Several new variants of SARS-CoV-2 virus have emerged in recent months. The U.K. variant B.1.1.7, the Brazil variant P.1, the South Africa variant B.1.351, and the most recent Indian variant B.1.617 are particular concerning because of their high prevalence. A subset of the mutations identified in the RBD domain of the spike protein occurs in more than one strain. These convergent mutations are of high interest because they may be the cause of the increased transmissibility. Sino Biological has launched RBD and Spike proteins of these variants.

The mutations in these strains also occur in the nucleocapsid protein, which is commonly used as the biomarker in rapid antigen tests. It’s critical to assess whether the current commercial antigen tests can detect the mutated N proteins with the same sensitivity and specificity as their WT counterpart.

By monitoring the evolution of SARS-CoV-2, WHO has published these Variants of Concern (VOCs) to accelerate the globe to make quick response to the ongoing COVID-19 pandemic.

Characteristics of VOCs can be:
• Increase in transmissibility or detrimental change in COVID-19 epidemiology
• Increase in virulence or change in clinical disease presentation
• Decrease in effectiveness of public health and social measures or available diagnostics, vaccines, therapeutics

Browse full list of variants

Mutations in the Spike Glycoprotein of Omicron

The Omicron variant is by far the most divergent variant characterized by a high number of mutations in the spike protein. Several mutations (e.g., N501Y, P681H) have been identified in previous variants and are associated with increased transmissibility, immune escape, or other properties.

• 30 amino acid changes: among which 15 are located in the RBD region
• Three deletions
• Three insertions in the spike protein NTD

other tags available His & AVI – please inquire

Recombinant Spike N501Y Mutant
Cat#: 40592-V08H82
Activity: Bind with ACE2

The spike protein of the Omicron Variant carries more than 30 mutations. It’s critically important to understand whether these mutations allow the Omicron variant to escape from neutralizing antibodies.

Mutations in the spike glycoprotein of Omicron

Source: Stanford University Coronavirus Database

After screening a panel of previously established spike antibodies, we identified one mAb, clone 40592-MM117, strongly reactive with the Omicron Spike protein. There is another antibody that demonstrates moderate reactivity against the Omicron variant. All other tested clones, including several broad-spectrum ACE2-competing neutralizing antibodies, don’t bind with the mutant at all.

It’s noteworthy that clone 40592-MM117 has non-ACE2 competing neutralizing activity against both the original strain and the Delta variant. Interestingly, the other mAb tested reactive to Omicron also has non-ace2 binding neutralizing activity against previous strains. Whether the same clones can also neutralize the Omicron variant is currently being tested.

Sino Biological has developed recombinant Omicron Spike RBD Protein and Antibody reacting with Omicron to support the study of Omicron variant. More Omicron antigens, including B.1.1.529 S-ECD, S1, and Nucleocapsid, are coming soon.

SARS-CoV-2 Spike Antibody, Omicron Reactive, Mouse MAb

Cat: 40592-MM117

• Ability to Recognize Omicron Spike Protein

40592-MM117 showed strong reactivity with Omicron spike protein.

• Binding Activity by ELISA Assay against SARS-CoV-2 Variants

40592-MM117 shows binding activity against spike proteins of the Omicron variant. It also detects the wild type and Delta variants. In comparison, the other two broad-spectrum ACE2-competing neutralizing antibodies, 40591-MM43 and 40150-D001, do not bind with the Omicron variant.

Omicron RBD Protein
(Cat#: 40592-V08H121)

Delta RBD Protein
(Cat#: 40592-V49H1)

RBD Protein
(cat#: 40592-V08H)

SARS-CoV-2 B.1.1.529 (Omicron) Spike RBD Protein (His Tag)

Cat: 40592-V08H121

• Mutations in RBD Region:

G339D、S371L、S373P、S375F、K417N、N440K、G446S、S477N、T478K、E484A、Q493R、G496S、Q498R、N501Y、Y505H

• High Purity

> 90 % as determined by SDS-PAGE

• High Binding Activity

Bind with ACE2 Validated by ELISA Assay

Immobilized human ACE2 protein (mFc tag)(Cat: 10108-H05H) at 2 μg/mL (100 μL/well) can bind Omicron RBD Protein (His Tag)(Cat: 40592-V08H121), the EC₅₀ is 13.7 ng/mL.

• More Omicron Variants is Developing by Sino Biological.

Please Contact Us for More Detailed Information.

Antibodies for the Detection of Nucleocapsid Mutants

Sino Biological has newly developed a series of antibodies that can detect nucleocapsid mutants found in Variants of Interest (VOIs) and Variants of Concern (VOCs).

