Preparation of 14C-labeled Tuberculin Purified

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APPLIED MICROBIOLOGY, July 1967, p 839-843 Copynight 1967 American Society for Microbiology Vol. 15, No. 4 Printed in U.S.A. Preparation of 14C-labeled Tuberculin Purified Protein Derivative S.

1 APPLIED MICROBIOLOGY, July 1967, p Copynight 1967 American Society for Microbiology Vol. 15, No. 4 Printed in U.S.A. Preparation of 14C-labeled Tuberculin Purified Protein Derivative S. LANDI, H. R. HELD, A. H. W. HAUSCHILD,l AND R. HILSHEIMER1 Connaught Medical Researchl Laboratories, University of Toronto, Toronto, Ontario, Canada Received for publication 20 February 1967 'IC-labeled tuberculin purified protein derivative ("4C-tuberculin PPD) has been prepared from culture filtrates of Mycobacterium tuberculosis var. hominis grown in a culture medium containing uniformly labeled 'IC-amino acids. With a mixture of "4C-amino acids (an acid hydrolysate of "4C-Chlorella protein) in the medium, the recoveries of 'IC in the final product were higher than with "4C-labeled-Lglutamic acid. 'IC-tuberculin PPD was separated into tuberculoprotein and nucleic acid by paper electrophoresis. The specific radioactivity of tuberculoprotein was substantially greater than that of the nucleic acid. 'IC-tuberculin PPD is advocated as a means to measure the adsorption of tuberculin to glass or other surfaces. It could also prove useful as a means to study the structure and mode of action of tuberculin. For some time, we noticed a partial loss of potency in diluted preparations of tuberculin purified protein derivative (PPD) used in the intracutaneous method of skin testing. An attempt was made by Landi et al. (4) to determine the reason for this loss by replacing conventional PPD with '4C-labeled PPD and correlating the changes in biological activity and radioactivity in solution. By use of '4C-labeled PPD, this loss was shown to result mainly from the adsorption of the tuberculoprotein to glass or plastic surfaces. The present paper describes in detail the preparation of 4IC-labeled PPD and some of its properties. MATERIALS AND METHODS Source and isolation of crude 14C-tuberculin. The "Johnston" strain of Mycobacterium tuberculosis var. hominis, isolated in 1920 in Toronto, Canada, from a case of human tuberculosis, was used for the production of 14C-tuberculin PPD. The "Johnston" strain showed the characteristic surface growth of M. tuberculosis var. hominis on Long's synthetic medium (6), as well as virulence for guinea pigs and mice and nonvirulence for rabbits. The cultures of the organism were maintained by weekly subculture on Long's synthetic medium. Long's medium was dispensed in 150-ml amounts into nine Roux bottles and autoclaved for 30 min at a steam pressure of 15 psi. These nine bottles were equally divided into three batches designated as batches 1 to 3. To batch 1, a total of approximately 200 j,c of uniformly labeled 14C-L-glutamic acid (New 1'Present address: Microbiology Division, Food and Drug Directorate, Department of National Health and Welfare, Ottawa, Canada. England Nuclear Corp., Boston, Mass.) was added. Batches 2 and 3 received a total of approximately 200 and 600 Ac, respectively, of a mixture of 14C-Lamino acids (acid hydrolysate of 14C-Chlorella protein, New England Nuclear Corp.). The actual radioactivities are expressed as counts per minute (Tables 1 to 3). The inoculum for the nine bottles was grown as a surface pellicle in a small Erlenmeyer flask on Long's synthetic medium at 37 C for 8 to 10 days. After inoculation, the Roux bottles were placed in a smahl incubator at 37 C for 6 weeks. NaOH pellets were placed in a glass pan adjacent to the Roux bottles to remove expired 14CO2 during growth. The incubator was kept in a fume hood. After cultivation, the Roux bottles were tested for purity (2) and steamed for 3 hr in a flowing-steam unit (100 C). The bottles were allowed to stand overnight at room temperature. The 839 contents of the bottles were then passed through a sterile flannelette cloth placed over a filter-paper pulp pad to separate the medium containing 14C-tuberculin from the bacterial growth. The filtrates of the three batches were kept in separate containers and designated "crude 14C-tuberculin solution," batches 1, 2 and 3. The '4C-bacterial cells were thoroughly washed with distilled water and vacuum-dried. Purification of crude 14C-tuberculin. Crude 14Ctuberculin solution was pi rifled essentially by methods previously described by Landi and Held (3). 14C-tuberculin PPD (batches 1 and 2) was prepared from crude "4C-tuberculin solution by trichloroacetic acid precipitation at 5 C [method D of Landi and Held (3)l. Details of this procedure are given in Table 1. "4C-tuberculin PPD (batch 3) was also prepared by precipitation with trichloroacetic acid and by redissolving the wet precipitate in 0.1 M Na2HPO4 to give a preparation designated ""14C-tuberculin PPD stock solution." This preparation was further purified

840 LANDI ET AL. APPL. MICROBIOL. by (NH4)2SO4 precipitation by use of method B of Landi and Held (3). Details of this procedure are given on both Tables 1 and 2. Radioactivity determinations.

