APPLIED MICROBIOLOGY, March, 1965Copyright © 1965 American Society for Microbiology

Vol. 13, No. 2Printed in U.S.A.

Physicochemical and Biological Studies on VariousPreparations of Tuberculin Purified Protein

DerivativeS. LANDI AND H. R. HELD

Connaught Medical Research Laboratories, University of Toronto, Toronto, On.tario. Canada

Received for publication 10 August 1964

ABSTRACTLANDI, S. (University of Toronto, Toronto, Ontario, Canada), AND H. R. HELD.

Physicochemical and biological studies on various preparations of tuberculin purifiedprotein derivative. Appl. Microbiol. 13:132-139. 1965.-Tuberculin purified proteinderivative (PPD) has been prepared by seven different precipitation methods fromculture filtrate of Mycobacterium tuberculosis var. hominis. It was found to contain48 to 99% tuberculoprotein, depending on the method of precipitation. The remain-ing percentage is represented by nucleic acid, polysaccharide, and ash. Activationanalysis on tuberculin PPD and on tubercle bacilli has revealed the presence of traceelements. The molecular weight of tuberculin PPD has been found to be of the orderof 14,800 to 27,800. The biological activity of tuberculin PPD varies from lot to lot andfrom method to method. A correlation between its molecular weight and its biologicalactivity seems to exist.

Tuberculin is a natural product of the growthof mycobacteria and is mainly found in the culturefiltrate. A commonly used method for the iso-lation of tuberculoprotein from the culturefiltrate of Mycobacterium tuberculosis of mam-malian origin is precipitation by trichloroaceticacid. Tuberculoprotein in this form is known astuberculin purified protein derivative (PPD),and is used as a skin-testing diagnostic agent intuberculosis prevention programs. Besides theactive tuberculoprotein, an appreciable amountof nucleic acid is also precipitated by the tri-chloroacetic acid. The presence of the nucleicacid makes it especially difficult to evaluate thepotency of a preparation of tuberculin PPD onthe basis of its nitrogen content and dry weight.In this investigation, suitable precipitationmethods for the preparation of highly purifiedtuberculin PPD were examined. This reportcompares various precipitation methods for thepreparation of tuberculin PPD, presents physico-chemical data on the preparations, and relatesthese findings to their biological activities. Inthis way, an evaluation of the various precipi-tation methods can be made on the basis of thequality, in addition to the yield, of the tuberculinPPD.

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, theculture was harvested and steamed for 3 hr in aflowing-steam cabinet. The contents of the bottleswere passed through a flannelette cloth placedover a filter-paper pulp pad to separate the filtratecontaining the tuberculoprotein from the micro-organisms. This filtrate was then sterilizedthrough a Berkefeld candle. The sterile filtrateis designated "crude tuberculin solution."

Tuberculin PPD stock solution. From the crudetuberculin solution, the total protein was pre-cipitated with trichloroacetic acid (4% finalconcentration) at 5 C (Landi, 1963). The washedprecipitate was dissolved in a phosphate-salinesolution to a volume of approximately 4% of theoriginal volume of the crude filtrate. This provideda stock solution of tuberculin PPD designated"tuberculin PPD stock solution."Elementary analysis. Analyses, including deter-

mination of the moisture and ash content of tuber-culin PPD samples obtained by various precipita-tion methods were performed by Carl Tiedeke,Laboratory of Microchemistry, Teaneck, N.J.

Carbohydrate determinations. The polysaccharidecontent of the tuberculin PPD calculated as glu-cose was determined by the anthrone method(Koehler, 1952). Since the nucleic acid and tuber-culoprotein in the tuberculin PPD preparationinterfered with the anthrone reaction, the prep-aration was first treated with 4% trichloroaceticacid (final concentration) to remove these substan-ces, and the polysaccharide was determined inthe supernatant liquid.

