Abstract
Immunologic tolerance to simple hapten bound to nonimmunogenic carriers has gained wide acceptance to study the mechanism of tolerance (1–6). Of particular interest is autologous IgG as a tolerance-inducing carrier because it is a physiologic protein naturally tolerated by the host (5). Although this phenomenon has been observed in several strains of mice (7), it is not known whether this is a general property of autologous IgG. For this reason, we have examined the tolerogenic properties of autologous IgG in a different system, the anti-fluorescein response of rabbits (8). This particular system was chosen for two reasons. Firstly, the rabbit anti-fluorescein (anti-Fl) response to an immunogenic hapten carrier conjugate (Fl-KLH) (Fl-keyhole limpet haemocyanin) has already been extensively characterized in this laboratory (8). Secondly, if one can induced tolerance to a fluorescent hapten it will offer distinct advantages to follow the fate of the tolerogen in vivo.
Materials and methods. Preparation of hapten carrier conjugates. Keyhole limpet haemocyanin (KLH) and purified isologous rabbit gamma globulin (RGG) were obtained commercially from Schwartz-Mann. Autologous RGG was purified from the serum of individual rabbits by starch block electrophoresis. Fluorescein isothiocyanate (Sigma Chemical Co., St. Louis, Mo.) was conjugated in different molar ratios to KLH, RGG (homologous), and RGG (autologous) by previously described methods (8).
Immunizations. Randomly bred New Zealand white rabbits (Gloucester Rabbitry) were used throughout these studies. The rabbits were injected subcutaneously (in the rear footpads) with 3–5 mg of hapten carrier conjugate in complete Freund's adjuvant (CFA), or intravenously (in the marginal ear vein) with 30–50 mg of the hapten carrier conjugate in phosphate-buffered saline (PBS). For iv injections the molar ratio of hapten to carrier was 8–10:1 based on molecular weights of 900,000 for KLH and 150,000 for RGG.
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