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Neutralization Assays

The outbreak of COVID-19 caused by SARS-CoV-2 has widely spread worldwide as continuous global public threats. To better control the pandemics, anti-SARS-CoV-2 drugs and vaccines are urgently to be developed. SARS-CoV-2 has four main structural proteins, namely S, M, N, E. S protein is responsible for binding to cellular receptors and prompting membrane fusion for virus entry to target cells. ACE2 is reported as the receptor of SARS-CoV-2, while the host cell serine protease TMPRSS2 primes the S protein of SARS-CoV-2 for entry. Because of the key functions, S protein is the main target for antibody-mediated neutralization and the focus of vaccine development.

Neutralization assays determine the ability of an antibody to inhibit virus infection of cultured cells and the resulting cytopathic effects of viral replication, which are important for drug discovery and vaccine development. Virus neutralization is a specialized type of immunoassay since it detects antibody that can block virus replication. Neutralizing antibodies generally recognize proteins or glycoproteins on the virus surface. There are several mechanisms for antibody neutralization including blocking attachment to the host cell, inhibiting penetration of the host cell membrane, or interfering with uncoating of the virus within the cell.

Neutralization AssaysFig. 1 Workflow of a pseudotype neutralization assay

Generally, replication-competent and infectious viruses are used in the traditional neutralization assays, which have the safety concerns in evaluating virus neutralizing activities. In comparison of these viruses, replication-defective pseudoviruses are widely used to mimic SARS-CoV-2 due to the safety features. Pseudoviruses with S glycoprotein as the envelope protein are developed based on the lentiviral, AAV and VSV systems. Neutralizing antibodies prevent the pseudovirus from entering host cells. The activity of neutralizing antibodies can be screened through luminescence of target cells.

Neutralization AssaysFig.2 In antibody-mediated viral neutralization, neutralizing antibodies binding to the receptor-binding domain of the viral spike protein, prevent virus from docking onto its entry receptor ACE2.

Advantages

  • High specificity
  • High sensitivity
  • Fast turnaround time
  • Multiple pseudoviruses based on lentiviral, AAV and VSV systems

Together with global researchers and scientists, Creative Biogene provides Pseudovirus neutralization assay services including generation of pseudovirus infected cells, immunofluorescence assay, flow cytometry-based receptor-binding inhibition assay, ELISA-based binding affinity assay and assessment of NAb Titers. In addition, related pseudoviruses and cell lines are also available for study of SARS-CoV-2.

Neutralization AssaysFig.3 Virus infection validation. SARS-CoV-2 S glycoprotein pseudotyped GFP lentivirus was added into HEK293T-ACE2 cells, capture cell image after 16 h.

References:

  1. Lei, C., Qian, K., Li, T., Zhang, S., Fu, W., Ding, M., & Hu, S. (2020). Neutralization of SARS-CoV-2 spike pseudotyped virus by recombinant ACE2-Ig. Nature Communications, 11(1).
  2. Iwasaki, A., & Yang, Y. (2020). The potential danger of suboptimal antibody responses in COVID-19. Nature Reviews Immunology.
  3. Tse, L.V., Meganck, R.M., Graham, R.L., Baric, R.S. (2020). The Current and Future State of Vaccines, Antivirals and Gene Therapies Against Emerging Coronaviruses. Front Microbiol, 11:658.
  4. Carnell, G. W., Ferrara, F., Grehan, K., Thompson, C. P., & Temperton, N. J. (2015). Pseudotype-Based Neutralization Assays for Influenza: A Systematic Analysis. Frontiers in Immunology, 6.
For research use only. Not intended for any clinical use.

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