Application of the two-dimensional technique to the 'crossing' paper electrophoresis of immunological reactions

The principle and procedure of two-dimensional crossing paper electrophoresis are described. In applying the technique to the antigen-antibody reaction, electrophoresis of the antiserum alone was carried out in one direction, then the antigen was applied on a line to encounter each fraction of the antiserum and electrophoresis was started in a direction at right angles to the first one. Peaks appeared on the line of antigen owing to the formation of complexes in the regions of antibody proteins contained in the antiserum, thus forming a “crossing diagram”. An anomaly was observed in several cases of the “crossing diagrams” of egg albumin-anti-egg albumin reaction; it was ascribed to mechanical disturbances of the paper surface. A “crossing diagram” of a diphtheria toxoid obtained by initial electrophoresis of the antiserum showed two peaks in the region of T-globulin and one in the region of γ-globulin of the antiserum. Reversing the order of electrophoresis (first toxoid, then antiserum) the “crossing diagram” showed two flat asymmetrical peaks in the γ-globulin zone. In general, the toxoid used must contain more than four antigens, of which two have their antibodies in T-globulin and the other two in γ-globulin. It is suggested that the antibodies have different equivalence zones for the same toxoid.