Separation of a water-soluble adjuvant (MAF3) from delipidated cells of Mycobacterium tuberculosis strain Aoyama B by hydrogenolysis and gel filtration.

A water-extract from hydrogenolyzed cells of Mycobacterium tuberculosis strain Aoyama B was separated into four portions (F-1 to F-4 fractions) by gel filtration with a Sephadex G-100 column. The third peak (called MAF3) eluted from the column was the most adjuvant-active fraction. The molecular weight determined by gel filtration was around 16 000 daltons. MAF3 consisted of heteropolymer(s) composed of approximately 76 to 79% neutral sugars (Ara, Gal, Man, and Glc) and 19% mucopeptide (MurN, GlcN, Glu, Ala, Dpm, Gly, Asp, Thr, Ser, Leu, Lys, Arg, His, Pro, Tyr, and Phe). The adjuvanticities of MAF3 and other fractions in water-in-oil emulsion were estimated by the enhancing effect on immune response to egg albumin (EA) in guinea pigs. MAF3 stimulated the production of humoral antibodies, particularly IgG2 antibody specific to the antigen, and induced delayed type hypersensitivity against EA in the skin and cornea of antigen-primed guinea pigs. These adjuvanticities of MAF3 were similar to the characteristics of mycobacterial cell wall in Freund's complete adjuvant.