132

on May 27, 2019 by guest

http://aem.asm

.org/D

ownloaded from

Paper electrophoresis. All samples were qualita- 220 260 300tively tested for protein and nucleic acid by Ir7Ipaper electrophoresis (Fig. 1). The paper used inthe electrophoresis runs was Whatman no. 1(38 by 3 cm, with 1% sodium borate, pH 7.7 buffer). 2The paper strip was loaded with 200 jig of tuber-

4culin PPD, in 20 ,Iliters of 0.033 M Na2HPO4. Elec- 40trophoresis was performed at a potential gradientof 10 v/cm for 1.5 hr, and then the paper stripswere dried in a current of air, stained, and washedas follows. The stain for protein was a mixture of50 ml of 0.4% aqueous Wool Fast Blue BL (Na-tional Aniline Division, Allied Chemical Corp.,Buffalo, N.Y.), 40 ml of methanol, and 10 ml ofglacial acetic acid. The stain for nucleic acidcontained 100 ml of methanol and 10 ml of 0.5%aqueous methylene blue chloride. Washing forboth 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 20iliters of 2% Na2B407.10H20 were applied to thepaper strip. Electrophoresis was performed in anelectrolyte consisting of 2%Na2B407-10H20 at apotential gradient of 5 v/cm for 4.5 hr; the paper

N

20

_-U)k

z

0

10

220 260 300- ~~~~~~~~~~~WAVELENGTH, m,ui

o~~~~~~~~~~~~ A

. ~~~~~~~FIG.2. Spectral absorption of tuberculin PPDN A B C and of nucleic acid (concentration, 200 ,ug/ml).(A) Trichloroacetic acid precipitate (E C); (B)

(NH4)2S04 precipitate; (C) CHCls precipitate;FIG. 1. Paper electrophoresis of 200 lag of tuber- (N) nucleic acid isolated from tuberculin PPD

culin PPD and of 200 lgof nucleic acid. stock solution.

VOL. 13, 1965 TUBERCULIN PPD 133

on May 27, 2019 by guest

http://aem.asm

.org/D

ownloaded from

LANDI AND HELD

strips were dried, stained, and washed as describedabove.

In all cases, the nucleic acid content was de-termined quantitatively by elution from unstainedpaper strips with 5 ml of 0.033 M Na2HPO4 andmeasurement of the ultraviolet absorption at 260m,u. A blank paper was similarly treated, and thissolution was used as a control. Both extractionswere carried out for approximately 0.5 hr.

Ultraviolet absorption. Ultraviolet spectra (Fig.2) were measured on all the described preparationsof tuberculin PPD, with a Beckman DU spectro-photometer. Tuberculin PPD stock solution con-tains two ultraviolet-absorbing substances, tu-berculoprotein and nucleic acid. Quantitativedeterminations were made of the nucleic acidseparated electrophoretically from the protein.

Activation analysis (metals). The activationanalysis was carried out by R. E. Jervis, Depart-ment of Chemical Engineering, University ofToronto. Samples of tuberculin PPD and ofkilled tubercle bacilli (Johnston strain) weresealed in clear polyethylene film and irradiatedwith neutrons for 2 days in an atomic reactor.After activation, weighed portions were separatedfrom the plastic wrappings and examined with amultichannel pulse height y-ray spectrometer.y-Ray spectra were taken at intervals as theactivated samples decayed during a 1-monthperiod.

Ultracentrifugation. The ultracentrifugation wascarried out by L. Pinteric, Connaught MedicalResearch Laboratories, University of Toronto.A 10-mg amount of the tuberculin PPD was dis-solved in 1 ml of 0.033 M Na2HPO4-0.5% NaCI(pH 8.9) and sedimented in a Spinco analyticalultracentrifuge (model E). The molecular weightof the tuberculin PPD was determined by themethod of Svedberg and Pedersen (1940).

