Fighting COVID-19 with specific research reagents

With the now global outbreak of the novel SARS-like coronavirus, SARS-CoV-2 (2019-nCoV, COVID-19 virus), access to research tools has become critical to better understand the pathophysiology of the disease and develop specific and efficient novel therapeutics and prevention methods. Scientists identified the novel coronavirus as a group 2B CoV, distinct from the SARS-CoV. Genome sequencing indicated that SARS-CoV-2 (2019-nCoV) shares 79.5% sequence identity with SARS-CoV. It is therefore essential to assess SARS-CoV-2 specificity of research reagents. RayBiotech, distributed by tebu-bio in Europe, has made very special efforts to allow fast release of SARS-CoV-2 validated tools. This post introduces this new range of detection reagents, proteins and antibodies now available to boost COVID-19 research in Europe.

Rec. COVID-19 Proteins

The nucleocapsid protein (N-protein) and spike protein (S-protein) are encoded by all coronaviruses, including the coronavirus (SARS-CoV-2, COVID-19) that was first detected in Wuhan City, China, in December 2019. Both recombinant forms of these proteins are now available from RayBiotech through tebu-bio in Europe to advance infectious disease research (Figures 1 & 2). This will save you lengthy cloning, expression and purification steps to enable you to move much faster in your research. Time is of the essence these days.

Nucleocapsid Protein (N-Protein)

The nucleocapsid protein (N-protein) is a structural protein that binds to the coronavirus RNA genome, thus creating a shell (or capsid) around the enclosed nucleic acid. The N-protein also 1) interacts with the viral membrane protein during viral assembly, 2) assists in RNA synthesis and folding, 3) plays a role in virus budding, and 4) affects host cell responses, including cell cycle and translation.

Fig. 1. COVID19 N-Protein domains:
RBD = RNA binding domain; IDR = intrinsically disordered region; SR = serine-arginine-rich; NLS = nuclear localization signal.

Spike Protein (S-Protein)

The spike protein (S-protein) performs two primary tasks that aid in host infection: 1) it mediates the attachment between the virus and host cell surface receptors, and 2) facilitates viral entry into the host cell by assisting in the fusion of the viral and host cell membranes.

Fig. 2. COVID-19 S-Protein domains
RBD = RNA binding domain; SP = signal peptide; SR = serine-arginine-rich; TM = transmembrane domain

ACE2

ACE2 is an endogenous membrane protein that enables COVID-19 infection. During infection, the extracellular peptidase domain of ACE2 binds to the receptor binding domain of spike protein, which is a surface protein on SARS-CoV-2 (Fig. 3).

Fog. 3COVID-19 ACE2 protein domains:
SP = Signal peptide; TM = transmembrane domain; ID = Intracellular domain

Selection of Purified COVID-19 Proteins…

This table below will guide you towards the different COVID-19 proteins available, whether produced in E.coli or HEK293, with information on exact protein sequence and tag location:

Protein Cat #  Domain Host Region Tag MW (kDa)
N protein 230-30164 Full length HEK293 Cell Met1 – Ala419 C-term His ⁓50 kDa
N protein 230-01104 Full length E.coli Met1 – Ala419 N-term His ⁓50 kDa
S Protein 230-01102 S1 subunit E.coli Arg319 – Phe541 N-term His ⁓25 kDa
S Protein 230-30162 S1 subunit HEK293 Cell Arg319 – Phe541 C-term His ⁓25 kDa
S Protein 230-01101 S1 subunit E.coli Val16 – Gln690 N-term His ⁓75 kDa
S Protein 230-01103 S2 subunit E.coli Met697 – Pro1213 N-term His ⁓58 kDa
Human ACE2 230-30165 Full-length HEK293 Cell Gln18-Ser740 C-terminal His-tag ⁓90 kDa

… and their matching SDS-PAGE QC controls

COVID-19 Proteins SDS-PAGE analysis (part 1)

SDS-PAGE analysis of the purified recombinant SARS-CoV-2 target proteins and human ACE2

COVID-19 Antibodies

The antibodies available below have been validated to bind to proteins from SARS-CoV-2 (COVID-19), but were developed originally to target proteins from SARS-CoV-1, the virus responsible for the 2003 outbreak. Monoclonal mouse and polyclonal rabbit antibodies specific to SARS-CoV-2 spike and nucleocapsid proteins are currently available.

