PRNT is an assay that test the ability of molecule to neutralize a virus. Serum or monoclonal antibody is mixed with a serial dilution of virus before being placed on a monolayer of cells. A semisolid agar is placed on top to restrict the spread of the virus from cell-to-cell and will create holes or “plaques” where the virus replicated, killing the host cells. After several days of incubation, the plaques are counted and the concentration of serum or antibody that reduced the number of plaques by 50% is denoted as the PRNT50 value. This is often seen as the gold standard for measuring antibody potency, but it is also labor intensive and takes several days to get results.

Like PRNT assays, neutralization assays test the ability of a compound to neutralize live virus. However, unlike PRNT assays they aren’t limited to viruses that cause plaques. These assays are typically performed in a 96-well or 384-well plates (microneutralization assay) and are generally less labor intensive that PRNT. To perform these assays, a monolayer of permissive cells are seeded in microwell plates overnight before infecting them with virus in the presence of absence of a test compound such as a small molecule or antibody. The wells treated with the compound are then compared to the wells without treatment, and well treated with a positive or negative control. The concentration of compound that reduced the virus by 50% is denoted IC50 or EC50.

A cell-based ELISA assay tests the ability of a compound to reduce the amount of viral antigen present in host cells. A monolayer of permissible cells is coated onto an ELISA plate and then virus is added to the monolayer in the presence or absence of the test article. The amount of viral antigen presence in compound treated and untreated cells is measured and to calculate the anti-viral potency of the test compound.

The recombinant Vesicular Stomatitis Virus (rVSV-ΔG) reverse genetics pseudotype system is widely used to generate rVSV particles that express the surface glycoprotein protein of a heterologous virus. This is a particularly powerful tool to study viruses that require high level biosafety containment (BSL-3 and BSL-4). At Eliteimmune, we have extensive experience generating rVSV-pseudotyped viruses for Arenaviruses, Filoviruses, and Coronaviruses which can be handled in BSL-2 containment.

This assay measures the ability of a therapeutic compound to inhibit the cytopathic effects (CPE) resulting from viral infection and can be used to measure a compound’s anti-viral potency. Virus is added to a monolayer of permissive cells in the presence or absence of an antiviral compound. After several days, the amount of CPE caused by the virus is compared to that of the cells treated with the antiviral compound. Reduction in CPE is visible under the microscope and can be quantified by luminescent signal.