INFECrION AND IMMUNITY, March 1971, p. 378-384Copyright © 1971 American Society for Microbiology

Vol. 3, No. 3Printed in U.S.A.

Separation of Mycobacterial Soluble Antigens byIsoelectric Focusing

R. G. MOULTON, T. M. DIETZ, AND S. MARCUS

Microbiology Research Unit, Veterans Administration Hospital, and University of Utah College of Medicine,Salt Lake City, Utah 84113

Received for publication 28 September 1970

Ion-exchange chromatography and gel filtration have been reported to yieldpartial separation of mycobacterial antigens. These procedures were used in com-bination with isoelectric focusing in an attempt to purify antigens of Mycobac-terium tuberculosis strain H37Ra. The fractionating action of isoelectric focusing isdependent upon differences in the isoelectric points of the proteins to be sepa-rated. Culture filtrate of M. tuberculosis H37Ra was chromatographed on Sepha-dex G-200. This resulted in two widely separated peaks. The first peak, presum-ably containing high-molecular-weight substances, was then fractionated on adiethylaminoethyl Sephadex anion-exchange column. Three peaks were collected,and each was subjected to isoelectric focusing. Each peak was further separatedinto two or more fractions. The serological reactivity of each fraction was deter-mined by immunodiffusion and immunoelectrophoresis. Sensitized guinea pigswere also skin-tested with the fractions. Two of the fractions contained only a singleprecipitinogen. One fraction contained two precipitinogens. A fourth fractioncontained three precipitinogens and was also the only fraction to display sensitinactivity. Four of the fractions were inactive either as precipitinogens or sensitins.The results suggest that the methods described are useful for the separation ofmycobacterial antigens.

It has been demonstrated by different investi-gators that culture filtrates and cell extracts fromMycobacterium tuberculosis contain a multi-plicity of antigens. Chemical techniques were usedby Seibert (15) to fractionate mycobacterialculture filtrate into three proteins and two poly-saccharides. Employing disc electrophoresis, it hassince been demonstrated that mycobacterialculture filtrates and cell extracts may contain asmany as 24 protein staining components (9, 14).Lind (12) has demonstrated that 17 such culturefiltrate components are active as precipitinogens.The techniques utilized in attempting to isolate

and purify mycobacterial antigens have variedand include ammonium sulfate precipitation(13, 16), acid and alcohol precipitation (15),organic solvent extraction (2, 6), disc electro-phoresis (1, 9), column chromatography (8, 11),ultrafiltration (3, 5), and ultracentrifugation (4).Chemical methods of extraction and fractiona-tion usually result in an incomplete separation ofantigens; in addition, some proteins may bedenatured during the fractionation process.Methods of fractionation which depend on differ-ences in the physical properties of the antigens

may be less destructive and in practice have agreater power of resolution.The technique of isoelectric focusing has been

recently developed (10). The process separatesproteins with dissimilar isoelectric points. In thework described in this paper, the technique ofisoelectric focusing has been used in combinationwith gel filtration and ion-exchange chromatog-raphy in an attempt to fractionate and purifymycobacterial antigens.

MATERIALS AND METHODSCulture filtrate. M. tuberculosis strain H37Ra was

grown for 10 weeks on modified Sauton liquid me-dium (17). At the end of the incubation period, phe-nol was added to a final concentration of 0.5%, andthe cultures were allowed to stand for 72 hr. The phe-nol-killed bacterial cell mass was separated from theculture fluid by Seitz filtration. The filtrate was con-centrated to one-tenth of the original volume by per-vaporation at room temperature.

Gel filtration. A Sephadex laboratory column (2.5by 100 cm) fitted with flow adaptors was filled withSephadex G-200. The elution buffer was 0.067 M phos-phate buffer (pH 7.0) containing 0.1% sodium azide.The buffer was pumped upward through the column.The light absorbance of the eluate was determined at

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254 nm, and the eluate was collected in 6-ml amounts.The void volume of the column was approximately127 ml.

