What is a Pseudovirus Neutralization Assay?

A Pseudovirus Neutralization Assay (also known as Pseudotyped Virus Neutralization Assay or Pseudotype-Based Neutralization Assay) is a laboratory method used to study the effect of antibodies or drugs to neutralize the capability of viruses to enter cells and thus prevent infection.

Pseudotypes or pseudotype particles are chimeric “viruses” consisting of a virus core (typically a lentiviral vector) surrounded by a lipid envelope with the surface glycoproteins of another virus (the virus of interest). By using a vector which can’t replicate itself and is hence not pathogenic, viruses can be studied in a safer system. In the case of SARS-CoV-2, the pseudovirus has to express the S glycoprotein, which mediates entry into the host cells by binding to human angiotensin-converting enzyme 2 (ACE2). In addition to the surface glycoproteins of the virus of interest, the pseudotyped virus contains the gene of a luciferase, which is only expressed after entering the cell. The more pseudoviruses enter the cells, the more luciferase is expressed, and the higher the intensity of emitted light.

The Pseudovirus Neutralization Assay involves incubating the cells and the pseudovirus in the presence of different concentrations of an antibody of interest and measuring light emission using a plate luminometer or a multimode reader. If the antibody is effective in neutralizing the surface glycoproteins and blocking the entry to the cells, a significant reduction in light emission will be measured. Antibody effect is displayed using one of the inhibitory concentrations (IC50, IC80, or others).

Why are Pseudovirus Neutralization Assays important?

Neutralization assays are very valuable tools to study human and animal antibody responses against viruses elicited by vaccination and other therapies or natural exposure. These assays routinely require using wild-type viruses, and this limits their application when the subject of study is a highly pathogenic human virus such as SARS-CoV-2.

However, replication-deficient viruses used in pseudotyped-virus neutralization assays can be a safe alternative. Pseudoviruses have a conformational structure of the surface proteins which closely resembles that of the native virus of interest and have the same ability to enter cells using the same mechanisms and receptors as the virus of interest, but they are much safer to handle than the virus from which they originated.

The unique properties of pseudoviruses allow for them to be safely handled in biosafety level (BSL) 2 laboratories, which typically work with agents that pose a moderate health hazard to humans. This is a great advantage, as highly pathogenic viruses such as SARS-CoV-2 require BSL-3 laboratories: while most research institutions have BSL-2 laboratories, BSL-3 labs are much less common.

And last but not least: as the method is based on luminescence, it has a high sensitivity and large dynamic range.

Why using Berthold instruments for your Pseudovirus Neutralization Assays?

Berthold Technologies is your reliable and competent partner when you want to perform pseudotyped virus neutralization assays. We are proud to count ourselves among the pioneers of luminescence measurement and have engineered and developed more than 20 different types of luminometers (from tube to microplate) in the past 4 decades. Renowned for precision, sensitivity and reliability Berthold Technologies is still the market leader for luminometers today.

The Centro Microplate Luminometer can be used to measure Luciferase activity. It is a versatile instrument for both Glow and Flash luminescence assays that can be equipped with up to 3 high-performance injectors. It is also characterized by high sensitivity thanks to a stringent selection of photomultipliers working in photon counting mode. Due to its crosstalk reduction design, the Centro delivers very reliable results. An example of its performance is shown in the following articles.

A human monoclonal antibody blocking SARS-CoV-2 infection

Naturally mutated spike proteins of SARS-CoV-2 variants show differential levels of cell entry 

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The Tristar 5 is a modular high-performance microplate reader equipped with independent, user-selectable filters and monochromators on both, the excitation and emission side. It includes advanced cell-friendly injector technology and One-4-All Optics for uncompromised performance of all detection modes. 

In the article below, Gaussia was used as reporter gene instead of firefly. The Tristar 5 performed the measurement of Gaussia luciferase expression.

Potential host range of multiple SARS-like coronaviruses and an improved ACE2-Fc variant that is potent against both SARS-CoV-2 and SARS-CoV-1

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Other publications

In the following articles, measurements were performed with earlier instrument versions of the Tristar system family but can be transferred 1:1 to the new Tristar systems.

Author and yearTitleInstrument
Prévost et al. (2020)

Cross-sectional evaluation of humoral responses against SARS-CoV-2 Spike

TriStar LB 941 Multimode Microplate Reader
Beaudoin-Bussières et al. (2020)Decline of humoral responses against SARS-CoV-2 Spike in convalescent individualsTriStar LB 941 Multimode Microplate Reader

 

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