Plaque reduction neutralization test

Plaque reduction neutralization test
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Purpose	To measure the neutralizing antibody response against viruses
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The Plaque Reduction Neutralization Test (PRNT) is a laboratory assay used to measure the ability of antibodies to neutralize viruses. It is considered the gold standard for quantifying the neutralizing antibody response to viral infections and is widely used in virology and immunology research.

Principle

The PRNT is based on the principle that antibodies present in a sample can inhibit the infectivity of a virus, thereby reducing the number of plaques formed in a cell culture. A plaque is a clear area on a layer of host cells where the virus has destroyed the cells.

Procedure

The procedure for a PRNT involves several key steps:

Preparation of Virus and Cells

1. A known quantity of virus is prepared and titrated to determine the concentration that produces a countable number of plaques. 2. A monolayer of susceptible host cells, such as Vero cells or BHK-21 cells, is grown in a multi-well plate.

Serum Sample Preparation

3. Serum samples from the subject are collected and heat-inactivated to destroy complement proteins that could interfere with the test. 4. The serum is then serially diluted, typically in a two-fold series.

Virus Neutralization

5. Each dilution of the serum is mixed with a fixed amount of virus and incubated to allow neutralization to occur.

Infection of Cell Monolayer

6. The virus-serum mixtures are added to the cell monolayers and incubated to allow infection. 7. After a period of incubation, the cells are overlaid with a medium containing agar or carboxymethylcellulose to restrict the spread of the virus.

Staining and Counting

8. After further incubation, the cells are fixed and stained with a dye such as crystal violet to visualize the plaques. 9. The number of plaques is counted, and the reduction in plaque number compared to a control without serum is calculated.

Interpretation

The results of a PRNT are expressed as a titer, which is the highest dilution of serum that results in a specified percentage reduction in plaque number, typically 50% (PRNT50) or 90% (PRNT90). A higher titer indicates a stronger neutralizing antibody response.

Applications

The PRNT is used in various applications, including:

• Evaluating the efficacy of vaccines by measuring the neutralizing antibody response.
• Diagnosing viral infections by detecting the presence of neutralizing antibodies in patient sera.
• Studying the immune response to viral infections in epidemiological studies.

Advantages and Limitations

Advantages

• High specificity: The PRNT specifically measures neutralizing antibodies, which are important for protective immunity.
• Quantitative: Provides a quantitative measure of antibody levels.

Limitations

• Labor-intensive: The test is time-consuming and requires skilled personnel.
• Requires live virus: Handling live virus necessitates appropriate biosafety measures.
• Variability: Results can vary depending on the virus strain and cell line used.

See Also

• Neutralizing antibody
• Viral plaque assay
• Vaccine efficacy

External Links

• [CDC Guidelines on PRNT](https://www.cdc.gov)
• [WHO Manual on PRNT](https://www.who.int)