What is 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 that neutralize the capability of a virus to enter cells and 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 not pathogenic the viruses can be studied in a safer environment. In the case of SARS-CoV-2, the pseudovirus must 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 so important?

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

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. They also have the same ability to enter cells using the same mechanisms and receptors as the virus of interest. They are much safer to handle than the virus from which they originated from.

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 BSL-3 labs are much less common most research institutions will have BSL-2 laboratory.

Finally, as the method is based on luminescence it will be highly sensitive with a large dynamic range.

Why is using Berthold instruments for your Pseudovirus Neutralization Assays the right choice?

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

The Centro LB 960 Microplate Luminometer can be used to measure Luciferase activity. It is a versatile instrument for both Glow and Flash luminescence assays and can be equipped with up to 3 high-performance injectors. It is also characterized by high sensitivity thanks to the stringent selection process of photomultipliers working in photon counting mode. The Centro delivers very reliable results due to its crosstalk reduction design. 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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Other publications

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

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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