Binding Activity by ELISA Assay (Partial List)

	40588-MM123	40143-MM08	40588-MM137	40143-R004	40588-R001	Antigen Mutation Description
40588-V07E	+++	+++	+++	+++	+++	Wild Type
40588-V07E1	++++	++++	++++	++++	++++	N (R203K, G204R), discovered in many lineages
40588-V07E2	++++	++++	++++	+++	++++	N (P13L), discovered in many lineages
40588-V07E3	++++	+++	++++	+++	+++	N (S194L), discovered in many lineages
40588-V07E4	++++	+++	++++	+++	+++	N (I292T), discovered in many lineages
40588-V07E7	++++	++++	+++	++++	++++	N (D3L, R203K, G204R, S235F), found in B.1.1.7
40588-V07E8	++++	++++	++++	+++	+++	N (D3L, S235F), found in B.1.1.7
40588-V07E9	++++	++++	++++	++++	++++	N (T205I), found in B.1.351
40588-V07E11	+++	+++	++	++++	+++	N (P80R), found in P.1
40588-V07E19	+++	+++	+++	+++	+++	N (A12G, T205I), found in the B.1.429 & B.1.351
40588-V07E16	+++	++	++	++	++	N (D377Y), found in B.1.617
40588-V07E20	++++	++++	++++	++++	++++	N (R203M, D377Y), found in B.1.617, B.1.617.1
40588-V07E29	++++	++++	+++	++++	++++	N (D63G, R203M, D377Y), found in B.1.617.2
40588-V07E30	++++	++++	++++	++++	++++	N (P67S, R203M, D377Y), found in B.1.617.3

+: Binding Capacity

Please kindly contact us for more antibodies.

Neutralization Efficacy Assay aganist SARS-CoV-2 Variants

Some of the above mutations may have allowed the virus to escape from neutralizing antibodies. To characterize these variants, a new panel of monoclonal antibodies have been launched this month. These antibodies demonstrate differential neutralizing activities against different variants validated by Competitive ELISA Assay. Notably, the B.1.351 and P.1 variants seem to be immune to a subset of the antibodies.

+: Neutralizing Capacity

The Aforementioned Recombinant RBD :
WT: 40592-V08B
B.1.1.7 | U.K. Variant: 40592-V08H82 (N501Y)
B.1.351 | South Africa Variant: 40592-V08H85 (K417N, E484K, N501Y)
P.1 | Brazil Variant: 40592-V08H86 (K417T, E484K, N501Y)
B.1.617 | India Variant: 40592-V08H88 (L452R, E484Q)

The Aforementioned Recombinant S1 :
B.1.617 | India Variant: 40591-V08H19 (E154K, E484Q, L452R, D614G, P681R)
B.1.429 | California Variant: 40591-V08H17 (W152C, L452R, D614G)

40150-D001

40592-MM57

All coronaviruses share very similar structures.

The viral genome encodes several proteins of unique functions, S protein

N protein

HE protein

papain-like proteases

M protein.

The two antigens of main pharmaceutical interest are the S (spike) protein and the N protein.

• The N (nucleocapsid) protein is often conserved, which can be used as a diagnostic marker.
• The Spike protein is mainly responsible for receptor binding, and is a common target for vaccines and antibodies. The size of the abundantly N-glycosylated S protein varies greatly between CoV species

The spike protein is particularly important as its interaction with the host cell receptor is the pivotal step during the infection. Different viruses may utilize different surface receptor for binding. The HCOV- NL63, SARS-COV, and the new SARS-COV-2 viruses all use the ACE2 receptor, while the MERS-COV virus selectively binds with the DPP4 receptor. The HCOV-229E virus targets APN receptor. The rest two common coronavirus, HKU1 and OC43 bind with O-ac Sia.

Spike Protein

The spike protein is a large type I transmembrane protein containing two subunits, S1 and S2. S1 mainly contains a receptor binding domain (RBD), which is responsible for recognizing the cell surface receptor. S2 contains basic elements needed for the membrane fusion. The S protein plays a key role in the induction of neutralizing-antibody and T-cell responses, as well as protective immunity.

The spike (S) glycoprotein of coronaviruses contains protrusions that will only bind to certain receptors on the host cell

Nucleocapsid Protein

The N protein constitutes the only protein present in the nucleocapsid. It is composed of two separate domains, an N-terminal and a C-terminal domains both capable of binding viral RNA in vitro

N protein is also heavily phosphorylated and binds to the viral genome

N protein also binds nsp3, a key component of replicase-transcriptase complex , and the M protein and subsequently packages the encapsidated genome into viral particles.

Angiotensin-converting enzyme 2 (ACE2)

Angiotensin-converting enzyme 2 (ACE2) is a membrane receptor expressed on the surface of airway epithelial cells.

The interaction between the viral spike protein and the ACE2 receptor is mediated by the RBD domain of the spike protein, and is believed to be the pivotal event in the membrane fusion process.