Tuberculin skin tests in guinea pigs. Potencycomparisons of various tuberculin preparationswere made with the National Institutes of HealthReference Standard Tuberculin PPD. The Tuber-culin PPD preparations used were stored in powderform, in small glass vials, at room temperature.From each preparation, a 4-mg sample was accu-rately weighed and dissolved in 4 ml of 0.033 MNa2HPO4. These solutions were further diluted inphosphate-buffered saline to give the followingtwo concentrations, 0.004 mg/ml and 0.001 mg/ml.The dose injected intradermally into BCG-sen-sitized guinea pigs was 0.1 ml at each of theseconcentrations of standard and unknown. At24 hr after injection, the diameter of each reac-tion zone was measured, and the result was re-corded as the sum of two perpendicular diametersin millimeters. The estimate of potency was madeby the method of Long, Miles, and Perry (1954).

Preparation of tuberculin PPD from tuberculinPPD stock solution. Tuberculin PPD was preparedfrom PPD stock solution by trichloroacetic acidprecipitation at 5 C (method A), by ammoniumsulfate precipitation (method B), and by the

chloroform method (method C). Details of theprocedures are given in Table 1.

Trichloroacetic acid was used by Seibert et al.(1934) for the isolation of the active principleresponsible for the tuberculin skin reaction. Theproduct was then called SOTT, meaning "syn-thetic medium old tuberculin trichloroacetic acidprecipitate"; the designation was later changedto PPD (purified protein derivative). A prepara-tion of tuberculin PPD by this method (method A)was made from each lot of tuberculin PPD stocksolution by trichloroacetic acid precipitation (4%final concentration) to obtain a representativesample in a dry state. In this form, it is believed tobe stable and, therefore, more suitable for com-parative studies with other preparations of tuber-culin PPD.

Seibert and Glenn (1941) reported a method forobtaining tuberculin PPD by (NH4)2SO4 precipita-tion at half saturation and neutral pH. They foundthat the precipitates obtained under those con-ditions contained only traces of nucleic acid andthat they were so pure that a standardization ofthe final solutions on the basis of their nitrogencontent was justified.

Isolation of nucleoproteins by a gel formationbetween protein and CHCl3 was described bySevag, Lackman, and Smolens (1938). In our useof the chloroform method (method C), precipita-tion was carried out at pH 4.6, and an optimalyield of tuberculin PPD containing a small amountof nucleic acid was obtained. The yield of tuber-culin PPD was highest when working with fairlyhigh concentrations (approximately 10 mg/mlof tuberculin PPD).

Preparation of tuberculin PPD from crude tuber-culin solution. Tuberculin PPD was preparedfrom crude tuberculin solution by trichloroaceticacid precipitation at 5 C (method D), by hot(60 C) trichloroacetic acid precipitation (methodE), by ammonium sulfate precipitation (methodF), and by trichloroacetic acid precipitation inthe presence of amnmonium sulfate (method G).For the last method, trichloroacetic acid (4%final concentration) was added to a crude tuber-culin solution which had been previously half-saturated with neutral (NH4)2SO4. Details ofthis procedure and the others are given in Table 1.

RESULTS AND DISCUSSION

In this investigation, seven different methodsof precipitation have been used to preparetuberculin PPD (Table 1). Tuberculin PPD pre-pared by precipitation with trichloroacetic acidat 5 C from either tuberculin PPD stock solutionor crude tuberculin solution was found to containconsiderable amounts of nucleic acid, varyingfrom lot to lot. The concentrations of nucleicacid, calculated on the basis of the dry weight ofthe trichloroacetic acid precipitate, were foundto vary from 14 to 47.5% (methods A and D,

134 APPL. MICROBIOL.

on May 27, 2019 by guest

http://aem.asm

.org/D

ownloaded from

10 C4 Cq

5 0r-

44c

._o t

Cd0"6 - xxxP4-.d o o C;

.00-ID oI

4)8o oC

*-ola

o0Q0.0*L 3

-~

Q

OQ.0_

0-

0;dV3-4

ao,-b

m 0

,§owKO ^v_ X 4l xv_ _- ___

i 0

02_ 02

0 a

-0_0

a)° 02

; rC .

,- - - -',-- l- - t.S ~ ~ :~97, .N CO-,4c O.- .II- f

0

0XoX 0

o-

S.-1.-I02r-

0a,

-

VCO

CII

.C.0 .a7,.0

*i4..

