VIROLOGICAL METHODS 279 



able end points. Increments between these two extremes have not been 

 popular, but there is no reason why they could not be used. 



If it is anticipated that either antibody or virus is to be used in high 

 dilution, a protective diluent should be used. This precaution is dis- 

 cussed under "Cultivation in Chick Embryos," infectivity titers. 



Incubation time. All viruses do not react with their antibodies at 

 the same speed. Whereas 15 min at room temperature may be an accept- 

 able time for the influenza virus and its antibody, vaccinia virus-antibody 

 mixtures require a longer period of time. As the exposure time is 

 extended, temperature should probably be reduced, for some viruses 

 are perishable rather quickly at higher temperature. The time-tem- 

 perature variable undoubtedly plays some role in altering the preciseness 

 of the test. Considerably more inactivation may occur with the virus 

 than with the antibody. Inactive virus may bind antibody, yet only 

 active virus produces the end point. Preliminary determination of the 

 lability of the virus and the use of adequate controls usually reduce these 

 variables to an insignificant level. To report an exposure time of virus 

 and antibody as 15 min is inaccurate if all dilutions are mixed at once and 

 it takes 1 hr to inoculate the experimental animals. If antibody and 

 virus dilutions, or vice versa, are mixed together at intervals parallel- 

 ing inoculation time, accuracy is increased. Longer exposure time, e.g., 

 overnight at 4°C, probably requires less or no adjustment of this poten- 

 tial error. 



Test system. Susceptible animals, embryos, or tissue cultures are 

 used depending on their suitability for achieving the objective in mind. 

 Certain precautions should be kept in mind when the outcome of the 

 neutralization test is being determined. The end point should be clearly 

 designated. For example, at least four end points could be used in a 

 mouse neutralization test where a mouse-adapted strain of influenza 

 virus is used: death, signs of illness, production of lung lesions, and no 

 infection on subsequent passage of lung tissue. Considerable disparity 

 would occur among these end points. If the production of antigen is 

 used as an indication of propagation, the antigen introduced with the 

 inoculum must be taken into consideration. Neutralized virus may not 

 grow or agglutinate red cells, but it will function in the complement- 

 fixation test. A sufficient amount of neutrahzed virus could make the 

 tissue into which it had been introduced appear anticomplementary. 

 Sufficient antibody to prevent viral growth need not prevent it from 

 adhering to susceptible cells (Hutlin and McKee, 1951). 



Generally speaking one should allow more incubation time on a 

 neutralization test than for determining infectivity. Antibody insuffi- 

 cient in quantity to neutralize a virus frequently will slow down its 

 progress. For example, viruses reaching their propagation peak in 



