Activity of InvivoGen's Anti-CoV2RBD Monoclonal Antibodies

Validation assays

InvivoGen's anti-CoV2RBD monoclonal antibodies (mAbs) have been tested in-house to compare their activity. The tests consist of Lucia luciferase-based ELISA, Luciferase immunoprecipitation system (LIPS) assays and FACS screening assays that have been performed using:

• RBD-Lucia proteins, a collection of Lucia luciferase-tagged Spike RBD proteins from SARS-CoV-2 variants (including the variants of concern), or  
• 293-SARS2-S-dfur cells, a series of HEK293-derived cells expressing at the surface the Spike protein of the variants of concern.

Below are the data obtained with the following anti-SARS-CoV2 Spike antibodies featuring a mouse IgG2a constant region:

   -  Anti-CoV2RBD-cas : REGN10933-derived mAb
   -  Anti-CoV2RBD-imd : REGN10933-derived mAb
   -  Anti-CoV2RBD-bam : LY-CoV555-derived mAb
   -  Anti-CoV2RBD-ete : LY-CoV016-derived mAb

Figure 1: Lucia Luciferase-based ELISA

The binding capacity of InvivoGen's anti-SARS-CoV2RBD mAbs to a set of Spike variants has been validated using a Lucia luciferase-based ELISA.
Anti-murine IgG F(ab’)2 fragment (2 µg/ml) was coated on an ELISA plate overnight. Anti-CoV2RBD-cas-mIgG2a, Anti-CoV2RBD-imd-mIgG2a, Anti-CoV2RBD-bam-mIgG2a, Anti-CoV2RBD-ete-mIgG2a along with RBD-Lucia proteins (original and V2 to V8; 1 µg/ml) were added and incubated for 2 hours at room temperature. After washing (3x times), binding affinity was assessed by measuring the activity of Lucia luciferase in the supernatant using QUANTI-Luc™. Data are shown as a fold change over no antibody.

Figure 2: Luciferase immunoprecipitation system (LIPS) assays

InvivoGen's RBD-Lucia proteins allow the testing of anti-Spike antibodies in the sera of COVID-19 patients or vaccinees.
Protein A beads, vaccinee serum, and RBD-Lucia proteins were co-incubated for 2 hours at room temperature. RBD-Lucia-antibody complexes bound to Protein A beads were then purified and extensively washed before quantification using  QUANTI-Luc™. Representative data are shown as a fold change over no serum.

Figure 3: FACS screening assays

InvivoGen's anti-SARS-CoV2RBD mAbs allow the detection of Spike surface expression by mammalian cells for a set of SARS-CoV-2 variants of concern.
Human embryonic kidney (HEK)-293 cells featuring stable cell surface expression of Spike variants were incubated with either Anti-CoV2RBD-cas-mIgG2a, Anti-CoV2RBD-imd-mIgG2a, Anti-CoV2RBD-bam-mIgG2a, Anti-CoV2RBD-ete-mIgG2a, or Anti-CoV2RBD-2130-hIgG1 mAbs, or hACE2-Fc fusion protein for 1 hour at 4°C. Cells were then washed and incubated with a PE-conjugated goat anti-murine IgG or PE-conjugated goat anti-human IgG for 1 hour at 4°C, and surface staining was analyzed by FACS. The grey line indicates the maximal fluorescence intensity for unstained cells.