Ion-exchange chromatography. To obtain reproduc-ible results, a prescribed method of preparing the col-umn was strictly adhered to. The system was patternedafter a method described by Knicker and Laborde(11). Three buffers were used. For the first wash buf-fer, a volume of 0.01 M Na2HPO4 was adjusted to pH8.1 by adding 0.01 M KH2PO4. The limiting buffer was0.01 M KH2PO4 (pH 4.5), and the final wash bufferwas 0.3 M NaH2PO4 (pH 4.5). All three buffers con-tained 0.1% sodium azide.The bed was prepared by soaking 7.0 g of diethyl-

aminoethyl (DEAE) Sephadex in 1,050 ml of the firstwash buffer for 24 hr. After soaking, the pH of thegel was 6.5. A Sephadex column (2.5 by 45 cm) waspacked with the gel, and the bed was washed with thefirst wash buffer until the eluate coming from the col-umn had a pH of 7.5. The sample was then applied.The flow was downward and no flow adapters wereused. During the first part of the fractionation run, asystem of constant molarity but decreasing pH wasused. A 600-ml round-bottomed flask was filled with300 ml of the first wash buffer (pH 8.1). An identicalround-bottomed flask was filled with 300 ml of limitingbuffer (pH 4.5). The flasks were connected with asmall siphon tube. The buffer in the first flask wasstirred gently with a magnetic stirrer. As the wash buf-fer in the first flask was pumped to the column, thelimiting buffer from the second flask was siphonedover and the pH of the buffer going to the columngradually decreased. The flow rate was 12 ml/hr. Atapproximately 17 hr the buffer was changed to limitingbuffer only. At 25 hr the buffer was changed to thefinal wash buffer. At 42 hr the final wash buffer wasmade 1.0 M in sodium chloride and used to elute thecolumn. The optimum time to change buffers variedfrom run to run, depending on the volume of sampleput on the column. The eluate was analyzed and col-lected as previously described.

Isoelectric focusing. An isoelectric focusing column(model 8101, LKB Instruments, Inc., Rockville, Md.)was filled as described in the LKB 8100 AmpholineElectro-focusing Manual. The column was usually op-erated with the cathode at the bottom. The dense elec-trode solution contained 0.4 ml of ethanolamine, 15.6 gof sucrose, and 14 ml of distilled water. The light elec-trode solution contained 0.1 ml of phosphoric acid in10 ml of distilled water. The dense ampholyte solutionwas comprised of 1.88 ml of Ampholine (40% stocksolution) and 28 g of sucrose diluted to a volume of 50ml with distilled water. The light ampholyte solutioncontained the sample and 0.62 ml of Ampholine (40%stock) diluted to 50 ml with distilled water. The denseand light ampholyte solutions were put on the columnby using a gradient mixer. The column was operatedat a constant temperature of 4 C. Throughout the op-eration of the column, the voltage was maintained atabout 400 v d-c. The amperage started at 6 ma anddropped to about 0.3 ma as the ampholytes and samplebecame stabilized. As the column was drained, thematerial was analyzed at 254 nm and collected in vol-

umes of 3.0 ml. The pH of each 3-ml volume was de-termined.

Immunoelectrophoresis and immunodiffusion. Thetechniques described by Zaman (21) for immunoelec-trophoresis and immunodiffusion were used with slightmodification; e.g., Na2HPO4 was used instead ofNa2HPO4-2H20, saponin was not used, and the welland trough dimensions varied from those describedby Zaman.

