電気泳動法,定量的沈降法による家兎抗体の研究

多種反応系,抗原抗体稀釈法,分離抗体について 免疫化学研究第3報

抄録

1. The quantitative precipitin curves of horse serum and its rabbit antibody showed two maximum precipitation zones (so-called double zone), and we were able to analyze them into the quantitative precipitin curves of its several components, namely, albumin, α, β and γ-globulin. α-globulin and γ-globulin showed higher antigenicity than albumin against the antihorseserum rabbit antibody.
Albumin, α, β and γ-globulin had different equivalence zones of their own and albumin and γ-globulin had no crossreactivity between them.
2. A quantitative precipitin curve was traced by the reaction of synthetic mixed antigen (egg albumin plus bovine serum γ-globulin), and we were able to analyze the curve into curves which could be attributed to the reaction of its components, egg albumin and γ-globulin against their respective antibodies.
It was proved that the precipitable antibody N produced by the reaction of the compound antigen against its antibody was equal to the sum total of each component of compound antigen, when its components have no crossreactivity among them. Native compound protein, bovine lens protein and its components α, β Crystalline was studied, and approximately identical results was obtained, as α and β crystalline had no crossreactivity between them.
The quantitative precipitin curves of the compound antigen and antibody system indicated a wide zone (or double zone).
From the double zone which appears with the initial reaction of synthetic mixed antigen against its antibody, it was inferred that the antibody produced by an injection of egg albumin and bovine serum γ-globulin contained egg albumin and γ-globulin antibody, as well as a very small amount of antibody reactive both to egg albumin and to γ-globulin.
3. The solubility of specific precipitate was measured by the quantitative precipitin method and it was recognized that the precipitate diminished in proportion as the solvent increases.
The precipitate quantity obtained by antigen antibody dilution method could be expressed by the following equation;
Z=(2R+1)x-R²x²/Ay
where
Z; total N precipitated
x; antigen concentration
y; antibody concentration
From this equation, it was proved that the isoprecipitate line drew approximate hyperbola and the quantitative prectpitin curve of constant antibody traced parabola, while the quantitative precipitin curve of constant antigen did hyperbola. The antibody titer in a given precipitate was proportional to the antibody N in original serum, where its solubility was at its lowest level.
The antigen titer in a geven precipitate was approximately proportional to the antibody/antigen ratio of equivalence zone in original serum.
During immunization process, the antigen titer in a given precipitate increased as the determinant group and antibody/antigen ratio increased, and the equivalence zone shifted from antibody excess side to antigen excess side.
4. The electrophoretical pattern of the specific precipitate in alkali dissociation (Urea-NaOH buffer, pH 12.3.) showed two separate antigen and antibody peaks. The peaks shown in the electrophoretical pattern of the soluble component by the specific precipitate in antigen excess zone was observed to appear in terms of mobility in the middle between the peak of antigen and that of antibody.
5. Antibody in 80% purity was isolated by peptic digestion of the specific precipitate and franctionation with 15% ethanol.