2 840 LANDI ET AL. APPL. MICROBIOL. by (NH4)2SO4 precipitation by use of method B of Landi and Held (3). Details of this procedure are given on both Tables 1 and 2. Radioactivity determinations. 14C-labeled material was assayed by evaporating portions of the liquid to dryness on stainless-steel planchets, which were then counted in a gas-flow Geiger Muller counter fitted with a Micromil window (Nuclear Chicago Corp., Des Plaines, 111.). The radioactivity is recorded as indicated in the text, either in counts per minute per total amount of or as specific radioactivity in counts per minute per milligram of substance. No correction was made for the absorption of radioactivity by the buffer salts or by the bacterial cells. Besides tuberculoprotein, tuberculin PPD contains various amounts of nucleic acid, depending upon the particular batch of tuberculin and on the method of precipitation. To determine the distribution of both weight and 14C between tuberculoprotein and nucleic acid, tuberculin PPD was separated by paper electrophoresis into the two groups of compounds (3). Fraction TABLE 1. The nucleic acid was extracted and assayed quantitatively by ultraviolet spectrophotometry. The difference in weight between the original tuberculin PPD and the nucleic acid was taken as the weight of the tuberculoprotein. The distribution of 14C was determined by autoradiography (1) and direct counting of the excised radioactive portion of the paper strips. From these data and the specific radioactivities of the tuberculin PPD preparations, the specific radioactivities of tuberculoprotein and nucleic acid were calculated. Tuberculin skin tests in guinea pigs. Comparisons of the potency of 14C-tuberculin PPD, batches 1, 2, and 3, were made with National Institutes of Health (NIH) reference standard tuberculin PPD, lot 69. A 4-mg of each batch was dissolved in 4 ml of M Na2HPO4. These solutions, as well as the standard, were further diluted in phosphate-buffered saline containing 0.3% phenol and % Tween 80 to give concentrations of mg/ml and mg/ml. Doses of 0.1 ml of each concentration for batches 1, 2, and 3 and the standard PPD were injected intradermally into a total of 48 BCG-sensitized Procedure for the purification of crude "4C-tuberculina 14C-L-glutamic acid batch 1 (132 X ) Crude 14C-tuberculin solution ml Precipitant (44% trichloroacetic acid) c ml Supernatant fluid ml (After precipitation and centrifugation, the precipitates were washed)d 4% trichloroacetic acid... 1% trichloroacetic acid... 8 ml Ethyl alcohol... Ethyl alcohol... Ether... 5 ml Ether... 8 ml 14C-tuberculin PPD, dry precipitate mg Total radioactivity measured in solutions and dry precipitates 7.0 X 3.1 X 0.04 X 0.07 X 0.02 X X X 0.50 X 260 ml 26 ml 280 ml Acid hydrolysate of 14C-CMlorelia protein Batch 2 (148 X )b 8 ml 5 ml 8 ml 91mg 19.7 X 11.2 X 0.14 X 0.23 X 0.03 X X X 1.42 X 10, Batch 3 (444 X )b 250 ml 25 ml 270 ml 25 ml Precipitatef 60.5 X 0.75 X a Procedure used corresponded to method D previously described by Landi and Held (3). b Initial radioactivity in culture medium (counts per minute). The added radioactivity in microcuries was approximately 200 Mc (batch 1) and approximately 200 and 600,c for batches 2 and 3, respectively. c A 44% (w/v) trichloroacetic acid solution (room temperature) was added to the crude 14C-tuberculin solution (5 C). d Washing was by stirring up the precipitate with a glass rod and centrifuging again. * The precipitate was ground in a mortar and vacuum-dried over CaC12. f The wet precipitate was redissolved in 20 ml of 0.1 M Na2HPO4 to give 20 ml of ""14C-tuberculin PPD stock solution," which was further purified as shown in Table 2.