1104

0,67-40 -14

-.4 (-'),= ceQ

. 4 Q$..,. -q

+. -4-'-)4) (34Qe Ca

'IF44"te

C.)

04

i- l)fz4

° : 0 c,

Oos. -111

Cq cq NC9C-ICO CO 0 CYD CO020o

I l:

Wi

3̂ C) lo

135

a)

ax)a1)

_

0)+ co

o0 t

4.cn uomO0

_0 o

00

00

0

.0

0

0

0

0i

:1

'AiX:3

a)

o

0

0.

ct-

QD

0

1.

,'l-

a)

0

,a¢)

"0a)0.

"00-P."00Q

H~1

0

0.2

0p40

0

En

i

04

00.

p4

I--, 1-1 1-11

000.6m m mr-4 r-4 r-i1. 11-1 I.-I CVD

(M cqa 00 com Cq 1114

on May 27, 2019 by guest

http://aem.asm

.org/D

ownloaded from

LANDI AND HELD

TABLE 2. Chemical composition of tuberculin PPD prepared by various precipitation methods

Method of precipitation

A

B

c

D

E

F

G

Lotno.

121126127128

121126127128

121128

133134136137139

133134

133134136137139

136137139

Nucleic acid

Per cent

27.840.636.647.5

0.72.81.1ND

3.5ND

27.835.021.614.018.7

4.53.1

0.40.40.60.50.6

2.92.93.5

Avg

38.2

1.5

3.5

23.4

3.8

0.5

3.1

Polysaccharide

Percent

NDt0.030.630.13

0.250.190.770.31

0.450.55

1.881.561.541.131.15

1.200.89

1.001.961.370.800.72

0.640.430.50

Avg

0.26

0.38

0.50

1.45

1.04

1.17

0.52

Ash

Per cent

ND4.18ND3.76

0.461.402.86ND

2.58ND

NDND2.49ND2.20

ND1.65

0.591.06NDND1.01

2.571.682.11

Avg

3.97

1.57

2.58

2.35

1.65

0.89

2.12

Protein* (by differencefrom 100%)

Per cent

67.9755.1958.8048.61

98.5995.6195.2796.62

93.4793.37

67.9761.0974.3782.5277.95

92.6594.36

98.0196.5897.1497.8197.67

93.8994.9993.89

Avg

57.66

96.52

93.42

72.78

93.50

97.44

94.26

Water

Per cent

ND0.0ND0.21

2.282.142.37ND

NDND

NDND0.68ND2.68

NDND

ND1.94NDND2.14

2.031.942.46

Avg

0.11

2.26

1.68

2.04

2.14

* When nucleic acid, polysaccharide, or ash content was not determined, the average figures for theparticular method were used, to obtain the approximate protein content.

t Not determined.

Table 2). Since nucleic acid is nontuberculin skin-reactive (Seibert, 1940), it is to be expected thatthe potency of the tuberculin PPD on a dry-weight basis would decrease as its nucleic acidcontent increases. Therefore various methodswere tested to obtain a nucleic acid-free tuberculinPPD. However, to compare the methods ofpreparation of tuberculin PPD, both the relativepotency and the yield are to be taken intoconsideration.A tuberculin PPD prepared by (NH4)2SO4

precipitation from tuberculin PPD stock solutioncontained concentrations of nucleic acid varyingfrom 0.7 to 2.8% (method B, Table 2). Whenthe same procedure of precipitation was used onthe crude tuberculin solution, it contained concen-trations of nucleic acid varying from 0.4 to 0.6%(method F, Table 2). In both cases, the yield of

tuberculin PPD was much lower than that ob-tained with trichloroacetic acid at 5 C, but theirrelative potency was much higher (Tables 3and 4).A tuberculin PPD of low nucleic acid content

(3.5%) can also be prepared by CHC13 gel pre-cipitation (method C, Table 2). This method wasfound to give a satisfactory yield only whenworking with a fairly concentrated solution oftuberculin (tuberculin PPD stock solution) atpH 4.6. However, the yield was much lower thanthat obtained by the trichloroacetic acid pre-cipitation method at 5 C, but its relative potencywas somewhat higher (Tables 3 and 4).A tuberculin PPD also of low nucleic acid

content (average 3.8%) was prepared by tri-chloroacetic acid precipitation at 60 C from acrude tuberculin solution (method E, Table 2).

