1. When immune sera of rabbits, injected with human or chicken erythrocytes, are heated at 75-90°C for 30 minutes, there occurs a peculiar power in the heated sera; that is, the heated serum destroys the erythrocytes at low temperatures, while it is innocuous to them at 37°C. For this peculiar sort of cold hemolysis, the aid of the hemolytic complement is not necessary.
2. The appearance of the cold hemolytic power in the heated immune sera is conditioned by the degree of the heating temperature, i.e., in the case of rabbits' immune sera, it is entirely negative in the immune sera heated at 60° and 65°C, and only a trace of hemolysis occurs in the serum heated at 70°C, the strongest cold hemolysis takes place in the serum heated at 75°C and 80°C, and further degrees of heating temperature (85-90°C) weaken the property of cold hemolysis of heated sera.
3. The temperature at which the hemolytic agent in the heated serum reacts with erythrocytes is the most characteristic. The lower the temperature, the stronger the hemolysis which takes place in the heated immune sera. The most part of th heated immune sera destroys the added erythrocytes only in the ice box. And the hemolysis is very weak and rare already at room temperature.
4. The production of the cold-hemotoxin does not go parallel with that of other immune bodies, such as agglutinin, hemolytin, etc.. The strength of the cold-hemotoxin is the greatest after the second or the third injevtion of antigen erythrocytes, and decreases, thereafter.
It disappears or comes to normal level after the fifth injection at the latest, though the titer of hemoagglutinin and hemolysin is still going up at this stage of immunization.
5. The cold-hemotoxin in the immune serum of rabbits, injected with chicken erythrocytes, shows a specificity as to its action; that is, it reacts only against chicken erythrocytes.
The cold-hemotoxin in the serum of rabbits injected with human erythrocytes, however, does not show such a strict specificity, and hemolyses not only the human blood cells but also the chicken erythrocytes in a fairly strong degree, and those of cattles and guinea pigs in a slight degree.
6. When the serum protein is separated into fractions by means of CO₂-method, the cold-hemotoxin comes in the albumin fraction.
I wish to express my indebtness to Prof. K. Yamakami for the guidance and encouragement, which he has extended to me in carrying out these experiments.