SARS-CoV-2 Spike RBD Nanobody

Code CSB-RA33245A2GMY
Size US$700
Image
  • Activity Assay- ELISA
    The Binding Activity of SARS-CoV-2 Spike RBD Nanobody with SARS-CoV-2-S1-RBD
    Activity: Measured by its binding ability in a functional ELISA. Immobilized SARS-CoV-2-S1-RBD (CSB-YP3324GMY1) at 2 μg/ml can bind SARS-CoV-2 Spike RBD Nanobody, the EC50 is 0.8674 ng/ml.

  • GICA
    In the Colloidal Gold Immunochromatography Assay detection system, the background of antibody (CSB-RA33245A2GMY) is clean, the detection limit can be as low as 25ng/ml (1.75ng/0.07ml), and the sensitivity is very good.

  • Binding signal of SARS-CoV-2-S1-RBD (CSB-YP3324GMY1) and ACE2 protein-HRP conjugate (CSB-MP866317HU) was inhibited by SARS-CoV-2 Spike RBD Nanobody (CSB-RA33245A2GMY) with the IC50 is 1.296 nM.
  • Binding signal of SARS-CoV-2-S1-RBD (CSB-YP3324GMY1) and ACE2 protein-HRP conjugate (CSB-MP866317HU) was inhibited by SARS-CoV-2 Spike RBD Nanobody (CSB-RA33245A2GMY) with the IC50 is 0.1074 μg/ml.
  • SARS-CoV-2 Spike protein RBD His/Sumostar Tag (CSB-YP3324GMY1) captured on COOH chip can bind SARS-CoV-2 Spike RBD Nanobody (CSB-RA33245A2GMY) with an affinity constant of 28.2nM as detected by LSPR Assay.
  • ELISA: Immobilize various types of SARS proteins at concentration of 2μg/ml on solid substrate, then react with SARS-CoV-2 Spike RBD Nanobody at concentration of 100μg/ml, 10μg/ml and 1μg/ml. It shows the SARS-CoV-2 Spike RBD Nanobody (CSB-RA33245A2GMY) is specific for SARS-CoV-2-S1-RBD protein, without any cross-reactivity with MERS-CoV, SARS-CoV, HCoV-OC43 or HCoV-229E.
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Product Details

Description

This SARS-CoV-2 S1-RBD (Spike Glycoprotein S1 receptor-binding domain) antibody is a recombinant monoclonal antibody (also a Nanobody) generated through the expression of a DNA sequence inserting a human IgG1 Fc domain at the C-terminus, in human embryonic kidney 293 cells (HEK293). The DNA sequence encodes the SARS-CoV-2 spike RBD. The antibody is purified by protein G in vitro. It has been validated with high reactivity towards SARS-CoV-2 S1-RBD by a functional ELISA and good sensitivity for human SARS-CoV-2 spike glycoprotein (S protein) via the Colloidal Gold Immunochromatography Assay (GICA).
It is also validated in Neutralizing and LSPR. In neu assay, the binding signal of the SARS-CoV-2 S1 RBD antibody was inhibited by ACE2 protein-HRP conjugated inhibitor, with a 0.1074 μg/ml IC50. In LSPR assay, the SARS-CoV-2 S1 RBD antibody showed a high affinity with SARS-CoV-2 Spike protein RBD (affinity constant: 28.2nM).
Specifically binding and recognizing the RBD of the SARS-CoV-2 spike glycoprotein, the SARS-CoV-2 S1 RBD antibody can react with samples infected with human coronavirus SARS-CoV-2. But it does not respond to MERS or SARS-CoV spike protein. Akin to other nanobodies, this recombinant nanobody is small and stable, which allows for its reaching to hidden epitopes such as crevices of target proteins.