136 APPL. MICROIBIOL.

on May 27, 2019 by guest

http://aem.asm

.org/D

ownloaded from

TUBERCULIN PPD

However, the relative potency was low as com-

pared with the tuberculin PPD prepared by(NH4)2S04 precipitation, whereas the yield was

much higher (Tables 3 and 4). This lower relativepotency could not be entirely accounted for bythe presence of nucleic acid. In comparison withthe trichloroacetic acid precipitate at 5 C, therelative potency was somewhat higher but theyield was not as great (Tables 3 and 4).When trichloroacetic acid was added to a crude

tuberculin solution previously half saturated withneutral (NH4)2S04, a tuberculin PPD of lownucleic acid content (average 3.1 %) was obtained(method G, Table 2). Its relative potency was

comparable to that obtained by the neutral(NH4)2S04 precipitation method alone (Tables 3and 4). Both these relative potencies are con-

siderably higher than that obtained with thetrichloroacetic acid precipitate at 5 C. In ad-dition, the yield by the (NH4)2S04 plus tri-chloroacetic acid method was higher than theyield after (NH4)2S04 precipitation only, butneither yield was as high as with the trichloro-acetic acid precipitation at 5 C (Tables 3 and 4).A tabulation of both the yield and relative

potency of the preparations is given in Table 4.

TABLE 3. Relative potencies of various preparationsof tuberculin PPD determined by skin tests on

BCG-sensitized guinea pigs*

Lot no.

121126127128133134136137139

121-139

Method of preparation from

Tuberculin PPDCrude tuberculin solution

stock solution

A

0.640.600.760.44

B

1.120.981.220.74

c

0.830.801.010.63

0.56 0.92 0.74

D E F G

0.680.750.811.010.970.85

0.800.810.871.050.860.92

1.221.231.341.711.251.30

1.141.171.351.521.151.19

* The relative potencies were determined bycomparison with National Institutes of Healthreference standard tuberculin PPD (taken as1.00), and are the result of statistical evaluation.Each figure represents the geometric mean fromtwo or three series of skin tests, each involvingeight guinea pigs.

TABLE 4. Comparison of the methods of preparation on the basis of the recovery of potency

Relative potency Recovery of potency

M ethod of Lot no. Yield"precipitation NIH standardb NIH standard' TU.yer prTprn(inTichloroaceticNIH standardb NIHTU prep (incid (5 C) method

1.00 0,000TU/mg mi ion TU)d taken as 100%

mg

A 121, 126, 127, 128 400 0.56 28,000 11.20 100B 192 0.92 46,000 8.82 78.8

A 121, 128 335 0.52 26,000 8.70 100B 173 0.88 44,000 7.61 87.5C 233 0.71 35,500 8.27 95.0

D 133, 134, 136, 137, 139 470 0.85 42,500 20.00 100F 231 1.30 65,000 15.00 75.0

D 133, 134 406 0.71 35,500 14.40 100E 328 0.80 40,000 13.10 90.9F 214 1.22 61,000 13.04 90.5

D 136, 137, 139 479 0.91 45,500 21.80 100F 243 1.47 73,500 17.87 81.9G 357 1.39 69,500 24.80 113.5

a Average yield of dry precipitate tuberculin PPD per 40 ml tuberculin PPD stock solution and per1,000 ml of crude tuberculin solution.

b The relative potency given in this column is a combined value (geometric mean) for the indicatedlots (Table 3). NIH, National Institutes of Health.

c The relative potency in tuberculin units (TU) per milligram of tuberculin PPD is the product of50,000 X relative potency.

d Average recovery of potency in million TU for the combined lots. It is the product of relative potency(in TU/mg) X yield (in mg).

