Research Solutions for Coronavirus – Lifespan Biosciences

LSBio’s library of coronavirus products includes numerous kits for the detection of SARS, SARS-CoV-2, MERS, HCoV, FIPV, and other common coronaviruses. We offer COVID-19 real-time quantitative PCR (RT-qPCR) kits for the direct detection of SARS-CoV-2 RNA and a wide range of ELISA kits to detect human IgG/IgM, coronavirus proteins, and host proteins relevant to COVID-19 disease progression.

Real-Time qPCR (RT-qPCR) is a technique that allows researchers to quantitatively detect the presence of specific RNA, such as SARS-CoV-2, within a sample. Following nucleic acid extraction and reverse transcription of RNA to cDNA, the polymerase chain reaction is used to amplify the target DNA, which is then detected using fluorescently labeled highly-specific probes. As the reaction progresses, the release of fluorescence can be monitored, providing a means of quantifying the target. In response to the COVID-19 pandemic, we offer both Real-Time qPCR kits and sample collection kits. The RT-qPCR kits require access to a real-time quantitative PCR instrument.

This rapid test is for research use only and has not been approved by the FDA for clinical diagnostic use. Samples collected and processed for this test are potentially infectious, and should be handled only by trained laboratory professionals in a BL-2 laboratory or equivalent setting with a laminar flow hood and personal protective equipment.

LSBio provides a range of Sandwich ELISA kits for the quantitative detection of numerous coronavirus proteins, including SARS-CoV and SARS-CoV-2 spike and nucleocapsid proteins. We also offer kits which detect the SARS-CoV-2 receptor ACE2, protease TMPRSS2, and numerous inflammatory proteins, such as IL-6, IL-10, and TNF-alpha.

ELISA for SARS-CoV-2 IgG/IgM Antibody Detection

ELISA for SARS-CoV-2 Viral Proteins

LSBio offers antibodies, proteins, and cDNAs specific to coronavirus proteins and host proteins involved in coronavirus disease progression. Reagents for investigating SARS-CoV-2, SARS-CoV, MERS, and HCoV coronavirus structural and non-structural proteins, as well as coronavirus-interacting host proteins such as ACE2, TMPRSS2, Cathepsin, and ADAM17 are available and approved for use in multiple applications. We have a wide selection of related recombinant proteins, native proteins, and over-expression lysates. Furthermore, we provide expression-ready ORF cDNA clones to coronavirus genes of prominent research interest.

LSBio offers over 150 antibodies to structural and non-structural coronavirus proteins, including antibodies for the detection of the spike (S), nucleocapsid (N), and envelope (E) proteins of both SARS-CoV and SARS-CoV-2. We also offer antibodies which detect human proteins known to interact with coronaviruses or to contribute to COVID-19 disease progression. Many of our antibodies are available in multiple conjugated forms and are designed for use in a range of assays including ELISA, IHC, Western Blot, IF, and Flow Cytometry.

View LSBio’s library of high-quality recombinant coronavirus proteins, produced in a variety of expression systems and ready for use in functional assays, high throughput screens, structural analysis studies, and a variety of other applications. Full length recombinant proteins to SARS-CoV-2 structural proteins are available for your COVID-19 research needs.

LSBio offers expression-ready ORF clones to a range of coronavirus genes intended for immediate transfection. SARS-CoV-2 clones are available for stable or transient mammalian and lentiviral expression.

LSBio offers a range of research-use-only biochemicals that show potential inhibitory action against various coronaviruses such as SARS-CoV-2. These include direct inhibitors of coronavirus spike, nucleocapsid, protease, and polymerase proteins. We also offer various chemicals that block host proteins utilized by coronaviruses for infection and replication.

SARS-CoV-2 Spike (S) Protein Inhibitors

The spike (S) protein is responsible for attachment to and fusion with the host cell. For both SARS-CoV and SARS-CoV-2, the host receptor is known to be angiotensin converting enzyme 2 (ACE2) (Hoffmann, 2020). The spike protein is proteolytically cleaved into two subunits, S1 and S2. The S1 subunit binds to ACE2 presented on the host cell surface, and the S2 subunit is responsible for fusion with the host cell. Target cells include pneumocytes and macrophages expressing ACE2 in the lung, as well as ACE2-positive epithelial cells in the lung, gastrointestinal tract, and liver (cholangiocytes). The spike protein subunits are of interest as targets of vaccines; the S2 subunit is highly conserved and may be an effective pan-coronavirus target. SARS-CoV studies in monkeys immunized with full-length spike protein showed successful protection against subsequent infection (Shang, 2020).