Uniprot No. P0DTC2
Target Names S (Spike glycoprotein)
Alternative Names Anti-coronavirus spike Antibody; Anti-cov spike Antibody; Anti-ncov RBD Antibody; Anti-ncov S1 Antibody;Anti-ncov spike Antibody; Anti-novel coronavirus RBD Antibody; Anti-novel coronavirus S1 Antibody; Anti-novel coronavirus spike Antibody; Anti-RBD Antibody; Anti-S1 Antibody; Anti-Spike RBD Antibody; E2 Antibody; E2 glycoprotein Antibody; Human coronavirus spike glycoprotein Antibody; S Antibody; SARS-CoV-2 S1 RBD Antibody; S glycoprotein Antibody; Spike glycoprotein Antibody
Species Reactivity Human Novel Coronavirus (SARS-CoV-2/ 2019-nCoV)
Immunogen Recombinant Human Novel Coronavirus Spike glycoprotein(S) (319-541aa) (CSB-YP3324GMY1 and CSB-MP3324GMY1b1)
Immunogen Species Human Novel Coronavirus (SARS-CoV-2/ 2019-nCoV)
Conjugate Non-conjugated
Clonality Monoclonal
Isotype VHH fusion with human IgG1 Fc
Clone No. A1
Purification Method Affinity-chromatography
Concentration It differs from different batches. Please contact us to confirm it.
Buffer Preservative: 0.03% Proclin 300
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Form Liquid
Tested Applications ELISA, GICA, Neutralising
Recommended Dilution
Application Recommended Dilution
ELISA 1:10000-1:100000
GICA 1:10000-1:40000
Neutralising 1:100-1:10000
Protocols ELISA Protocol
CSB-RA33245A2GMY LSPR protocol
Troubleshooting and FAQs Antibody FAQs
Storage Upon receipt, store at -20°C or -80°C. Avoid repeated freeze.
Lead Time Basically, we can dispatch the products out in 1-3 working days after receiving your orders. Delivery time maybe differs from different purchasing way or location, please kindly consult your local distributors for specific delivery time.

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 Q&A
Q:

Where was CSB-RA33245A2GMY purified from? cell culture / hybridoma or serum / ascites?

A:
It is a Nanobody that expressed from 293 cells (HEK293).
Q:

What is the concentration of this antibody, do you have this antibody in stock?

A:
The concentration of CSB-RA33245A2GMY is 1mg/ml, we have stock for this antibody and the lead time is 1-2 working days.
Q:

I am interested in this product. What is the detection limit? Is it below 100-1000 SARS-CoV-2 particles?

A:
We do not have information on the exact number of SARS-CoV-2 particles detected by the SARS-CoV-2 Spike RBD Nanobody. Please refer to the attached data sheet for the detection limit.
Q:

Is there any advantage of Nanobody compared with traditional hybridoma monoclonal antibody?

A:
Compared with traditional hybridoma monoclonal antibody, the Nanobody has many advantages as below.
1. The homology between sequences of VH and VHH is very high, so the Nanobody can be humanized easily.
2. The Nanobodies are more likely to bind with antigens specifically, so the Nanobodies have a high affinity.
3. The Nanobodies are very stable on quality, because they can resistance high temperature, as well as have good solubility and strong penetration.
4. The Nanobodies have low immunogenicity and can recognize hidden epitopes.
Q:

What's the meaning of "Nanobody"?

A:
The antibody contains only one heavy-chain variable region (VHH) and two regular CH2 and CH3 regions, but it does not stick to each other or even clump together as easily as the artificially engineered single-chain antibody fragment (scFv). More importantly, the independently cloned and expressed VHH structure has the same structural stability as the original heavy chain antibody and the binding activity with the antigen, which is the smallest unit of known binding target antigen, with the molecular weight of only 15KDa, also known as Nanobody (Nb).
Q:

What is the research significance of SARS-CoV-2 Spike RBD Nanobody?

A:
The Nanobodies are very small in size, with a molecular weight of only 12-15 kDa, furthermore, Nanobodies have good biophysical properties, so they can be administered by inhalation, which makes Nanobodies particularly suitable for the treatment of respiratory diseases. Therefore, the SARS-CoV-2 Spike RBD Nanobody plays a very important role and has significance in the research of novel coronavirus.
Q:

In the Colloidal Gold Immunochromatography Assay, the detection limit was as low as 25ng/ml (1.75ng). Does it mean a concentration of 25ng/ml?

A:
In the Colloidal Gold Immunochromatography Assay, the loading volume of sample is 70ul per test, and the concentration of sample is 25ng/ml, so the loading quantity of sample is 1.75ng.

