Preparation of an Adjuvant‐Active, Tuberculin‐Free Peptidoglycolipid from Human Tubercle Bacilli

Wax D isolated from human tubercle bacilli is a macromolecular peptidoglycolipid having adjuvant activity [21] and adjuvant polyarthritogenicity [2, 22]. However, it was shown by Tanaka et al [18-20] that wax D prepared according to the classical procedure of Aebi et al [1], could be fractionated after acetylation by column chromatography on silicic acid; Jolles and his associates [8] demonstrated its polydispersity by use of differential ultracentrifugation in organic solvents; and Kumazawa et al [9] demonstrated the presence of several components in wax D and described the isolation of an adjuvantactive fraction by preparative thin-layer chromatography. The present report deals with the isolation of an active peptidoglycolipid by organic solvent fractionation of wax D followed by treatment with a detergent and proteinase. Preparation of wax D. A culture of Mycobacterium tuberculosis, strain Aoyama B, was supplied by Dr. H. Nemoto, National Institute of Animal Health, Tokyo. The cells were cultured on Sauton's synthetic medium at 37 C for 7 weeks, and after sterilization by heating at 100 C for 10 min, they were collected by centrifugation and washed repeatedly with water, followed by ethanol, and then dried. The preparation of wax D was done principally by the method of Aebi et al [1]. Three-hundred grams dried cells were extracted three times with 5 liters