Camostat Mesylate

Camostat mesylate is an inhibitor of the protease TMPRSS2. This host protease is used by SARS-CoV-2 for spike protein priming, and inhibition with camostat mesylate has been shown to block SARS-CoV-2 infection in lung cell lines (Hoffmann, 2020).

Emodin

Emodin is an anthraquinone derivative extracted from Rheum tanguticum. It has been found to inhibit the SARS-CoV ORF3a protein and also block interactions between the SARS-CoV spike protein and the ACE2 receptor. It is of interest as a potential inhibitor of SARS-CoV-2 infection (Zhou, Y., 2020).

SARS-CoV-2 Membrane (M) Protein Inhibitors

The membrane (M) protein determines the shape of the SARS-CoV-2 viral envelope and organizes viral assembly through interaction with each additional structural protein (Chang, 2014).

Toremifene

Toremifene is a nonsteroidal selective estrogen receptor modulator (SERM) used in the treatment of metastatic breast cancer. It has also been found to block the fusion of viral and endosomal membranes by destabilizing the viral membrane glycoprotein and has been demonstrated to inhibit Ebola, MERS-CoV, and SARS-CoV viral replication in vitro using established cell lines. It is of interest as a potential inhibitor of SARS-CoV-2 infection (Zhou, Y., 2020).

SARS-CoV-2 Envelope (E) Protein Inhibitors

The envelope (E) protein is the smallest of the SARS-CoV-2 structural proteins, and it is incorporated into the virion envelope, though this represents only a small amount of total expressed envelope protein. A high proportion is also expressed inside the infected cell, where it is involved in viral assembly, budding, maturation, and propagation (Schoeman, 2019; Chang, 2020). The E protein sequence of SARS-CoV-2 has 90% amino acid overlap with other human coronavirus envelope proteins, which may allow for the development or repurposing of pan-coronavirus antiviral drugs that target this protein.

Belachinal, Macaflavanone E, and Vibsanol B are phytochemicals which have been shown in in silico models to potentially inhibit the activity of E protein (Gupta, M.G., 2020).

SARS-CoV-2 Papain-like Protease Inhibitors

There are two types of proteases expressed by SARS-CoV and SARS-CoV-2, the papain-like protease (PLpro, NSP3), and the CL-like protease (NSP5A & B, 3CLpro, Mpro). These proteases are responsible for cleaving the viral polyprotein and releasing nonstructural proteins (NSPs). These NSPs are vital for SARS-CoV-2 viral replication, maturation, and its overall life-cycle. In SARS-CoV, the papain-like protease (PLpro) inhibits type I interferon (IFN) by blocking IRF3 phosphorylation, which results in downstream inhibition of interferon induction and a reduction in the host’s innate immune response. This is thought to contribute to higher viral titer and leads to an increase in cell death and damage to infected and surrounding tissue (Matthews, 2014). The SARS-CoV-2 papain-like protease is thus of interest as a drug target to prevent viral replication and potentially reduce tissue damage (Báez-Santos, 2015).

Lopinavir

Lopinavir is the most powerful inhibitor of CoV protease and Saquinavir is the least powerful. As per current guidelines, Lopinavir + Ritonavir is the recommended protease inhibitor combination for the treatment of COVID-19.

Cytokines are small proteins produced in the body to mediate cell signaling and regulation of the immune system. Cytokine Release Syndrome is characterized by an immune over-response, where the majority of the damage done to the host is due to an attack by the immune system rather than a direct cytotoxic effect by the pathogen. This systemic inflammatory response is mediated by the release (by immune cells) of inflammatory cytokines such as IFN-alpha, IFN-gamma, IL-1, IL-1 beta, IL-2, IL-2R, IL-6, IL-8, IL-10, IL-12, IL-17D, IL-18, IL-33, TNF-alpha, TGF-beta, and chemokines such as CCL2, CCL3, CCL5, CXCL8, CXCL9, and CXCL10. The overproduction of inflammatory cytokines leads to an attack on multiple organ systems resulting in ARDS, multi-organ failure, and hypotensive shock (Li, X., 2020; Huang, C., 2020). Both severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome CoV (MERS-CoV) cause a severe and highly lethal respiratory disease in humans. These diseases are also characterized by a prominent pro-inflammatory response and mediated by release of cytokines (Chan, J.F., 2015).