Antisera preparation. Young adult rabbits were in-jected with an inoculum made of a mixture of liveH37Ra cells plus cell extract components obtained bydisrupting tubercle bacilli in a vortex mixer with glassbeads. A glass test tube containing 1 g of bacilli perml of water was vibrated vigorously for 2 to 3 hr em-ploying the mixer and a heavy ring stand. The beadsstrike sharply against the glass tube and against eachother. The process was considered completed when theaqueous phase had a protein concentration of approxi-mately 4 mg/ml. The cells (1.0 mg per ml of water)and cell extract were mixed with Freund's incompleteadjuvant at a ratio of 1:1:1. The mixture was homoge-nized by sonic treatment for 2 min at 20,000 cyclesper sec. Several rabbits and one goat received subcu-taneous injections given at 0, 1, and 10 weeks. Thesera were collected 1 week after the final injection.

Skin test. Adult Hartley strain guinea pigs weresensitized by subcutaneous injection of 1.0 mg of livingH37Ra cells. After 3 weeks, skin tests were performedby injection of 0.1 ml of test and control antigen solu-tions intradermally into the flanks of the guinea pigs.The reactions were read as the diameter in millimetersof erythema.

Protein determination. Protein concentration wasdetermined by using the technique of Lowry et al. asdescribed by Williams and Chase (19).

RESULTS

Analysis of whole culture filtrate. The proteinconcentration of the pervaporated culture filtrateswas 8.0 mg/ml. In immunodiffusion, the culturefiltrate produced 10 precipitin lines when reactedagainst rabbit or goat anti-H37a sera (Fig. 1).Eight precipitin lines were seen in immunoelectro-phoresis (Fig. 2). Not all of the precipitin linesare visible in the photographs.Sephadex G-200 chromatography. A volume of

16.0 ml of culture filtrate was put on the SephadexG-200 column. Two peaks were eluted. The firstpeak came off with the void volume. The secondpeak was completely eluted after 570 ml. The twopeaks were concentrated by pervaporation andsubjected to further analysis.

Six precipitin lines were seen when the first peakwas analyzed by immunoelectrophoresis (Fig. 3).Three components were anodic, one was neutral,and the fifth and sixth components were cathodic.In immunodiffusion, seven bands were seen (Fig.4). The second G-200 peak also contained severalserologically reactive components as did thematerial between the two peaks. All three areas

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(peak 1, the area between peaks, and peak 2)contained overlapping components.DEAE Sephadex chromatography. The first

Sephadex G-200 peak was concentrated by per-vaporation from 110 ml to 15 ml and put on theDEAE Sephadex column. Three peaks wereeluted. Each was subjected to immunoelectro-phoresis (Fig. 5). The first DEAE peak containedtwo precipitinogens; one produced a heavy band,and the other produced a faint band. Both bandswere located at the cathode end of the slide. Nobands were seen with the material of the secondpeak. The third peak yielded one anodic line.Immunodiffusion demonstrated one precipitinline for the first peak, none for the second peak,and two lines for the third peak.

Isoelectric focusing. The three DEAE peaks

FIG. 1. Immunodiffusion of whole culture filtrate.The center well contained H37Ra culture filtrate. Thetop two wells contained rabbit anti-H37Ra. The bottomwells contained goat anti-H37Ra.

FIG. 2. Immunoelectrophoresis of whole culture fil-trate. The well contained H37Ra culture filtrate. Thetroughs contained goat anti-H37Ra. The anode was tothe left.

FIG. 4. Immunodiffusion of peak I from SephadexG-200. Wells 1 and 2 contained material from peak 1.The center well was filled with rabbit anti-H37Ra.

-_..IIZ.

FIG. 3. Immunoelectrophoresis of peak I fromSephadex G-200. The well contained materialfrom peakL The troughs contained rabbit anti-H37Ra. The anodewas to the left.

FIG. 5. Immunoelectrophoresis of DEAE peaks Iand III. The well in the top diagram contained DEAEpeak I. The well in the bottom diagram contained DEAEpeak III. The troughs were filled with rabbtt anti-H37Ra. The anode was to the left.

