A water-soluble tuberculin-active peptidoglycan (TAPG) with a molecular weight of ca. 28,000 to 30,000 was isolated from the culture filtrate of Mycobacterium tuberculosis. TAPG was approximately four to five times more potent than tuberculin purified protein derivative S in guinea pigs sensitized with M. tuberculosis or M. bovis (freeze-dried BCG). It showed little or no cross-reactivity at a dose of 0.1 to 0.4 ,jg in guinea pigs sensitized with M. kansasii, M. scrofulaceum, M. intracellulare,

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... or M. avium. TAPG did not show any adjuvant activity when injected in guinea pigs in a water-in-oil emulsion containing ovalbumin. TAPG, in Freund incomplete adjuvant, proved to be an effective immunogen for inducing delayed hypersensitivity in guinea pigs. Chemical analysis of TAPG showed that it contains proline, glutamic acid, alanine, diaminopimelic acid, tyrosine, threonine, glucosamine, and the reducing sugars, arabinose and galactose. In immunoelectrophoretic studies with reference M. tuberculosis H37Rv antiserum, TAPG did not show any precipitin bands. Tuberculin purified protein derivative (PPD) is capable of eliciting a delayed hypersensitivity reaction in humans and animals infected with Mycobacterium tuberculosis, but, due to its lack of specificity, it also elicits delayed hypersensitivity cross-reactions in humans and animals infected with mycobacteria other than M. tuberculosis or M. bovis. These cross-reactions obscure the interpretation and limit the significance of tuberculin skin testing as an aid to the diagnosis of tuberculosis (8). Mycobacterial antigens that contain few or no cross-reacting components are therefore needed to differentiate between tuberculous and nontuberculous infections. Although we have previously reported (10) on the isolation of "specific" tuberculin peptides from PPD, the need for specific antigens with tuberculin activity is still great. The present studies were, therefore, undertaken to find out whether specific antigens could be isolated from the culture filtrate of M. tuberculosis after its PPD contents had been removed. MATERIALS AND METHODS Isolation of a tuberculin-active peptidoglycan (TAPG) from the culture filtrate of M. tuberculosis. The outline of the method used for the isolation of a peptidoglycan fraction from the culture filtrate of M. tuberculosis Johnston is shown in a flow diagram (Fig. 1) . Precipitation of PPD. From the culture filtrate of M. tuberculosis, tuberculin PPD was precipitated by employing the method described by Landi (15). Essentially, the method consists of growing M. tuberculosis Johnston on the synthetic medium of Long and Seibert (17) for 6 weeks at 370C. The culture is then steamed at 1000C for 3 h and filtered through a Berkfeld candle. Tuberculin PPD is precipitated from the culture filtrate by trichloroacetic acid, at a final concentration of 4%, and separated by centrifugation. PTA precipitate. To the trichloroacetic acid supernatant, phosphotungstic acid (PTA) was added to give a 0.5% final concentration, resulting in a heavy white precipitate, which was sedimented by centrifugation at 17,300 x g for 30 min. A saturated solution of barium hydroxide was added slowly to the precipitate and stirred until the pH of the suspension was 7.2. After centrifugation at 30,900 x g for 30 min, the supernatant was collected and neutralized with 0.1 N H2SO4. The acid precipitate was removed by centrifugation and then discarded. The addition of 10 volumes of acetone to the supernatant resulted in the formation of a white precipitate, which was washed twice with acetone, dried in vacuo, and designated "PTA precipitate." Fractionation of PTA precipitate by ion-exchange chromatography on Dowex 50W-X8. The ion-exchange chromatographic method using volatile buffers was chosen for the fractionation of the PTA precipitate. Dowex 50W-X8 resin was prepared by the method of Moore and Stein (20). Six hundred milligrams of PTA precipitate was dissolved in 5 ml of 0.2 M pyridine-acetic acid buffer (pH 3.1). The sample was carefully layered on the top of the resin bed. The column (100 by 2.5 cm) was connected to a gradient vessel. The mixer and the reservoir contained 500 ml each of 0.2 M pyridine-acetic acid (pH 3.1) and 2 M pyridine-acetic acid buffer (pH 5), respectively. The 344 on May 9, 2020 by guest http://iai.asm.org/ Downloaded from