Target Data

Function Spike glycoprotein comprises two functional subunits responsible for binding to the host cell receptor (S1 subunit) and fusion of the viral and cellular membranes (S2 subunit). For many coronavirus (CoVs), S is cleaved at the boundary between the S1 and S2 subunits, which remain non-covalently bound in the prefusion conformation. The distal S1 subunit comprises the receptor-binding domain(s) and contributes to stabilization of the prefusion state of the membrane-anchored S2 subunit that contains the fusion machinery. S is further cleaved by host proteases at the so-called S2' site located immediately upstream of the fusion peptide in all CoVs. This cleavage has been proposed to activate the protein for membrane fusion via extensive irreversible conformational changes. However, different CoVs use distinct domains within the S1 subunit to recognize a variety of attachment and entry receptors, depending on the viral species. Endemic human coronaviruses OC43 and HKU1 attach via their S domain A to 5-N-acetyl-9-O-acetyl-sialosides found on glycoproteins and glycolipids at the host cell surface to enable entry into susceptible cells. MERS-CoV S uses domain A to recognize non-acetylated sialoside attachment receptors, which likely promote subsequent binding of domain B to the entry receptor, dipeptidyl-peptidase 4. SARS-CoV and several SARS-related coronaviruses (SARSr-CoV) interact directly with angiotensin-converting enzyme 2 (ACE2) via SB to enter target cells.
Gene References into Functions
  1. Study presents crystal structure of C-terminal domain of SARS-CoV-2 (SARS-CoV-2-CTD) spike S protein in complex with human ACE2 (hACE2); hACE2-binding mode similar overall to that observed for SARS-CoV. However, details at the binding interface show that key residue substitutions in SARS-CoV-2-CTD slightly strengthen the interaction and lead to higher affinity for receptor binding than SARS-CoV receptor-binding domain. PMID: 32378705
  2. crystal structure of the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 bound to the cell receptor ACE2 PMID: 32365751
  3. crystal structure of the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 (engineered to facilitate crystallization) in complex with ACE2 PMID: 32320687
  4. Out of the two isolates from India compared to the isolates from Wuhan, China, one was found to harbor a mutation in its receptor-binding domain (RBD) at position 407 where, arginine was replaced by isoleucine. This mutation has been seen to change the secondary structure of the protein at that region and this can potentially alter receptor binding of the virus. PMID: 32275855
  5. Structural modeling of the SARS-CoV-2 spike glycoprotein show similar receptor utilization between SARS-CoV-2 and SARS-CoV, despite a relatively low amino acid similarity in the receptor binding module. Compared to SARS-CoV and all other coronaviruses in Betacoronavirus lineage B, an extended structural loop containing basic amino acids were identified at the interface of the receptor binding (S1) and fusion (S2) domains. PMID: 32245784
  6. crystal structure of CR3022, a neutralizing antibody from a SARS patient, in complex with the receptor-binding domain of the SARS-CoV-2 spike (S) protein to 3.1 A; study provides insight into how SARS-CoV-2 can be targeted by the humoral immune response and revealed a conserved, but cryptic epitope shared between SARS-CoV-2 and SARS-CoV PMID: 32225176
  7. SARS-CoV and SARS-CoV-2 spike proteins have comparable binding affinities achieved by balancing energetics and dynamics. The SARS-CoV-2-ACE2 complex contains a higher number of contacts, a larger interface area, and decreased interface residue fluctuations relative to the SARS-CoV-ACE2 complex. PMID: 32225175
  8. Interaction interface between cat/dog/pangolin/Chinese hamster ACE2 and SARS-CoV/SARS-CoV-2 S protein was simulated through homology modeling. Authors identified that N82 of ACE2 showed closer contact with receptor-binding domain of S protein than human ACE2. PMID: 32221306
  9. SARS-CoV-2 S glycoprotein harbors a furin cleavage site at the boundary between the S1/S2 subunits, which is processed during biogenesis and sets this virus apart from SARS-CoV and SARS-related CoVs; determined cryo-EM structures of the SARS-CoV-2 S ectodomain trimer. PMID: 32201080
  10. Study demonstrates that SARS-CoV-2 uses the SARS-CoV receptor ACE2 for entry and the serine protease TMPRSS2 for S protein priming. PMID: 32155444
  11. The ACE2-B0AT1 complex exists as a dimer of heterodimers. Structural alignment of the RBD-ACE2-B0AT1 ternary complex with the S protein of SARS-CoV-2 suggests that two S protein trimers can simultaneously bind to an ACE2 homodimer. PMID: 32142651
  12. study demonstrated SARS-CoV-2 S protein entry on 293/hACE2 cells is mainly mediated through endocytosis, and PIKfyve, TPC2 and cathepsin L are critical for virus entry; found that SARS-CoV-2 S protein could trigger syncytia in 293/hACE2 cells independent of exogenous protease; there was limited cross-neutralization activity between convalescent sera from SARS and COVID-19 patients PMID: 32132184
  13. study determined a 3.5-angstrom-resolution cryo-electron microscopy structure of the 2019-nCoV S trimer in the prefusion conformation; provided biophysical and structural evidence that the 2019-nCoV S protein binds angiotensin-converting enzyme 2 (ACE2) with higher affinity than does severe acute respiratory syndrome (SARS)-CoV S PMID: 32075877

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Subcellular Location Virion membrane, Single-pass type I membrane protein, Host endoplasmic reticulum-Golgi intermediate compartment membrane, Single-pass type I membrane protein, Host cell membrane, Single-pass type I membrane protein
Protein Families Betacoronaviruses spike protein family

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