3 VOL. 15, 1967 PREPARATION OF 'IC TUBERCULIN PPD 841 TABLE 2. Procedure for the purification of 14Ctuberculin PPD stock solutiona Total radioactivity measured in solutions and dry precipitate of acid hydrolysate of 14C-Chlor- Fraction ella protein (batch 3) Co Countslminb 4C-tuberculin PPD stock solution ml 7.68 X Precipitant [saturated (NH4)2SO4]J ml Supernatant fluid.. 40 ml 3.78 X After precipitation and centrifugation the precipitates were washedd Half-saturated (NH4)2SO4 solution... 1 ml 0.02 X 4% trichloroacetic acid. 1 ml 0.02 X 4% trichloroacetic acid. 1 ml 0.02 X 4% trichloroacetic acid. 1 ml 0.01 X 4% trichloroacetic acid. 1 ml X 4%0 trichloroacetic acid. 1 ml X Acetone... 2 ml 0.4 X Acetone... 2 ml X Ether X 4C-tuberculin PPD, dry precipitate mg 3.43 X a Procedure used corresponded to method B previously described by Landi and Held (3). binitial radioactivity in culture medium was 444 X counts per min. The added radioactivity in microcuries was approximately 600, c. c Saturated solution of (NH4)2SO4 (room temperature), previously adjusted to ph 7.2 with K2HPO4, was added to the "4C-tuberculin PPD stock solutions (5 C). d Washing was by stirring up the precipitates with a glass rod and centrifuging again. e The precipitate was ground in a mortar and vacuum-dried over CaCl2. female guinea pigs. At 24 hr after injection, the size of each reaction was recorded as the sum of two perpendicular diameters in millimeters. The estimate of potency was made by the method of Long, Miles, and Perry (5). RESULTS AND DIscussIoN Recovery of radioactivity. The recovery of 14C in tuberculin PPD in method D was greater when glutamic acid was replaced with a mixture of amino acids as 14C source (Table 3). Similar trends were found in the total 14C content of the bacterial cells. After removal of the bulk of nucleic acid by method B (Table 3), the per cent recovery of 14C in the final product was still higher with the amino acid mixture than with L-glutamic acid as 14C source. The differences between batches 1 and 2 in the radioactivity of cells and tuberculin PPD, and in the amount of 14C unaccounted for (approximately 82% in batch 1 and approximately 59% in batch 2) show that the amount of 4IC incorporated into tuberculoprotein was larger in batch 2 than in batch 1. Therefore, to favor the production of 14C-labeled tuberculin PPD with a higher specific radioactivity, it is preferable to use a mixture of radioactive amino acids as represented by the 14C-Chlorella protein hydrolysate, rather than the highly metabolizing amino acid, glutamic acid, labeled with "C. The radioactivity unaccounted for was lost during the growth and steaming process. Distribution of radioactivity between tuberculoprotein and nucleic acid in "C-tuberculin PPD. Table 4 shows the distribution of weight and radioactivity between tuberculoprotein and nucleic acid in the three batches of tuberculin PPD. In batches 1 and 2, the nucleic acid comprised 27 to 37% of the total weight, but contained only 16 to 18% of the total radioactivity. In method B, most of the nucleic acid remained in the supernatant fluid after (NH4) 2S04 precipitation. The nucleic acid of batch 3, therefore, comprised only 4.5% of the total weight and 1.8% of the total "IC. The data show that the "IC-content relative to weight (specific radioactivity) was considerably higher in the tuberculoprotein than in the nucleic acid. Biological activity of "4C-tuberculin PPD. Skin tests on 48 BCG-sensitized guinea pigs used to compare the three batches of "4C-tuberculin PPD with the NIH reference standard PPD showed that the relative potency of batches 1 and 2 was 50 to 60% compared with the standard, whereas batch 3 showed the same relative potency as the standard (Table 5). However, the recovery of potency in tuberculin units was higher for the trichloroacetic acid precipitates (batches 1 and 2) than for the ammonium sulfate precipitate (batch 3; Table 5). The results are in agreement with our previous work with nonlabeled tuberculin PPD (3), and show that the skin test is not affected by the low level of 14C of the tuberculin PPD used in these studies. Use of radioactive tuberculin. 14C-tuberculin PPD has been used to measure the adsorption of tuberculoprotein to glass and other surfaces (4). Since the original concentration of tuberculoprotein in tuberculin solutions used for the skin test (Mantoux) is only 50 units/ml, that is, 1 ppm of tuberculoprotein, the loss by adsorption could

