV neutralizing mAb (CR3022) against the S-RBD was rapidly found to cross-react with SARS-CoV-2 [2]. However, CR3022 does not neutralize SARS-CoV-2 possibly because, unlike clone H4, it does not target the ACE2 binding motif in the RBD [3]. Interestingly, synergistic neutralization ability was observed when clone H4 was used in combination with a different epitope-targeting antibody (clone B38) identified in the same study [1]. This makes them a potentially promising virus-targeting antibody cocktail for therapeutic and/or vaccine purposes.

InvivoGen's Anti-CoV2RBD-c1 mAbs (clone H4) have been generated by recombinant DNA technology, produced in CHO cells, and purified by affinity chromatography, ensuring lot-to-lot reproducibility. Furthermore, Anti-CoV2RBD-c1-hIgG1 and Anti-CoV2RBD-c1-mIgG2a have been functionally validated by ELISA and/or neutralizing assays (see data below). The absence of bacterial contamination has been confirmed by cellular assays.

Of note, InvivoGen also offers Anti-CoV2RBD-c2-hIgG1 and Anti-CoV2RBD-c2-mIgG2a, derived from another SARS-CoV-2 neutralizing mAb (Clone B38)  [1]. In-house data suggests that clone H4 has greater sensitivity for detection by ELISA, and clone B38 displays better inhibition of HEK-Blue™ hACE2 cell infection by Spike-pseudotyped lentiviral particles. However, depending on your application both mAbs may be suitable for the detection of Spike-RBD and neutralization of SARS-CoV-2 infection.

Applications

• Detecting the presence of SARS-CoV-2 in culture supernatant and/or in serum (ELISA)
• Flow cytometry binding assays
• Developing neutralizing antibody cocktails against SARS-CoV-2

Quality control

• Functionally validated by ELISA using a coated Spike-RBD-His fusion peptide

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References

1. Wu, Y. et al. 2020. A non-competing pair of human neutralizing antibodies block the COVID-19 virus binding to its receptor ACE2. Science 368, 1274-1278.
2. 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.
3. 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.

Figures

Validation of binding to SARS-CoV-2 RBD by ELISA. SARS‑CoV-2 Spike-RBD-His fusion peptide (5 μg/ml) was coated onto ELISA plates overnight. A serial dilution of Anti‑CoV2RBD-c1-hIgG1 (red curve) or Anti-βGal-hIgG1 (control antibody; grey curve) was added to the pre-coated plate and incubated for 1 hour. Subsequently, binding was detected with an HRP-labelled anti-hIgG1 antibody (1/1000 dilution) and the HRP substrate OPD (o-phenylenediamine dihydrochloride). Absorbance was read at 490 nm.

Validation of binding to SARS-CoV-2 spike-expressing cells. HEK293 wild-type (WT) and HEK293-expressing SARS-CoV-2 spike (S) cells were incubated with Anti-CoV2RBD-c1-hIgG1 (Blue and Red curve) or with a negative control mAb (Anti-βgal-hIgG1; orange curve) for 1 hour at 4°C (10 μg/ml per 5 x 105 cells). Following this, the cells were washed and incubated with an anti-human IgG-Fc PE-conjugated mAb for 1 hour at 4°C. The binding affinity was then measured using flow cytometry.

Inhibition of SARS-CoV-2 spike (G614-variant) pseudotyped lentiviral particles to infect human (h)ACE2-expressing cells. Anti-CoV2RBD-c2-hIgG1 (clone B38), Anti‑COV2RBD‑c1‑hIgG1 (clone H4), or a cocktail of both mAbs was incubated with SARS-CoV-2 spike-(G614-variant)-pseudotyped lentiviral particles for 1 hour at 37°C. Following this, HEK-Blue™ hACE2 cells were added and incubated for a further 72 hours. Infection by the pseudotyped particles (GFP-expression) was then measured using flow cytometry. Data are presented as % inhibition compared to the antibody-negative control.

Figure 1: Validation of binding to SARS-CoV-2 RBD by ELISA.
SARS‑CoV-2 Spike-RBD-His fusion peptide (5 μg/ml) was coated onto ELISA plates overnight. A serial dilution of Anti‑CoV2RBD-c1-mIgG2a (red curve) or Anti-β-Gal-mIgG2a (control antibody; grey curve) was added to the pre-coated plate and incubated for 1 hour. Subsequently, binding was detected with an HRP-labelled anti-mIgG2a antibody (1/1000 dilution) and the HRP substrate OPD (o-phenylenediamine dihydrochloride). Absorbance was read at 490 nm.

Specifications

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

• hIgG1: purified by affinity chromatography with protein G
• mIgG2a: purified by affinity chromatography with protein A

Quality control:

• The complete sequence of each antibody construct has been verified.
• Antibody binding 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.

 Upon receipt, store lyophilized antibody at -20 °C.

Details

Learn more about the SARS-CoV-2 Spike protein.