Neutralization tests for dengue and Japanese encephalitis viruses by the focus reduction method using peroxidase-anti-peroxidase staining.

Neutralization tests were made on 4 types of dengue (DEN) virus and Japanese encephalitis (JE) virus by incubation of serially diluted antisera and constant amounts of the viruses and then focus assay of surviving virus infectivity with peroxidase-anti-peroxidase (PAP) staining. Neutralization reactions were virtually completed in 2 hr on incubation of serum-virus mixtures at 28 C. A straight regression line was obtained on a probit chart by plotting the focus reduction rates at various dilutions of a given serum against the logarithm of the serum dilution used in the test. The slopes of the probit regression lines for the neutralization for DEN types 1 and 3 were similar, but differed somewhat from those for DEN type 2 and type 4. The slope of the line for JE virus was quite different from those for DEN viruses. Using these relations, the fifty percent focus reduction titer (FR50) of neutralizing antibodies of a given serum could be estimated from the focus reduction rates at several dilutions of the test serum when the latter was between 25-75% of the value of the control.