Plaque Reduction Neutralization Test (PRNT) Protocol

Plaque Reduction Neutralization Test (PRNT) is a serological test used to measure the presence and level of neutralizing antibodies against specific viruses. This test is commonly used to determine the immune response to viral infections and vaccine efficacy.

PRINCIPLE

• The PRNT assay measures the ability of neutralizing antibodies in a serum sample to prevent virus replication in cell culture.
• The serum sample is diluted and mixed with a known quantity of the virus.
• The mixture is then added to a monolayer of susceptible cells, allowing virus infection and replication.
• After a suitable incubation period, a semi-solid overlay is added to the cells, allowing plaques (visible areas of cell destruction) to form.
• Plaques are counted and compared with control samples to determine the neutralizing antibody titer.

Fig. 1 PRNT detection of neutralizing antibodies against SARS-CoV-2. (Shi, 2021)

Material

Procedure

1. Sample collection: Collect blood samples and separate the serum by centrifugation. Serum samples are used for the PRNT as they contain antibodies against the virus.
2. Virus preparation: Cultivate the virus of interest in appropriate cell culture systems until a sufficient virus titer is achieved. Then, prepare a working virus stock by diluting the virus in a cell culture medium.
3. Serum dilution: Prepare a series of serial dilutions of the serum in a 96-well microtiter plate. Typically, a two-fold dilution series is created starting from an initial dilution of 1:10. Prepare duplicate wells for each dilution.
4. Virus-serum incubation: Add an equal volume of the working virus stock to the diluted serum in each well of the microtiter plate. Mix gently and incubate the plate at a suitable temperature for a specific time period. This allows the neutralizing antibodies in the serum to interact with the virus.
5. Cell seeding: Prepare a cell monolayer of permissive cells for the virus in a separate cell culture plate. The specific cell line used depends on the virus being tested. Ensure the cells have reached an appropriate confluence before the viral infection step.
6. Virus inoculation: Remove the virus-serum mixture from the incubation step and transfer it onto the cell monolayer in the cell culture plate. Incubate the plate to allow the virus to infect the cells.
7. Overlay and incubation: Cover the infected cells with a gel-like substance (e.g., agarose overlay) containing nutrients and an indicator dye. This creates an environment to facilitate plaque formation. Incubate the plate under suitable conditions for virus replication and plaque formation.
8. Plaque counting: After an appropriate incubation time, carefully remove the overlay and stain the cell monolayer. Count the number of visible plaques formed in each well under a microscope.
9. Data analysis: Calculate the plaque reduction percentage for each serum dilution in comparison to control without serum. Generate a neutralizing antibody titer based on the highest serum dilution that yields a significant reduction in plaque formation.

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REFERENCE

1. Shi, A.C.; Ren, P. SARS-CoV-2 serology testing: progress and challenges. J Immunol Methods. 2021, 494: 113060.