It’s been shown that SARS-COV-2 also uses ACE2 as the receptor for host cell entry. This finding suggests that blocking the interaction between the S protein and ACE2 is a valid approach to prevent or treat coronavirus infection.

Mammalian cells CHO, HEK293

• Protein folding
• Post-translational modification
• Flexible and rapid production of proteins.
• Lower production yield than bacterial systems
• Higher cost than bacterial systems
• Complex culture media

Insect cells expression systems

• Robust
• Simple culture media
• High expression levels
• Able to perform a variety of post-translational modifications similar to mammalian cells

Bacterial Systems

• High protein expression yield
• Most economical
• No disulfite bonds
• Additional modifications are required post production

Tags

IgG-Fc-tag (mFc = mouse IgG – rFc = rabbit IgG)

• allows for rapid and simple detection of protein expression by ELISA
• allows for simple affinity purification of the Fc-tagged protein by protein A, protein G, and protein L affinity purification resins.
• increases protein expression yield.

His Tag

• 6xHis-tag is one of the simplest and most widely used purification tags
• The his-tag has a high affinity for metal ions  Ni2+or Co2+immobilized on beads or a resin for such as IMAC columns

An unprecedented mink cull has been ordered in Denmark amid the outbreak of SARS-CoV-2 in these farmed animals. The plan was announced after scientists discovered a widespread Y453F mutation in the spike protein, that has been passed from animal to humans.

This mutation is of particular concern, because it occurs at a conservative domain of the receptor binding domain (RBD) directly involved in ACE2 binding. Results from some preliminary studies suggest the Y453F mutation affects the ability of the Spike protein to bind with ACE2, while others demonstrate that the mutated spike can escape from detection from a commercial anti-S antibody.

Although there is still no clear evidence indicating this mutation, or any other mutation like the popular D614G, has any clinical significance, the characteristics of the mutations need to be thoroughly investigated in the context of vaccine and antibody therapy.

Sino Biological has launched the recombinant Y453F RBD protein. This product is the newest addition to a large library of recombinant spike variants (full list here). These proteins can be used to evaluate the efficacy of the antibodies and vaccination.

IF (40150-R007) – Pseudovirus Validated

SARS-CoV-2 Spike in ACE2-overexpressed 293T cells, infected (left) or noninfected (right) by 2019-nCOV pseudovirus

WB (40143-MM05)

1-2: SARS-CoV N Protein
3-4: 2019-nCoV N Protein

IHC (40143-R019)

Renal tissue from COVID-19 Patient. N antigens in kidney tubules is validated by IHC

Neutralization Activity Assay
Neutralizing antibodies can effectively inhibit the infection of SARS-CoV-2 Spike pseudovirus on ACE2 overexpression 293T cells.

S1 Neutralizing Antibody

RBD Neutralizing Antibody 

RBD Neutralizing Antibody Rabbit MAb

Competitive-ELISA Assay
Neutralizing ability was detected by SARS-CoV-2 Inhibitor Screening ELISA Kit (Catalog# KIT001) using competitive ELISA.

S1 Neutralizing Antibody

The IC50 is typically 0.857 nM

RBD Neutralizing Antibody

The IC50 is typically 3.694 nM

RBD Neutralizing Antibody Rabbit MAb

The IC50 is typically 0.066 nM

Specificity Assay

SARS-CoV-2 shares 80% sequence identity to the 2003 SARS-CoV. Hence, antibodies raised against SARS-CoV-2 antigens often shows various degree of cross-reactivity with the corresponding antigens of 2003 SARS-CoV. On the other hand, the similarities between SARS-CoV-2 and other members of human coronavirus are very limited. The following two clones of anti-spike antibodies have been tested against all seven coronavirus, and are shown to be specific to SARS-CoV-2 spike.

The reactivity of the antibodies against the known coronavirus is assessed in ELISA. The reactivity (OD450) is illustrated in the figure below.

S1 Neutralizing Antibody

RBD Neutralizing Antibody

Inihibitor Screening ELISA kits

Antibody Titer Assay Kits

The S and N detection kits employ a standard sandwich ELISA, allowing rapid quantification of the N and S antigens.

• All antibodies used in the kits are in-house developed monoclonal antibodies, ensuring batch-to-batch consistency.
• These assays may be used to quantify antigens on the surface of SARS-CoV-2 and/or pseudovirus, as well as antigens expressed recombinantly.

2019-nCoV N Detection Kit
(KIT40588)

• Sino Biological provides ready-to-use expression ORF clones without subcloning. The expression ORF clones can be use for transfecting immediately.
• Codon Optimized clones for high expression, no tag
• Datasheet contains complete information on on plasmid, gene and quality control