The standard procedure has been an incubation period of 1 hour at room temperature followed by subcutaneous inoculation into newborn mice. However, this time and temperature are not critical, for the virus in neutralized within 10 minutes of mixing with immune serum and remains neutralized for long periods. During the variable incubation periods used, the control virus remained active, even in dilute suspensions.

The neutralization test is not affected by the presence or absence of complement.

Neutralizing antibody is stable at 65°C. for 30 minutes, and immune serum has to be heated to 80°C. for 30 minutes before the antibody is no longer detectable.

As the quantity of virus is increased, the quantity of serum required for neutralization likewise increases, but not in a regular or predictable fashion.

Neutralized mixtures of the virus can be made infective again by simple dilution before inoculation.

The neutralization test is a satisfactory means for typing Coxsackie viruses. At least seven antigenic types have been identified. Similar antigenic types have been found to be scattered over wide areas. Thus the Conn.-5 type was present in 1948 in Massachusetts, Connecticut, New York, and North Carolina. The Texas-1 type was present in 1943 in Connecticut and in 1948 in North Carolina and Texas.

Further information on the specificity of the neutralizing antibody response has been obtained from a study of the occurrence and development of antibodies in 6 patients who contracted infections with one or another of the C viruses while working with them in the laboratory. From each patient a virus was isolated during the illness. No patient had detectable antibodies to his strain before his illness, but each soon thereafter developed antibodies to his own strain and to the prototype strain to which it was related.

By means of the neutralization test, it has been shown that a family epidemic may include two different immunological types of virus.

Neutralizing antibodies appear at the time of or soon after onset of illness, increase rapidly to titers of about 1:1000 which are maintained during the period of 1 to 3 months following infection, and are still present 2 years later, although at lower levels.

Neutralizing antibodies are present in the normal population. In North Carolina, over 80 per cent of the children have antibodies at birth. The level falls rapidly to a minimum of 14 per cent at the age of 1, and then it quickly rises to reach the adult level at the age of 7. Gamma globulin collected in various parts of the United States between 1944 and 1949 and in Denmark in 1949 neutralizes at least four antigenically different Coxsackie viruses.