Epsilon Variant - Luciferase-tagged RBD protein

Recombinant RBD fusion protein (B.1.429 variant - Californian origin) for ELISA & LIPS

RBD-LuciaV4 (B.1.429) (~52 kDa) is a soluble fusion protein composed of the Spike Receptor Binding Domain (RBD) from the SARS-CoV-2 Epsilon variant (B.1.429) fused to a C‑terminal Lucia luciferase tag. RBD-LuciaV4 (B.1.429) 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

SARS-CoV-2 Spike RBD

Key mutation in the Epsilon variant (B.1.429) RBD

RBD-LuciaV4 (B.1.429) contains the Spike RBD domain, including the receptor-binding motif (RBM) from the SARS-CoV-2 Epsilon variant, first reported in the California USA in September 2020. [4]. This variant is classified as a member of Clade 21C / B.1.429 lineage (Nextstrain/Pango lineage classification). It is characterized by the presence of a key mutation within the Spike RBD coding region, which is of concern [4].

• L452R

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

Binding affinity of Anti-Spike mAbs using
Spike variant RBD-Lucia proteins

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.

Importantly, using InvivoGen's expanding collection of Spike variant RBD-Lucia proteins, it can be seen that the SARS-CoV-2 variants display varying binding affinities to the different clinically relevant anti-Spike mAbs (see right). RBD‑LuciaV4 (B.1.429) 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. Deng, X. et al. 2021. Transmission, infectivity, and antibody neutralization of an emerging SARS-CoV-2 variant in California carrying a L452R spike protein mutation. medRxiv doi: 10.1101/2021.03.07.21252647

Figures

Luciferase-based ELISA using RBD-LuciaV4 (B.1.429). 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-LuciaV4 (B.1.429) (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.

Detection of Spike antibodies in vaccinee sera by LIPS. RBD-LuciaV4 (B.1.429) (10 μg/ml) was mixed with either diluted serum from individuals vaccinated against SARS-CoV-2 (Sample 1-3) or Anti-CoV2RBD-imd-mIgG2a (mAb) diluted in ‘negative’ serum. Protein A beads were added to the mixture and incubated at room temperature for 2 hours with gentle shaking. After extensive washing (6x times), detection of anti-Spike antibodies was assessed by measuring the activity of Lucia luciferase using QUANTI-Luc™. Data are shown as a fold change over ‘negative’ serum (or 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-LuciaV4 (B.1.429)

• Protein construction: RBD [R319-F541] from the Spike glycoprotein fused to a C-terminal Lucia luciferase reporter
• Origin: Epsilon Variant (B.1.429 lineage) | Californian origin
• Sequence Reference: GISAID EPI_ISL_753546
• 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-LuciaV4 (B.1.429) 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 fusion protein for ELISA & LIPS

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.