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A method of titrating influenza virus-neutralizing antibodies is described. This method is based on the technique for cultivating virus in fragments of chick chorio-allantoic membrane using a special plastic tray with cups for 100 replicates. The neutralization test cannot be made directly by adding the antiserum to the cups in which the virus is growing in the membrane fragments because there is a significant non-specific inhibitory effect. Therefore, use is made of the observation that virus added to the cups is rapidly fixed by the membrane so that, after a short time, the membrane may be washed and transferred to a fresh cup which will later be found to contain the new virus released from the membrane.
In the neutralization test the membrane fragments are exposed to virus-serum mixtures for 18 hr. and then transferred to other cups without serum to determine which pieces have become infected. The titre of neutralizing antibodies is defined as the serum dilution which will protect from infection with 100 ID 50 of virus, half of a large number of replicate pieces of membrane.
The neutralization technique has been applied to the antigenic comparison of six strains of influenza virus. The results support the relationships disclosed between the six strains by a complement-fixation technique. It is suggested, however, that the neutralization technique, because of its greater specificity, and because of its dependence on the infectivity of the strain, is not as satisfactory as the complement-fixation test for strain comparisons. Apart from its use for titrating neutralizing antibodies, the technique may also be of value for proving the virtual identity of two virus strains and in the selection of strains for a vaccine.
It is a pleasure to acknowledge the help given by Dr Isaacs of the WHO Influenza Centre. Not only did he provide many of the virus strains and antisera, but he also undertook the purification of the WS strain.