Composition of Antigen-precipitin Precipitate.

Practically nothing is known about the composition of the antigen-precipitin precipitate beyond the fact that the amount of the precipitate produced may be much more than the amount of the antigen used. 1 Just how much of the antigen is present in the precipitate does not seem to have been determined. This is due to the fact that there exists no chemical method of determining the antigen (protein) and the precipitin (protein) separately.

One of us has worked out a method for the determination of minute amounts of hemoglobin. 2 The method is based on the benzidine reaction, and as little as 0.02 mg. hemoglobin suffices for a quantitative determination. When hemoglobin is used as the antigen its amount in the antigen-precipitin precipitate can be determined directly by means of that method. The amount of the precipitin in the precipitate can be determined by the difference between the total protein and the hemoglobin. The assumption is here made that the precipitate contains only the antigen and the precipitin and no other protein.

The word hemoglobin is used here in the generic sense, including oxyhemoglobin and its immediate derivatives. Oxyhemoglobin on standing is slowly converted into methemoglobin. This is, however, a matter of no consequence since oxyhemoglobin and methemoglobin have the same immunological properties 3 as well as the same chemical properties utilized for their quantitative determination in the above mentioned method.

Sheep oxyhemoglobin solution was prepared from washed cells by laking with 4 volumes of water and shaking with 2.5% suspension of freshly prepared aluminium hydroxide. The mixture was centrifuged and the clear supernatant liquid was filtered through a Seitz filter. Dog's oxyhemoglobin solution was prepared from crystals obtained by electrodialysis according to the method of Stadie. 4 Alumina cream was also used. The final solutions contained about 5 % hemoglobin.