Abstract
For the production of highly specific complement fixing antigen of neurotropic viruses the extraction of non-specific substance by lipid solvents has often been used. These extraction procedures were investigated for their effect upon the hemagglutinin (for chick erythrocyte) of Japanese B encephalitis virus. Extraction of mouse brain was performed by 11 different lipid solvents: petroleum ether, ligroine, ethylether, amylalcohol, toluene, pyrigine, acetone, benzene, chloroform, carbon disulfide, and carbon tetrachloride. When extraction were carried out at low temperature (not above 10°C) either with or without previous lyophilization of the virus suspension, the difference between the specific Japanese B encephalitis virus hemagglutinin and the non-specific hen red cell hemagglutinin were as previously reported.
Specifically, on successive extraction by benzene, chloroform, acetone, and ethylether after lyophilization the non-specific hen red cell hemagglutinin was almost completely removed while the specific Japanese B encephalitis virus hemagglutinin and the specific complement fixing antigen were decreased only slightly inpotency. In this extracted samples with a relatively high specific hemagglutinin titer but without significant amounts of non-specific hemagglutinin (for hen red cell), inactivation of the specific hemagglutinin was attempted by treatment with carbolic acid, formalin, and trypsin. In addition by chick erythrocytes was tried. While specific Japanese B encephalitis virus hemagglutinin was inactivated by the above mentioned reagents and adsorbed by chick red cells, the complement fixing antigen potency was undiminished. Extraction by toluene resulted in a similar decrease in specific hemagglutinin titer without effecting complement fixing titer. Direct extraction of virus suspension by chloroform had same effect as the successive extraction described above. Ultracentrifugation of this sample at 23, 000r.p.m. (average g: 33, 000) resulted in almost complete sedimentation of the specific hemagglutinin, while the complement fixing antigen remained almost undiminished in the supernatant fluid.
These experiments confirm the difference between the specific hemagglutinin of Tapanese B encephalitis virus and the non-specific hen erythrocyte hemagglutinin, and present evidence that the specific hemagglutinin, and the complement fixing antigen are seperate entities.
