Priming of mice with complexes of antigen coupled to muramyl dipeptide and monoclonal anti-muramyl dipeptide antibodies enhanced the adjuvant activity of muramyl dipeptide on the humoral response to the antigen. The enhancement did not occur with free (uncoupled) muramyl dipeptide and required the presence of an adjuvant-active hapten within the complex as well as the Fc fragment of the monoclonal antibody. This system proved highly effective in eliciting antibodies to synthetic weak immunogens

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... whereas muramyl dipeptide, on its own, exerts very little or no adjuvant activity. The effect was not due to a general polyclonal activation and was restricted to the antigen coupled to the synthetic adjuvant. Possible pathways involved in this phenomenon are discussed. * Corresponding author. 35-amino-acid subpeptide fragment of M24 (S-CB7) were prepared as described elsewhere (7, 16). Haptens, synthetic adjuvants, and analogs. 2,4-Dinitrophenol-lysine (DNP-Lys) was purchased from Serva, Heidelberg, West Germany. MDP was obtained from the Pasteur Institute ( Paris, France). Derivatives of MDP, namely, N-acetylmuramyl-D-alanyl-D-isoglutamine [MDP(D-D)], N-acetylmuramyl-L-alanyl-D-isoglutamine-Llysine (MDP-Lys), MDP(D-D)-L-Lys, and multi-poly(MDP)poly(DL-alanyl)-poly(L-lysine) (MDP-A-L) and the neutral A-L chain were kindly provided by P. Lefrancier at the Choay Institute (Paris, France) and were described elsewhere (9, 24, 26, 35). Preparation of antigen-hapten conjugates. Conjugates of HA with MDP-Lys, MDP(D-D)-Lys, or DNP-Lys were prepared by using glutaraldehyde (Sigma Chemical Co., St. Louis, Mo.) as the coupling reagent. Briefly, 5 mg of HA and 20 mg of the hapten were dissolved in 2.4 ml of phosphatebuffered saline (PBS) at pH 7.4. To the mixture 50 or 100 p.l of 8% glutaraldehyde was then added under constant stirring, and coupling occurred over a 30to 60-min period at 4°C. The conjugate was then extensively dialyzed against PBS, centrifuged at 1,000 x g for 30 min, filtered through a 0.22-,um membrane filter (Millipore Corp., Bedford, Mass.), and stored in working samples at -20°C. The MDP or DNP content in each conjugate was determined as previously described (33, 34) , and the molar ratio of hapten to antigen was calculated. Similarly, HA on its own was treated with glutaraldehyde and used as the antigen control. M24-MDP-Lys conjugates were prepared as described above. The S-CB7-MDP conjugate was kindly provided by M. Jolivet and was prepared as previously described (21). Conjugates of MDP-Lys or DNP-Lys with horseradish peroxidase (HRPO) were prepared by the periodate technique of Nakane and Kawaoi (30). Two such conjugates, HRPO-MDP-Lys2 and HRPO-DNP-Lys8, were used to assess the avidity of the monoclonal anti-MDP and anti-DNP antibodies described below. Murine monoclonal antibodies. The preparation and characterization of monoclonal anti-MDP antibodies have been 312 on May 8, 2020 by guest http://iai.asm.org/ Downloaded from 28. Morrison, S. L., and G. Terres. 1966. Enhanced immunologic sensitization of mice by the simultaneous injection of antigen INFECT. IMMUN. on May 8, 2020 by guest http://iai.asm.org/ Downloaded from