Methods have been devised whereby the persistence of foreign antigens in mice can be detected. A highly diffusible, blue azo dye, echt-säure-blau was coupled to bovine γ-globulin and human serum albumin and injected into the animals. In this way deep blue tracer antigens were obtained. These were promptly stored in cells widely distributed throughout the body, especially in the reticulo-endothelial elements of the liver, spleen and mesenteric lymph nodes. The dye as such was not stored but rapidly excreted.
A blue coloration in the organs just mentioned was still perceptible after 85 to 120 days in the case of the azoglobulin and 36 to 44 days in that of the azoalbumin. To determine whether these substances had actually persisted, as well as to learn how long uncoupled globulin and albumin remained after injection, recourse was had to the phenomenon of reversed passive anaphylaxis, which was found to be characterized by extraordinary changes in the vessels of the ears (EVR) in the mouse, plainly visible under the microscope when called forth by an antiserum specific for the antigen to which the animal had been sensitized. So sensitive is the vascular response that as little as 0.5 to 0.1 µg. of protein as antigen, previously injected into the peritoneal cavity of a 30 gm. mouse, can be detected a few days later by an intravenous injection of antiserum.
By means of the EVR the globulin antigen has been detected in the blood and livers of injected mice for as long as 56 and 101 days, respectively; the albumin and azoalbumin for only 21 and 36 days. In the mesenteric lymph nodes of injected mice the albumin and azoalbumin antigens were found after 42 and 44 days, respectively.
The hepatic tissue and that of the mesenteric lymph nodes of mice injected with azoalbumin, containing in consequence stored blue material, when transferred to recipient mice yielded positive tests for antigen (EVR) as long as blue color could be perceived in these tissues with the unaided eye, or at low magnification. After the color had disappeared from the tissues the transfer tests were found to be negative. This fact speaks for the antigenicity of the colored material.
In summary it is plain that certain antigenic proteins, after introduction into the blood stream of mice, are stored in certain tissues and that they may persist there for weeks or even months, far longer than has generally been supposed. This persistence of antigen within the body, especially after detectable amounts of antigen have apparently disappeared from the blood, provides a reason for prolonged antibody formation, a phenomenon for which no adequate explanation has hereto been offered.