Induction of delayed hypersensitivity to protein antigens in mice without Freund adjuvants

Three ways for inducing tuberculin-type delayed hypersensitivity (DH) in mice to purified protein antigens without Freund adjuvant are described. Four strains of mice compared for susceptibility to sensitization with several antigens ranked SJL > CF-1 > CAF₁ = C57B1. Females were more easily sensitized than males. The first way of sensitizing without Freund adjuvant was simply to inject antigen intradermally in saline, but it could be used only with the potent antigen methylated human serum albumin (HSA). Six-mercaptopurine injections during the first 5 days of sensitization enhanced such sensitization in CF-1 but inhibited it in CAF₁ mice. The second way sensitized with a weaker antigen, chicken conalbumin (CA), and thus is more versatile. It consisted of injecting a saline solution of antigen into a 24-hr strong DH dermal reaction to an unrelated antigen (dextran). The third way was simplest and best: Freund adjuvant was replaced with aqueous Evans blue dye. Thus, distilled water solutions of antigen (CA or chicken ovalbumin) and dye injected subcutaneously induced DH indistinguishable from that induced with the same antigens in Freund adjuvant. Neither antigen in distilled water alone sensitized. The dye also intensified Arthus sensitization. This diazo dye therefore may be a practical, harmless water-soluble substitute for Freund adjuvant for inducing DH or cell-mediated immunity and for intensifying Arthus sensitization.