A Simple Method for the Serum Neutralization With Japanese Encephalitis (JE) Virus Utilizing Plaque Inhibition by Agar-diffused Antibody

Abstract

The cup method commonly used in antibiotic assay was applied to the serum neutralization with JE virus. A cup was fixed in the overlay covering chick embryo cells and filled with a test serum, and diffusion of antibody was allowed to take place at 20 C before addition of virus onto the agar surface. When the overlay contained 0.6% agar, 0.1 to 0.27% of the virus penetrated the overlay and formed plaques leaving a plaque inhibition zone around the cup. A clear inhibition zone was produced when the interval between the addition of serum and virus inoculation was 2 days, the amount of virus given per dish being 5×105 PFU, and the dishes were subsequently incubated at 20 C for one day and then at 35 C for 3 days before staining with neutral red. The plaque inhibition zone diameter was in linear relation to the logarithm of the antibody concentration used. The sensitivity of the test was such that positive results were obtained with sera possessing endpoint titers higher than 1:10 as determined by the ordinary 50% plaque reduction test. When sera of 83 children with different vaccination histories were tested by the present cup method and by the 50% plaque reduction method in parallel, a good correlation was observed between the two titers.