Plaque Reduction Assay Normal cells can take up reactive dyes during the metabolic process, but when they are infected with viruses, the cells will lose their ability to take up the dye and thus form colorless plaques. After the virus is neutralized by specific antibodies and then infects the cells, the number of cell plaques formed decreases accordingly. Therefore, the antibody titer for neutralizing the virus can be calculated according to the number of cell plaques. The plaque reduction assay measures the plaque-forming efficiency of a virus in the presence of different concentrations of a test article. The time required for the plaque to become visible depends on the kinetics of virus replication, which can vary from 24 hours to several weeks. Since plaques need to be at least 1 mm wide for accurate scoring (especially with the naked eye), this analysis is usually performed on 24 or 6-wells plates, as any smaller holes can affect readability and resolution. The plaque reduction neutralization test (PRNT) is a variant of plaque reduction assay and is considered the gold standard for detecting neutralizing antibodies to certain viruses (i.e., dengue viruses).
The virus PRNT assay was first described in the 1950s and later applied to the dengue virus (DENV). The basic design of PRNT is to generate virus-antibody interactions in test tubes or microtiter plates and then