FURTHER STUDIES ON A LABILE IMMUNOGENIC PARTICULATE SUBSTANCE ISOLATED FROM MYCOBACTERIUM TUBERCULOSIS

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Youmans, Anne S. (Northwestern University Medical School, Chicago, Ill.), and Guy P. Youmans. Further studies on a labile immunogenic particulate substance isolated from Mycobacterium tuberculosis. J. Bacteriol. 87:278–285. 1964.—A particulate fraction which was highly immunogenic for mice was collected by ultracentrifugation from mycobacteria disrupted in 0.25 m sucrose buffer. The active immunogenic material was present in the gelatinous pellet obtained after centrifugation at 40,000 rev/min (144,000 × g) for 3 hr. This active material could be prepared free from whole cells and cell walls. This was done either by several centrifugations at lower speeds, or by filtering the supernatant fluid from the 10,000 rev/min centrifugation through a Millipore filter (porosity 0.5 μ). No microorganisms were found on slides or in cultures made from these filtrates. The immunogenic moiety in the particulate fraction was found to be very labile. Temperatures higher than 0 to 4 C inactivated the immunogenic activity. There was an irreversible linear decrease in activity as the temperature increased. If fractions were frozen or lyophilized, the activity remained as high as the original material for 4 weeks, and then rapidly decreased. The immunogenic material also was very sensitive to the hydrogen-ion concentration; the optimal activity was found at pH 6.8 to 7.0. The activity decreased rapidly at more acid or alkaline pH values. Also, particulate fraction prepared in sucrose buffer at pH 7.3 and 7.6 was much less active than that prepared in sucrose buffer at pH 7.0. Immunogenic activity was decreased if the particulate fraction was dialyzed overnight against 0.01 m phosphate buffer or distilled water at 4 C. The detergent sodium lauryl sulfate inactivated immunogenic activity. Moreover, the use of a Waring Blendor to blend the ruptured cell mass before centrifugation decreased the activity. Finally, a markedly lower activity resulted if both the 20,000 and 40,000 rev/min centrifugations were done the day after the rupture of the cells. Some refinements in technique which are used now in the preparation of the particulate fraction are detailed.

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