Gamma Variant - Luciferase-tagged RBD protein

Recombinant RBD fusion protein (P.1 variant - Brazilian origin) for ELISA & LIPS

RBD-LuciaV5 (P.1) (~52 kDa) is a soluble fusion protein composed of the Spike Receptor Binding Domain (RBD) from the SARS-CoV-2 Gamma variant (P.1) fused to a C‑terminal Lucia luciferase tag. RBD-LuciaV5 (P.1) has been specifically designed to assess the binding affinity of anti-Spike antibodies using either ELISA or LIPS (luciferase immunoprecipitation systems) assays [1-3].

 More details about RBD-Lucia

RBD-LuciaV5 (P.1) fusion protein for ELISA & LIPS

SARS-CoV-2 Spike RBD

RBD-LuciaV5 (P.1) contains the Spike RBD domain, including the receptor-binding motif (RBM) from the SARS-CoV-2 Gamma variant, first reported in Brazil in December 2020 [4]. This variant is classified as a member of Clade 20J / P.1 lineage (Nextstrain/Pango lineage classification). It is characterized by the presence of a number of mutations within the Spike RBD coding region, which are of concern [4].

• K417T, E484K, N501Y

Learn more about the emerging SARS-CoV-2 variants around the world

Applications

Luciferase-tagged RBD proteins are ideal for studying the binding of anti-spike monoclonal antibodies (mAbs) by solid-phase ELISA and/or solution‑phase LIPS assays, as well as anti‑spike polyclonal antibodies in the sera of recovered COVID‑19 patients and/or vaccinees by LIPS [1-3].

• ELISA: the Lucia luciferase tag provides a larger dynamic range than the commonly used HRP detection.
• LIPS: for the detection of antibodies, against both linear and conformational epitopes.

RBD‑LuciaV5 (P.1) has been generated by recombinant DNA technology, produced in CHO cells, and purified by IMAC (Immobilized Metal Affinity Chromatography) using a C‑terminal histidine tag. Protein size and purity (>90%) have been validated by SDS‑PAGE and the absence of endotoxin contamination has been confirmed using cellular assays. 

References:

1. Burbelo, P.D. et al. 2010. Antibody-profiling technologies for studying humoral responses to infectious agents. Expert Rev Vaccines 9, 567-578.
2. Haljasmagi, L. et al. 2020. LIPS method for the detection of SARS-CoV-2 antibodies to spike and nucleocapsid proteins. Eur J Immunol 50, 1234-1236.
3. Liang, Y. et al. 2021. A luciferase immunosorbent assay for quantitative detection of IgG antibodies against SARS-CoV-2 nucleoprotein. J Virol Methods 292, 114141.
4. Faria, N.R. et al. 2021. Genomics and epidemiology of the P.1 SARS-CoV-2 lineage in Manaus, Brazil. Science. doi:10.1126/science.abh2644.

Figures

Luciferase-based ELISA using RBD-LuciaV5 (P.1). 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, or the negative control Anti-βGal-mIgG2a, along with RBD-LuciaV5 (P.1) (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.

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.

Specifications

RBD-LuciaV5 (P.1)

• Protein construction: RBD [R319-F541] from the Spike glycoprotein fused to a C-terminal Lucia luciferase reporter
• Origin: Gamma Variant (P.1 lineage) | Brazilian origin
• Sequence Reference: GISAID EPI_ISL_811149
• Tag: C-terminal 6x Histidine tag
• Total protein size: 461 amino acids (including the Lucia luciferase)
• Molecular weight: ~52 kDa (SDS-PAGE)
• Purification: Immobilized metal affinity chromatography (IMAC)
• Purity: >90% (SDS-PAGE)
• Quality control:
  - The protein has been validated by ELISA upon incubation with a coated Anti-murine IgG (Fab')2 and a clinically relevant anti-Spike mAb.
  - The absence of bacterial contamination (e.g. lipoproteins and endotoxins) has been confirmed using HEK-Blue™ TLR2 and HEK-Blue™ TLR4 cellular assays.

Contents

RBD-LuciaV5 (P.1) contents:

• 50 μg of lyophilized protein
• 1.5 ml of endotoxin-free water
• 1 tube of QUANTI-Luc™ 4 Reagent, a Lucia luciferase detection reagent (sufficient to prepare 25 ml)

 The product is shipped at room temperature.

 Lyophilized protein should be stored at -20°C.

 Resuspended protein is stable for up to 1 month when stored at 4°C, and 1 year when stored at -20°C.

Avoid repeated freeze-thaw cycles.

Details

RBD-Lucia in ELISA

RBD-Lucia proteins can be used in a luciferase-based ELISA. Unlike a conventional ELISA, the plate is coated overnight with an Anti-human IgG F(ab')2 fragment. Upon addition of anti-spike monoclonal antibodies (mAb), they will bind to this 'capture' fragment through their Fc region, and RBD-Lucia will bind to the variable region. The luciferase activity is then used to assess the mAb binding affinity to the Spike RBD.

RBD-Lucia in LIPS

Currently, to perform a LIPS assay, soluble crude cell lysates or culture media of the luciferase tagged recombinant protein are extracted from transfected cells and directly used for the assay. InvivoGen's RBD-Lucia proteins streamline the protocol even further. Simply add the RBD-Lucia protein to either anti-spike mAbs or to anti‑spike polyclonal antibodies in the sera of recovered COVID‑19 patients and/or a vaccinee. Following this, antibody-protein complexes are purified using Protein A beads. Quantification of either binding affinity (mAb) and/or antibody levels (sera) is easily determined by assessing the Lucia luciferase activity.