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were concentrated by pervaporation and sub-jected to isoelectric focusing. To avoid confusion,a code name was given to each peak from iso-electric focusing. Given the symbol I-EF 1-3:I = from DEAE peak I, EF I = isoelectricfocusing run number 1, 3 = third peak of focus-ing run number 1 (Fig. 8). DEAE peak I contain-ing a total of 2.62 mg of protein was put on thefocusing column. The pH range of the ampholytesused was from 3.0 to 10.0. The anode was at thebottom of the column; the cathode was at the top.When the run was completed, the material comingfrom the column was analyzed by the ultravioletanalyzer. Three peaks were seen (Fig. 6). Thecloseness of the peaks at the cathode indicatedthat a more narrow pH range should be used.Therefore, peaks 2 and 3 were pooled and put ona column having a pH range of 7.0 to 10.0 (EFII). The polarity was reversed in comparison tothe initial focusing run; i.e., the cathode was at thebottom and the anode was at the top. The pooledmaterial from peaks 2 and 3 was treated as asample containing no ampholytes. By using themore narrow pH range, greater separation wasachieved (Fig. 7).DEAE peaks 11 and III were focused in a simi-

lar fashion. The entire electrofocusing scheme isseen in Fig. 8. The tubes within each peak were

40 80eluate volume (ml)

FIG. 6. Isoelectric focusing run number I. DEAEpeak number I was focused. The pH range was from3.0 to 10.0.

'I

40 80 120

eluate volume (ml)

FIG. 7. Isoelectric focusing run number II. Thepooled peaks I and 2 from electrofocusing run numberI were refocused. The pH range was from 7.0 to 10.0.

peak I from Sephadex G-200

DEAE SeDhadex

1i3

EF I

(pH 3 -10)

2

EF fl

(pH 7-10)

kAAEF m3--pH 3 -10 )

1 2 3

EF 3(pH 3 -10 I

EF I )(pH 5-8 )

FIG. 8. Diagrammatic scheme of the isoelectricfocusing of DEAE) peaks I, II, and III.

pooled. This material was dialyzed for 7 days at4 C against 0.067 M phosphate buffer (pH 7.0)containing 1.0% sodium chloride. When thedialysis was completed, the fractions wereanalyzed for protein concentration and immuno-logical activity.

Analysis of fractions. The dialyzed fractionswere tested for precipitinogen and sensitin ac-tivity. The skin test reactions shown in Table 1are the mean diameters of the reactions from 14sensitized guinea pigs. Each guinea pig receivednine intradermal 0.1-ml injections including oneinjection of PPD-S (purified protein derivativefrom M. tuberculosis). Only fraction IT-EF111-3 was positive. This fraction was also reactivein normal animals. However, statistical analysiswith a t test for nonpaired data indicated that the24- and 48-hr reactions of sensitized guinea pigsto this fraction were significantly greater (P =

0.01) than the reactions of the normal animals.Previous experience with chromatographic frac-tions had shown that toxicity inducing non-specific skin reactions can sometimes be removedby dialysis. Therefore fraction II-EF III-3 wassubjected to further dialysis in an ultrafiltrationcell (model 52, Amicon Corp., Lexington, Mass.).A membrane was used which passes only mole-cules smaller than 10,000 in molecular weight.After being dialyzed, the ultrafiltered fraction wastested again for its sensitin activity. Nine sensi-tized and 10 normal guinea pigs were used. Theresults of the previous skin tests were sub-stantiated; that is, the fraction had sensitinactivity as well as some toxicity which was notreduced by the ultrafiltration procedure.Four of the peaks from electrofocusing demon-

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TABLE 1. Reaction of sensitized and normal guinea pigs to intradermal injectionsof isoelectric focusing fractions

