Preparations of Tuberculin Purified Protein Derivative

LANDI, S. (University of Toronto, Toronto, Ontario, Canada), AND H. R. HELD. Physicochemical and biological studies on various preparations of tuberculin purified protein derivative. Appl. Microbiol. 13:132-139. 1965.-Tuberculin purified protein derivative (PPD) has been prepared by seven different precipitation methods from culture filtrate of Mycobacterium tuberculosis var. hominis. It was found to contain 48 to 99% tuberculoprotein, depending on the method of precipitation. The remaining percentage is represented by nucleic acid, polysaccharide, and ash. Activation analysis on tuberculin PPD and on tubercle bacilli has revealed the presence of trace elements. The molecular weight of tuberculin PPD has been found to be of the order of 14,800 to 27,800. The biological activity of tuberculin PPD varies from lot to lot and from method to method. A correlation between its molecular weight and its biological activity seems to exist. Tuberculin is a natural product of the growth of mycobacteria and is mainly found in the culture filtrate. A commonly used method for the isolation of tuberculoprotein from the culture filtrate of Mycobacterium tuberculosis of mammalian origin is precipitation by trichloroacetic acid. Tuberculoprotein in this form is known as tuberculin purified protein derivative (PPD), and is used as a skin-testing diagnostic agent in tuberculosis prevention programs. Besides the active tuberculoprotein, an appreciable amount of nucleic acid is also precipitated by the trichloroacetic acid. The presence of the nucleic acid makes it especially difficult to evaluate the potency of a preparation of tuberculin PPD on the basis of its nitrogen content and dry weight. In this investigation, suitable precipitation methods for the preparation of highly purified tuberculin PPD were examined. This report compares various precipitation methods for the preparation of tuberculin PPD, presents physicochemical data on the preparations, and relates these findings to their biological activities. In this way, an evaluation of the various precipitation methods can be made on the basis of the quality, in addition to the yield, of the tuberculin PPD. MATERIALS AND METHODS Crude tuberculin solution. The "Johnston" strain of M. tuberculosis var. hominis was grown on Long's synthetic medium (Long and Seibert, 1926) in Roux bottles. After 6 to 6.5 weeks, the culture was harvested and steamed for 3 hr in a flowing-steam cabinet. The contents of the bottles were passed through a flannelette cloth placed over a filter-paper pulp pad to separate the filtrate containing the tuberculoprotein from the microorganisms. This filtrate was then sterilized through a Berkefeld candle. The sterile filtrate is designated "crude tuberculin solution." Tuberculin PPD stock solution. From the crude tuberculin solution, the total protein was precipitated with trichloroacetic acid (4% final concentration) at 5 C (Landi, 1963). The washed precipitate was dissolved in a phosphate-saline solution to a volume of approximately 4% of the original volume of the crude filtrate. This provided a stock solution of tuberculin PPD designated "tuberculin PPD stock solution." Elementary analysis. Analyses, including determination of the moisture and ash content of tuberculin PPD samples obtained by various precipitation methods were performed by Carl Tiedeke, Laboratory of Microchemistry, Teaneck, N.J. Carbohydrate determinations. The polysaccharide content of the tuberculin PPD calculated as glucose was determined by the anthrone method (Koehler, 1952). Since the nucleic acid and tuberculoprotein in the tuberculin PPD preparation interfered with the anthrone reaction, the preparation was first treated with 4% trichloroacetic acid (final concentration) to remove these substances, and the polysaccharide was determined in the supernatant liquid. 132 on A uust 4, 2017 by gest ht://aem .sm .rg/ D ow nladed fom Paper electrophoresis. All samples were qualita220 260 300 tively tested for protein and nucleic acid by Ir7I paper electrophoresis (Fig. 1). The paper used in the electrophoresis runs was Whatman no. 1 (38 by 3 cm, with 1% sodium borate, pH 7.7 buffer). 2 The paper strip was loaded with 200 jig of tuber4 culin PPD, in 20 ,Iliters of 0.033 M Na2HPO4. Elec40 trophoresis was performed at a potential gradient of 10 v/cm for 1.5 hr, and then the paper strips were dried in a current of air, stained, and washed as follows. The stain for protein was a mixture of 50 ml of 0.4% aqueous Wool Fast Blue BL (National Aniline Division, Allied Chemical Corp., Buffalo, N.Y.), 40 ml of methanol, and 10 ml of glacial acetic acid. The stain for nucleic acid contained 100 ml of methanol and 10 ml of 0.5% aqueous methylene blue chloride. Washing for both stains was 1% (v/v) acetic acid. Electrolyte (pH 7.7) contained 3 liters of 1% (w/v) boric 3.0 acid (H3B03) and 1 liter of 1% (w/v) borax (Na2B407 10H20). This method was also used with larger amounts of tuberculin PPD; 2,000,g of tuberculin in 20 iliters of 2% Na2B407.10H20 were applied to the paper strip. Electrophoresis was performed in an electrolyte consisting of 2%Na2B407-10H20 at a potential gradient of 5 v/cm for 4.5 hr; the paper N