Rabbit Anti-SARS-CoV-2 N Protein in Action

The Anti-SARS-CoV Nucleocapsid (N) Protein (RABBIT) Antibody (cat. nr 200-401-A50 – Rockland Immunochemicals) is validated for ELISA (recommended dilution range: 1:10,000 – 1:50,000) and Western Blot : (recommended dilution range 1:2,000 – 1:10,000). It has also been used for Immuno-Fluorescence (IF) has described in the publication of Zhao B. et al. “Recapitulation of SARS-CoV-2 Infection and Cholangiocyte Damage with Human Liver Organoids” – doi: 10.1101/2020.03.16.990317.

It has also been cited in the publication of Thao, T.T.N., Labroussaa, F., Ebert, N. et al. “Rapid reconstruction of SARS-CoV-2 using a synthetic genomics platform”. Nature (2020). doi: 10.1038/s41586-020-2294-9

Localization of SARS-CoV-2 infection in human liver ductal 330 organoids. SARS-CoV-2 N is indicated in red using our Anti-SARS CoV Nucleocapsid Antibody (cat. nr 200-401-A50), while E-cadherin is in green and DAPI staining of chromatin in blue. Souce: Zhao et al. doi: https://doi.org/10.1101/2020.03.16.990317.

Another antibody reacting with N-Protein from SARS-CoV-2 (2019-nCoV) as well as SARS-CoV has been tested in western blot as illustrated below. The negative control are 293T cell lysate (lane 2), host cells for the production of the positive control N-Protein (lane 1).

Lane M: Protein Marker; Lane 1: N-Protein, 100 ng; Lane 2: 293T cell lysate, 12 µl

Rabbit Anti-SARS-CoV-2 Spike Protein

Lane M: Protein Marker; Lane 1: Spike S2 (230-20408) 12 µl; Lane 2: 293T cell lysate, 12 µl

COVID-19 N-protein IgG/IgM Detection kits

The detection kit presented below will provide precious information as to the presence of Coronavirus (SARS-CoV-2 / COVID-19) N-Protein IgM / IgG antibodies in human serum, plasma, whole blood, or finger prick samples for research projects. Please note this product is for research use only by qualified health professionals. These kits should not be used as the sole basis to diagnose or exclude SARS-CoV-2 (COVID-19) infection or to inform infection status. Due to high demand, delivery may take up to 2 weeks for a minimum order of 3 kits (60 tests).

Rapid Detection of SARS-CoV-2 N-Protein Antibodies

This lateral flow kit detects IgG and IgM antibodies to the coronavirus N-protein in serum, plasma, and peripheral blood. It is very easy to use and provides rapid results.

Detection principle: The detection kit uses the principle of immunochromatography: the separation of components in a mixture through a medium using capillary force and the specific and rapid binding of an antibody to its antigen. Each cassette is a dry medium that has been coated separately with novel coronavirus N protein (“T” test line) and goat antichicken IgY antibody (“C” control line) (Figure 1). Two free colloidal gold-labeled antibodies, mouse anti-human IgG (mIgG) and chicken IgY, are in the release pad section. Once diluted serum, plasma, or whole blood is applied to the release pad section, the mIgG antibody will bind to coronavirus IgG antibodies if they are present, forming an IgG-IgG complex. The sample and antibodies will then move across the cassette’s medium via capillary action. If coronavirus IgG antibody is present in the sample, the test line (T) will be bound by the IgG-IgG complex and develop color. If there is no coronavirus IgG antibody in the sample, free mIgG will not bind to the test line (T) and no color will develop. The free chicken IgY antibody will bind to the control line (C); this control line should be visible after the detection step as this confirms that the kit is working properly

FIg. 1. Lateral Flow Detection principle

Product performance Index

  1. Confirmation of Positive Reference samples per batch: 3 individual positive references samples were tested, and the result should identify all as positive samples. Results found 3 of 3 to be a positive and valid result.
  2. Confirmation of Negative Reference samples per batch: 20 negative reference samples and products were tested, and the results should find all samples as negative. Results found 20 of 20 samples to show a negative and valid result.
  3. Minimum detection limit: 3 samples at different concentrations of antibodies were tested, whereby a correct dilution (L3) and a lower dilution (L2) should be positive, while a too far diluted sample (L1), should be negative. Results confirmed L3, and L2 as positive, while L1 was negative.
  4. Repeatability: 10 Detection Cassettes for the sample positive sample across 2 different lots of Detection Cassettes were probed simultaneously. All 10 showed a positive and valid result.

If you are currently working in a COVID-19 project and are looking for readily available reagents to boost your project, visit our dedicated COVID-19 webpage.

Isabelle Nobiron, PhD
Written by Isabelle Nobiron, PhD
Isabelle is a Product Manager at tebu-bio, and also the company's ISO Quality Manager.