Summary Attempts were made to devise a simple test model for studying the termination of acquired immunologic tolerance in mice. Animals rendered tolerant to HGG after birth were challenged with one of several azo-HGG preparations in corporated into incomplete Freund’s adjuvant. As judged by the trace-labeled antigen elimination method, a single challenge injection had only a marginal, if any, effect on tolerance, except in experiments where mice were injected with p-A, p-S-HGG or with o-S-HGG. Two challenge injections with azo-HGG (4 to 5 weeks apart) invariably caused a significant increase in the number of partially tolerant and non-tolerant mice. In the altered proteins, the number of azo-groups per molecule of HGG ranged from 2.6 to 13.6 and all the azo-HGG preparations used cross-reacted to a high degree (80 to 100%) with native HGG. Mice injected with either HGG or azo-HGG at a time (i.e., at the age of 13 weeks) when tolerance was disappearing spontaneously, formed precipitating antibodies to both HGG and azo-HGG without further stimulus 10 weeks later. In animals injected with either native HGG or modified HGG at the age of 23 weeks, precipitating antibodies to both antigens were found shortly after challenge. Similarly, PCA was positive with HGG, azo-HGG, and also, in half of the sera tested, with the hapten linked to a non-cross-reacting carrier protein. However, if still fully tolerant mice were challenged with HGG at the age of 10 weeks and rechallenged 5 weeks later, no precipitating antibodies to HGG or azo-HGG were detected 20 weeks after the second challenge. In the group injected with azo-HGG, precipitating antibodies only to azo-HGG, but not to native HGG, were found. Correspondingly, PCA reactions revealed that mice challenged early with HGG had no skin-fixing antibodies to HGG or azo-HGG, but sera from mice challenged with azo-HGG gave, with a few exceptions, only positive PCA reactions with azo-HGG.

Dietrich, F. M. (1966). The Immune Response to Azo-Human γ-Globulin Conjugates in Mice Tolerant to Human γ-Globulin. The Journal of Immunology, 97(2), 216–223.