4 842 LANDI ET AL. APPL. MICROBIOL. Batch' TABLE 3. Recovery of 14C in tuberculin PPD Location of radioactivity Total radioactivityb Percentage of radioactivity added 1 Initial radioactivity in the medium 132 X 100 Bacterial cells (4.26 g) 17.1 X Crude "4C-tuberculin solution 7.0 X C accounted for 24.1 X 18.3 "4C-tuberculin PPD (99 mg) 0.50 X Initial radioactivity in the medium 148 X 100 Bacterial cells (3.84 g) 40.7 X 27.5 Crude "4C-tuberculin solution 19.7 X C accounted for 60.4 X 40.8 "4C-tuberculin PPD (91 mg) 1.42 X Initial radioactivity in the medium 444 X 100 Bacterial cells (2.13 g) 79.8 X 18.0 "4C-tuberculin PPD stock solution 7.7 X 1.73 Supernatant fluid after (NH4)2SO4 precipi- 3.8 X 0.86 tation "4C-tuberculin PPD (37 mg) 3.43 X 0.77 Batches 1 and 2 were prepared from crude 14C-tLberculin solution by trichloroacetic acid precipitation at 5 C, according to method D of Landi and Held (3). Batch 3 was prepared from ""14C-tuberculin PPD stock solution" by (NH4)2SO4 precipitation, according to method B of Landi and Held (3). b The 14C source was L-glutamic acid for batch 1 and amino acid mixture (acid hydrolysate of 14C- Chlorella protein) for batches 2 and 3. TABLE 4. Distribution of weight and radioactivity between tuberculoprotein and nucleic acid in "4C-tuberculin PPD Radio- Specific Batch Fraction Wt (%)' activity radio- (%)a activityb 1 Tuberculin PPD ,050 Tuberculoprotein ,700 Nucleic acid ,310 2 Tuberculin PPD ,600 Tuberculoprotein ,800 Nucleic acid ,750 3 Tuberculin PPD ,800 Tuberculoprotein ,400 Nucleic acid ,100 a Percentage of tuberculin PPD. b Expressed as counts per minute per milligram. not be measured accurately by conventional analytical methods other than the skin test itself. '4C-tuberculin PPD should prove helpful in the study of the mechanism of the tuberculin skin reaction, in the elucidation of the structure of tuberculoprotein, in the evaluation of the efficacy of antiadsorption agents, and in the study of interactions between tuberculoprotein and other ingredients in PPD solutions. ACKNOWLEDGMENTS We are grateful to Mrs. S. Ober anc Mrs. L. Jailos for excellent technical assistance. We also wish to express our gratitude to Mrs. R. L. McClure for cooperation in the statistical analyses. LITERATURE CITED 1. BLOCK, R. J., E. L. DURRUM, AND G. ZWEIG Paper chromatography and paper electrophoresis, p Academic Press, Inc., New York. 2. LANDI, S Preparation, purification, and stability of tuberculin. Appl. Microbiol. 11: LANDI, S., AND H. R. HELD Physicochemical and biological studies on various preparations of tuberculin purified protein derivative. Appl. Microbiol. 13: LANDI, S., H. R. HELD, A. H. W. HAUSCHLLD, AND R. HILSHEIMER Adsorption of tuberculin PPD to glass and plastic surfaces. Bull. World Health Organ. 35:

5 VOL. 15X 1967 PREPARATION OF 'IC TUBERCULIN PPD 843 TABLE 5. Relative potency of "4C-tuberculin PPD and comparison of the methods of precipitation on the basis of recovery of potency Relative potency Recovery of potency Batch Method of prepn Yield of PPD Trichloroacetic NIH standard" = 1.00 NIH standard TU per prepn (in =50,OOOTUJ/mg million TU) c method acid (5taken C) as 100% mg 1 2 Dd Dd ( ) ( ) 29,000 27, Avg Be ( ) 53, athe relative potencies were determined by comparison with National Institutes of Health reference standard tuberculin PPD (taken as 1.00), and are the results of statistical evaluation. The figures in brackets represent 95% limits. b The relative potency given in tuberculin units (TU) per milligram of tuberculin PPD. c Obtained by multiplication of relative potency (in TU/mg) X yield (in mg). d Prepared by precipitation with trichloroacetic acid. 6 Prepared by precipitation with trichloroacetic acid and reprecipitation with ammonium sulfate. 5. LONG, D. A., A. A. MILEs, AND W. L. M. PERRY The essay of tuberculin. Bull. World Health Organ. 10: LONG, E. R., AND F. B. SEIBERT The chemical composition of the active principle of tuberculin I. A non-protein medium suitable for the production of tuberculin in large quantity. Am. Rev. Tuberc. 13: Downloaded from on July 13, 2018 by guest

5 Efrotomycin. [Summary of efrotomycin] ET A 2 ET A 1 ET B 2 ET B 1 C 59 H 88 N 2 O 20 MW: 1145

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