Cytokine Release Syndrome Targets

Interleukin-1 Beta (IL-1B)

IL-1B is a potent pro-inflammatory cytokine. Initially discovered as the major endogenous pyrogen, it induces prostaglandin synthesis, neutrophil influx and activation, T-cell activation and cytokine production, B-cell activation and antibody production, and fibroblast proliferation and collagen production. It promotes differentiation of the TH17 subset of T-cells.

Interleukin-2 (IL-2)

Produced by T-cells in response to antigenic or mitogenic stimulation, this protein is required for T-cell proliferation and many other activities crucial to regulation of the immune response. It can stimulate B-cells, monocytes, lymphokine-activated killer cells, natural killer cells, and glioma cells.

Interleukin-2 Receptor (IL-2R)

The increased expression of IL-2R and IL-6 in serum is expected to predict increased severity of COVID-19 pneumonia and a poorer patient prognosis (Chen, L., 2020).

Interleukin-2 Receptor Gamma (IL-2RG)

IL-2RG is a common subunit for the receptors for a variety of interleukins. Probably in association with IL-15RA, it is involved in the stimulation of neutrophil phagocytosis by IL-15.

Interleukin-6 (IL-6)

IL-6 is a cytokine with a wide variety of biological functions. It is a potent inducer of the acute phase response, plays an essential role in the final differentiation of B-cells into immunoglobulin-secreting cells, and is involved in lymphocyte and monocyte differentiation. IL-6 acts on B-cells, T-cells, hepatocytes, hematopoietic progenitor cells, and cells of the central nervous system (CNS). It is also required for the generation of TH17 cells.

Increased expression of IL-2R, D-Dimer, and IL-6 in serum was associated with increased severity of 2019-nCoV pneumonia and poorer patient prognosis (Russell, B., 2020) (Chen, L., 2020).

Interleukin-8 (IL-8)

IL-8 is a chemotactic factor that attracts neutrophils, basophils, and T-cells, but not monocytes. It is also involved in neutrophil activation. IL-8 is released from several cell types in response to an inflammatory stimulus.

Interleukin-10 (IL-10)

IL-10 inhibits the synthesis of a number of cytokines, including IFN-gamma, IL-2, IL-3, TNF, and GM-CSF. IL-10 is produced by activated macrophages and by helper T-cells.

Interleukin-17D (IL-17D)

IL-17D induces expression of IL-6, CXCL8/IL-8, and CSF2/GM-CSF from endothelial cells.

Interleukin-17A (IL-17A)

IL-17A is part of the ligand for IL-17RA and IL-17RC. The heterodimer formed by IL-17A and IL-17F is the ligand for the heterodimeric complex formed by IL-17RA and IL-17RC. It is involved in inducing stromal cells to produce proinflammatory and hematopoietic cytokines.

Interleukin-18 (IL-18)

IL-18 augments natural killer cell activity in spleen cells and stimulates interferon gamma production in T-helper type I (TH1) cells. IL-18 belongs to the IL-1 family.

Interferon Gamma Receptor 1 (IFNGR1)

IFNGR1 associates with IFNGR2 to form the receptor for interferon gamma (IFNG). Ligand binding stimulates activation of the JAK/STAT signaling pathway.

Tyrosine-Protein Kinase JAK2

JAK2 is a non-receptor tyrosine kinase involved in various processes such as cell growth, development, differentiation, and histone modification. The JAK2 pathway mediates essential signaling events in both innate and adaptive immunity. In the cytoplasm, JAK2 plays a pivotal role in signal transduction via its association with type I receptors, such as growth hormone (GHR), prolactin (PRLR), leptin (LEPR), erythropoietin (EPOR), and thrombopoietin (TPOR); or with type II receptors, including IFN-alpha, IFN-beta, IFN-gamma, and multiple interleukins. JAK2 mediates angiotensin-2-induced ARHGEF1 phosphorylation, which may lead to the hypertension observed in some COVID-19 patients (Guilluy C, 2010).