VOL. 13,2 1965 137

on May 27, 2019 by guest

http://aem.asm

.org/D

ownloaded from

TABLE 5. Activation analysis and ash determination on preparations of tuberculin PPD (93.3 to 98.6%tuberculoprotein) and tuberculosis cells

Method of preparation (according to Table 1)

Metal* Johnston tuberculosisB C E F cells

(lot 121) (lot 128) (lot 134) (lot 133)

Iron ............ 9.1 X 10-3 9.2 X 10-1 2.5 X 10-1 1.9 X 10-1 1.1 x 10-1Sodium .......... 3.1 X 10-1 3.3 X 10-1 6.0 X 10-1 1.3 X 10-1 6.0 X 10-2Zinc ............ 2.0 X 10-4 7.9 X 10-2 8.1 X 10-3 1.4 X 10-3 2.6 X 10-2Copper.......... 2.0 X 10-3 3.0 X 10-3 2.0 X 10-' 7.3 X 10-3 1.6 X 10-3Chromium ....... NDt 4.7 X 10-3 1.3 X 10-3 1.6 X 10-3 3.4 X 10-5Gold ............ 1.6 X 10-6 4.3 X 10-5 1.7 X 10-6 2.7 X 10-4 5.4 X 10-6Lanthanum ...... 1.3 X 10-4 1.4 X 10-4 2.8 X 10-4 4.6 X 10-4 6.2 X 10-4Antimony........ Few ppm Few ppm Less than 1 ppm Few ppm Few ppmn2 (metals) ....... 0.32 1.04 0.85 0.33 0.19Ash (by ignitionmethod) ....... 0.46 2.58 1.65 0.59 2.34

* Looked for and not detected: Br, Sc, Co, Rb, Cs, and Pt-metals (all judged to be less than 1 ppm);Hg, Cd, Mo, As, Ag, Ge (all judged to be less than 10 ppm). The activation technique is not sensitivefor the detection of: Ca, Pb, Si, S, P, I, Ni, Mg, and other light elements.

t Not detected.

FIG. 3. Ultracentrifugal peaks of tuberculin PPDprepared by different methods of precipitation.t = 24 minutes. (1) PPD-121 (method C); (2)PPD-133 (method E); (3) PPD-126 (method B);(4) PPD-121 (method B); (5) PPD-139 (method G);(6) PPD-139 (method F).

From these results, the recovery of potency fromtuberculin PPD stock solution or crude tuberculinsolution has been calculated as the product ofrelative potency in tuberculin units per milligramby the yield in milligrams.

Studies on the chemical composition haveshown that impurities in terms of polysaccharidesand ash are of the order of 0.2 to 1.4% and 0.9to 4.0% (Table 2), respectively, and thus would

TABLE 6. Molecular weight of tuberculin PPD(92.6 to 98.6% tuberculoprotein)*

RelativeMethod Lot S2 Molecular potency ac-of prepn no. wt cording to

Table 3

C 121 1.32 8.70 14,800 0.83E 133 1.00 6.25 15,600 0.80B 126 1.815 8.50 20,800 0.98B 121 1.625 6.85 22,900 1.12G 139 1.44 5.81 24,200 1.15F 139 1.54 5.38 27,800 1.25

* S2 represents the sedimentation coefficientat 20 C (X 10-13). D2o represents the diffusioncoefficient at 20 C (X 10-7). The partial volume ofthe samples was assumed to be 0.75. Centrifugedat 59,870 rev/min (259,000 X g).

not be considered of importance in respect tothe biological activity of the tuberculin PPD ob-tained by the various methods of preparationdescribed.

It is also interesting to note that activationanalysis (Table 5) has revealed the presence ofseveral metals in these preparations and in thetubercle bacilli. The importance of these metalsto the constitution of the tuberculoprotein isunknown.

