Original strain - Luciferase tagged RBD protein

Key amino acids in the RBD (original)

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

Recombinant RBD fusion protein (Original strain - Wuhan origin) for ELISA & LIPS

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

RBD-Lucia contains the Spike RBD domain, including the receptor-binding motif (RBM), from the original SARS-CoV-2 isolate first reported in Wuhan in December 2019 [4]. This protein is characterized by the presence of a number of key amino acid residues (below) and is classified as the root of the pandemic in Clade 19A/ Lineage A (Nextstrain/Pango lineage classification).

• K417, L452, S477, T478, E484, and N501

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 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‑Lucia 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. Zhou, P. et al. 2020. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 579, 270-273.

Figures

Luciferase-based ELISA using RBD-Lucia. 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-Lucia (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-Lucia (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 in the supernatant 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-Lucia

• Protein construction: RBD [R319-F541] from the Spike glycoprotein fused to a C-terminal Lucia luciferase reporter
• Accession sequence: YP_009724390 (codon-optimized)
• Origin: SARS-CoV-2 Wuhan-Hu-1 (D614) isolate
• 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-Lucia 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.