Tumor Necrosis Factor-alpha (TNFa)

The TNF family of receptors and cytokines is very large and this family is frequently targeted by drugs. TNFa is a pro-inflammatory cytokine involved in autoimmune and immune-mediated disorders such as rheumatoid arthritis, ankylosing spondylitis, inflammatory bowel disease, psoriasis, hidradenitis suppurativa, and refractory asthma. TNFa inhibitors act by suppressing the physiologic response to TNFa. Inhibition of TNFa can be achieved with a monoclonal antibody, such as infliximab (Remicade), adalimumab (Humira), certolizumab pegol (Cimzia) and golimumab (Simponi), or with a receptor fusion protein, etanercept (Enbrel). Low-dose prednisolone and tacrolimus may have beneficial impacts on COVID-19 (Russell B., 2020).

NACHT, LRR and PYD domains-containing protein 3(NLRP3)

As the sensor component of the NLRP3 inflammasome, NLRP3 plays a crucial role in innate immunity and inflammation. The SARS-CoV open reading frame 3a (ORF3a) accessory protein activates the NLRP3 inflammasome by promoting TNF receptor-associated factor 3 (TRAF3)-mediated ubiquitination of apoptosis-associated speck-like protein, containing a caspase recruitment domain (ASC). Since the ORF3a domain in SARS-CoV-2 is approximately 73% similar to SARS-CoV, it is likely to be involved in a similar mechanism (Siu, K.L., 2019).

C-C motif chemokine 2 (MCP1/CCL2)

C-C motif chemokine 2 (MCP1/CCL2) is a chemotactic factor that attracts monocytes and basophils but not neutrophils or eosinophils. CCL2 augments monocyte anti-tumor activity, and has been implicated in the pathogenesis of diseases characterized by monocytic infiltrates like psoriasis, rheumatoid arthritis, and atherosclerosis.

Granulocyte Colony-Stimulating Factor (MIP1A/GCSF)

Granulocyte/macrophage colony-stimulating factors are cytokines that act during hematopoiesis by controlling the production, differentiation, and function of granulocytes, monocytes, and macrophages. GCSF induces granulocytes and belongs to the IL-6 superfamily.

References

• Chan, J. F., Lau, S. K., To, K. K., Cheng, V. C., Woo, P. C., & Yuen, K. Y. (2015). Middle East respiratory syndrome coronavirus: another zoonotic betacoronavirus causing SARS-like disease. Clinical Microbiology Reviews, 28(2), 465–522.

• Chen, L., Liu, H. G., Liu, W., Liu, J., Liu, K., Shang, J., Deng, Y., & Wei, S. (2020). Zhonghua jie he he hu xi za zhi = Zhonghua jiehe he huxi zazhi = Chinese Journal of Tuberculosis and Respiratory Diseases, 43(0), E005. Advance online publication.

• Guilluy, C., Brégeon, J., Toumaniantz, G., Rolli-Derkinderen, M., Retailleau, K., Loufrani, L., Henrion, D., Scalbert, E., Bril, A., Torres, R. M., Offermanns, S., Pacaud, P., & Loirand, G. (2010). The Rho exchange factor Arhgef1 mediates the effects of angiotensin II on vascular tone and blood pressure. Nature Medicine, 16(2), 183–190

• Huang, C., Wang, Y., Li, X., Ren, L., Zhao, J., Hu, Y., Zhang, L., Fan, G., Xu, J., Gu, X., Cheng, Z., Yu, T., Xia, J., Wei, Y., Wu, W., Xie, X., Yin, W., Li, H., Liu, M., Xiao, Y., … Cao, B. (2020). Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet (London, England), 395(10223), 497–506.

• Russell, B., Moss, C., George, G., Santaolalla, A., Cope, A., Papa, S., & Van Hemelrijck, M. (2020). Associations between immune-suppressive and stimulating drugs and novel COVID-19-a systematic review of current evidence. Ecancermedicalscience, 14, 1022.

• Siu, K. L., Yuen, K. S., Castaño-Rodriguez, C., Ye, Z. W., Yeung, M. L., Fung, S. Y., Yuan, S., Chan, C. P., Yuen, K. Y., Enjuanes, L., & Jin, D. Y. (2019). Severe acute respiratory syndrome coronavirus ORF3a protein activates the NLRP3 inflammasome by promoting TRAF3-dependent ubiquitination of ASC. FASEB journal: Official Publication of the Federation of American Societies for Experimental Biology, 33(8), 8865–8877.