The reversibility of the antigen and amboceptor union is proved to be practically complete, so far as the immune hemolysin of the rabbit against sheep's blood is concerned.
The isolation of hemolytic amboceptor from its antigen union is accomplished by a simple method: When the hemolytic power of the original immune serum is 1:10,000, it is diluted to 100 times its volume with physiological salt solution; 5 cc. of this diluted serum is poured into 4 cc. blood cells, which are washed free from serum protein with physiological salt solution. After 15 to 20 minutes at room temperature all of the hemolytic amboceptor has been adsorbed by blood cells; and the antigen-amboceptor union is thus obtained. After the sensitized corpuscular sediment is washed with physiological salt solution several times, till the last trace of serum protein has been removed, this pure antigen-amboceptor combination is mixed with an isotonic or slightly hypertonic watery solution of saccharose, glucose or lactose and left at 55°C. for fifteen to thirty minutes, during which time the vessel is shaken several times. The sugar extract, which contains nearly all of the hemolysin used to sensitize the cells, is obtained by centrifugation.
In order to purify this sugar extract, which contains substances from destroyed blood cells, it is placed in a separatory funnel and shaken for one to two hours with 5 to 10 volumes of ether, this treatment, if necessary, being repeated twice or thrice, till at last the solution becomes quite colorless. This colorless solution is dialysed in parchment against running water in order to eliminate the sugar and traces of ClNa.
The solution thus obtained is concentrated in vacuo to the required volume.