Separation of a Water‐Soluble Adjuvant (MAF3) from Delipidated Cells of Mycobacterium tuberculosis Strain Aoyama B by Hydrogenolysis and Gel Filtration

Abstract

A water-extract from hydrogenolyzed cells of M. tuberculosis strain Aoyama B was separated into 4 portions (F-1 to F-4 fractions) by gel filtration with a Sephadex G-100 column. The 3rd peak (called MAF3) eluted from the column was the most adjuvant-active fraction. The MW determined by gel filtration was .apprx. 16,000 daltons. MAF3 consisted of heteropolymer(s) composed of .apprx. 76-79% 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 [immunoglobulin G2] 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.