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 measure the effect of antibodies on virus infectivity by spreading the mixture on virus-sensitive cells. The cells are covered with a semi-solid medium to limit the transmission of the progeny virus. Every virus that causes a productive infection produces a local infection area (plaque). Plaques are counted and compared with the initial concentration of the virus to determine the percentage reduction in total viral infectivity. In PRNT, the serum sample to be tested usually undergoes a serial dilution before mixed with a standard amount of virus. The virus concentration remains unchanged so that when virus-sensitive cells are added and covered with semi-solid media, individual plaques can be identified and counted. In this way, the PRNT endpoint titer of each serum sample at any selected percent reduction of virus activity can be calculated. For example, the virus is first diluted to an appropriate concentration so that every 0.2 ml virus contains 80 ~ 100 PFU (plaque-forming units). Then, it is mixed with the same amount of serum with different dilutions, incubating at 37°C for 1 to 2 h. Finally, the PFU at different serum dilutions is calculated, and the serum dilution that reduces plaque by 50% is the neutralization titer of the serum.
1. Detect antibodies from the serum to be tested or detect viruses from the diseased materials to diagnose viral infectious diseases.
2. Use antitoxin serum to check the toxins in the pathological material or identify the type of bacteria toxins.
3. Determine antiviral serum or antitoxin titers.
4. Identify and type newly isolated viruses.
Creative Diagnostics provides antiviral testing and customized early detection solutions for other life science companies working in the field of antiviral and infectious disease diagnosis. We offer different opportunities to meet the needs of our clients, including a wide range of anti-virus testing options (e.g., plaque reduction assays), as well as opportunities to establish cooperative efforts.
(Other viruses or microorganisms may be available on request)
We combine infection and analytical expertise to provide our clients with the most powerful portfolio of antiviral and antimicrobial in vitro testing services. Facing an increasing demand for new antiviral and antimicrobial compounds for the treatment of infectious diseases, Creative Diagnostics can test these compounds in vitro to determine their potential efficacy in vivo models.