Tuberculin preparations containing 92.6 to98.6% tuberculoprotein were found to be homo-geneous according to the ultracentrifugationmethod (Fig. 3), and to have a molecular weightranging from 14,800 to 27,800, depending on themethod of preparation (Table 6). Seibert, Peder-

138 LANDI AND HELD APPL. MICROBIOL.

on May 27, 2019 by guest

http://aem.asm

.org/D

ownloaded from

TUBERCULIN PPD

sen, and Tiselius (1938) obtained a molecularweight ranging from 9,000 to 32,000. We obtainedthe highest molecular weight by the (NH4)2SO4method on crude tuberculin solution and thelowest molecular weight by the CHC13 methodon tuberculin PPD stock solution. A correlationbetween the molecular weight and the biologicalactivity of the tuberculin PPD exists (Table 6).

For a tuberculin PPD containing 92.6 to 98.6%tuberculoprotein, values ranging from 44.35 to45.79%, 6.52 to 6.99%, 13.44 to 14.14%, 0.23 to0.30% were found for C, H, N, and S, respec-tively. The average nitrogen content was foundto be 13.73%. Taking into account the averagemoisture content of 2.11 %, an average valueof 14.03% N has been arrived at, which corre-sponds to a nitrogen factor of 7.13. This is inagreement with the factor 7 proposed by Green(1946), but differs from the factor of 6.14 usedby Seibert (1940) and 6.25 used by Affronti(1959).

ACKNOWLEDGMENTSWe wish to express our sincere appreciation to

C. 0. Siebenmann for his constant interestthroughout the course of this work. We are grate-ful to Mrs. S. Ober and Mrs. L. Jailos for theirexcellent technical assistance and we acknowledgethe help of M. Goldner in the preparation of thismanuscript. We also wish to express our gratitudeto Mrs. R. L. McClure for her co-operation in thestatistical analyses of data. Thanks are also ex-tended to M. H. Brown for his comments on thismanuscript.

LITERATURE CITEDAFFRONTI, L. F. 1959. Purified protein derivatives(PPD) and other antigens prepared from

atypical acid-fast bacilli and Nocardia as-teroides. Am. Rev. Tuberc. 79:284-295.

GREEN, H. H. 1946. Weybridge P. P. D. tuber-culins. Brit. Vet. J. 102:267-278.

KOEHLER, L. H. 1952. Differentiation of carbo-hydrates by anthrone reaction rate and colorintensity. Anal. Chem. 24:1576-1579.

LANDI, S. 1963. Preparation, purification, andstability of tuberculin. Appl. Microbiol. 11:408-412.

LONG, D. A., A. A. MILES, AND W. L. M. PERRY.1954. The assay of tuberculin. Bull. WorldHealth Organ. 10:989-1002.

LONG, E. R., AND F. B. SEIBERT. 1926. The chemi-cal composition of the active principle of tuber-culin. Am. Rev. Tuberc. 13:393-397.

SEIBERT, F. B., J. D. ARONSON, J. REICHEL,L. T. CLARK, AND E. R. LONG. 1934. The isola-tion and properties of the purified proteinderivative of tuberculin. Am. Rev. Tuberc.30:705-768.

SEIBERT, F. B. 1940. Removal of the impurities,nucleic acid and polysaccharide, from tuber-culin protein. J. Biol. Chem. 133:593-604.

SEIBERT, F. B., AND J. T. GLENN. 1941. Tuber-culin purified protein derivative. Am. Rev.Tuberc. 44:9-25.

SEIBERT, F. B., K. 0. PEDERSEN, AND A. TISELIUS.1938. Molecular weight, electro chemical andbiological properties of tuberculin protein andpolysaccharide molecules. J. Exp. Med. 68:413-438.

SEVAG, M. G., D. B. LACKMAN, AND J. SMOLENS.1938. The isolation of the components of strepto-coccal nucleoproteins in serologically activeform. J. Biol. Chem. 124:425-436.

SVEDBERG, T., AND K. 0. PEDERSEN. 1940. Theultracentrifuge. Oxford University Press, Lon-don.

VOL. 13, 1965 139

on May 27, 2019 by guest

http://aem.asm

.org/D

ownloaded from