The plaque assay is a terrific method for determining virus titers, but it doesn’t work for all viruses. Fortunately there are several alternative methods available, including the end-point dilution assay.
The end-point dilution assay was used to measure virus titer before the development of the plaque assay, and is still used for viruses that do not form plaques. Serial dilutions of a virus stock are prepared and inoculated onto replicate cell cultures, often in multi-well formats (e.g. 96 well plastic plates). The number of cell cultures that are infected is then determined for each virus dilution, usually by looking for cytopathic effect.
In this example of an end-point dilution assay, 10 monolayer cell cultures were infected with each virus dilution. After an incubation period, plates that displayed cytopathic effects were scored with a +. At high dilutions, none of the cell cultures are infected because no particles are present. At low dilutions, every cell culture is infected. Half of the cell cultures showed cytopathic effects at the 10-5 dilution. This is the end point: the dilution of virus at which 50% of the cell cultures are infected. This number can be calculated from the data and expressed as 50% infectious dose (ID50) per milliliter. The virus stock in this example contains 105 ID50 per ml.
In real life, the 50% end point does not usually fall exactly on a dilution as shown in the example. Therefore statistical procedures are used to calculate the end point of the titration.
End-point dilution methods can also be used to determine the virulence of a virus in animals. The same approach is used: serial dilutions of viruses are made and inoculated into multiple test animals. Infection of the animal can be determined by death or clinical symptoms such as fever, weight loss, or paralysis. The results are expressed as 50% lethal dose (LD50) per ml or 50% paralytic dose (PD50) per ml when lethality or paralysis are used as end points.
The following example illustrates the use of end point dilution to measure the lethality of poliovirus in mice. Eight mice were inoculated per virus dilution, and the end point was death. The statistical method of Reed and Muench was used to determine the 50% end point. In this method, the results are pooled, and the mortality at each dilution is calculated. The 50% end point, which falls between the fifth and sixth dilutions, is calculated to be 10-6.5. Therefore the virus sample contains 106.5 LD50 units.
Reed, L.J., & Muench, H. (1938). A simple method of estimating fifty percent endpoints. Am. J. Hygiene, 27, 493-497
