Invivogen
Menu

Original strain - Luciferase tagged RBD protein

RBD-Lucia Unit size Cat. code Docs Qty Price
Luciferase tagged original RBD protein
50 µg
rbd-lucia
+-
$284.00

You may also need : QUANTI-Luc™ | View more associated products

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

Key amino acids in the RBD (original)
Key amino acids in the RBD (original)

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

Binding affinity of Anti-Spike mAbs using InvivoGen's RBD-Lucia proteins
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 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
Luciferase-based ELISA using RBD-Lucia

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
Detection of Spike antibodies in vaccinee sera by LIPS

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).

Back to the top

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.
Back to the top

Contents

RBD-Lucia contents:

  • 50 μg of lyophilized protein
  • 1.5 ml of endotoxin-free water
  • 1 pouch of QUANTI-Luc™

room temperature The product is shipped at room temperature.

store Lyophilized protein should be stored at -20 ̊C.

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

Avoid repeated freeze-thaw cycles.

Back to the top

Details

RBD-Lucia fusion protein for ELISA & LIPS
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.

Back to the top
Customer Service
& Technical Support
Shopping cart is empty