Sensitized guinea pigse Normal guinea pigsbFraction no. ProteiD concn

(,Ug/ml)5 hr 24 hr 48 hr 5 hr 24 hr 48 hr

I-EF I-1 57.0 2.8 0.3 0.0 3.3 1.4 0.0I-EF II-1 32.0 6.3 1.7 0.2 5.7 2.3 1.7I-EF II-2 .54.0 3.8 0.5 0.0 5.3 2.0 0.0II-EF III-1 66.0 0.7 0.2 0.0 2.3 0.0 0.0II-EF III-3 93.0 10.3 9.0 4.3 9.0 4.7 1.7II-EF IV-1 73.0 2.7 0.4 0.0 2.7 1.4 0.0II-EF IV-2 302.0 2.8 1.3 0.0 0.0 0.0 0.0II-EF IV-3 362.0 5.5 4.4 1.1 4.3 4.3 2.3PPD-S (25TU) 5.0 6.6 13.4 10.6 6.0 4.0 0.0

a Mean diameter in millimeters; 14 guinea pigs.Mean diameter in millimeters; three guinea pigs.

TABLE 2. Analytical data on electrofocusingfractions"

Protein Approx Sensitin Precipitin-Fraction no. concn isoelectric activity actvity

I-EF I-1 57.0 1.4 Neg 2 LinesI-EF II-1 32.0 9.6 Neg 1 LineI-EF 11-2 54.0 8.6 Neg NegII-EF III-1 66.0 10.1 Neg 1 LineII-EF 111-3 93.0 2.9 Pos 3 LinesII-EF IV-1 73.0 9.6 Neg NegII-EF IV-2 302.0 5.7 Neg NegII-EF IV-3 362.0 4.8 Neg Neg

Neg, negative; pos, positive.

strated precipitinogenic activity (Table 2). Theprecipitinogens in peaks I-EF I-1, II-EF 111-1,and II-EF 11-1 were demonstrated by immuno-diffusion. Three of the fractions contained pre-cipitinogens which did not act as sensitins. Onefraction contained material which demonstratedthe activity of both a sensitin and a precipitino-gen. Four fractions contained neither precipi-tinogens nor sensitins. One of the fractions(I-EF IV-4) was not analyzed.The activity of the material in the peaks from

the electrofocusing run number V is not known.In immunodiffusion, a nonspecific cloudy pre-cipitate appeared which obscured any precipitinlines which might have occurred. The sensitinactivity of these fractions was not determined.

DISCUSSIONAs noted by others (12, 13) and as has been

described here, the culture filtrate of M. tubercu-losis contains numerous antigenic factors. Itseems feasible and has also been shown (7, 11)-that these components can be separated.

The fractionating action of Sephadex G-200depends upon having a mixture of constituentswithin a sample which vary in molecular size.Molecules differing from one another in theirelectric charge and in their isoelectric points canbe separated by ion-exchange chromatographyand isoelectric focusing. It has been suggested(11) that column chromatography is a usefultechnique for isolation of mycobacterial antigens,although some of the resulting fractions may con-tain overlapping antigens. Such overlapping wasseen in the results of the work reported here.However, fewer antigenic components were foundin Sephadex G-200 peak number I than werecontained in the whole culture filtrate. AfterDEAE chromatography, some fractions con-tained as few as two components. These obser-vations indicate that DEAE Sephadex andSephadex G-200 may be useful as a preparatorystep to a method with higher resolving power.When the three fractions obtained from ion-

exchange chromatography were subjected toisoelectric focusing, 12 peaks were detected by aultraviolet analyzer at a wavelength of 254 nm.This wavelength should detect both proteins andnucleic acids when they are present in the eluate.Three of the peaks were not extensively analyzedbecause of the presence of a nonspecific precipi-tate. Of the nine remaining peaks, four containedantigenically active material. All four of theseactive peaks contained precipitating antigens asdemonstrated either by immunodiffusion or byimmunoelectrophoresis. Only one of the activepeaks contained material reactive as a sensitin.

It was not demonstrated that the precipitino-gens found in the peaks from electrofocusingwere nonidentical. The precipitinogens did notform strong enough precipitin lines to provenonidentity. Several well sizes and distances be-

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tween wells were tried. