Neutralization Assay

  • Authors: Thomas Walton, Erica Suchman
  • Citation: Thomas Walton, Erica Suchman. 2009. Neutralization assay.
  • Publication Date : December 2009
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FIG. 1. Plaque assays used to quantify Venezuelan equine encephalomyelitis virus stocks and to perform viral neutralization assays.

Many viruses can be quantified using plaque assays. Viral stocks are serially diluted and then plated on a suitable cell culture system that will develop predictable cytopathic effect (CPE) upon infection. (Fig. 1). Quantification is achieved when the virus is diluted to the point where the number of foci of CPE (plaques) per well can be counted. Using the dilution, the number of plaque forming units/ml can be determined. The Spearman-Karber or Reed-Muench methods are often used to determine mathematically the cell culture infectious dose-50 or CCID50, the dilution that is used to achieve infection of 50% of cell culture wells. These formulae use data from the infection of multiple wells of serially diluted virus used to infect cell cultures. Each well is assayed for the presence of CPE, and the dilution at which 50% of wells show infection is determined.  

During an immune response to viral infection, the host often produces neutralizing antibody that binds to viral antigens and may inhibit the virus from initiating infection of host cells. There are many diagnostic tests that have been developed to assay host serum for the presence of antibodies, including enzyme-linked immunosorbent assay, complement fixation test, hemagglutination inhibition test, neutralization assays, and the agar gel immunodiffusion test (radial and double immunodiffusion) (1). 
Diagnosis of viral infection can be achieved using paired serums (acute and convalescent; 2 weeks apart) assayed for a four-fold increase in serum neutralizing antibody titers. This assay detects antibodies in the serum; if animals have had an immune response, antibodies will neutralize the ability of the virus to bind to cellular receptors and, therefore, produce plaques in a plaque assay (this titer should go up at least four-fold in the convalescent serum if the infection is current) (2). 

1.  Kuby, J. 1997. Immunology, 3rd ed. W. H. Freeman and Company, New York, N.Y. 

2.  Strauss, E., and J. Struass. 2002. Viruses and human disease. Academic Press, San Diego, Calif.

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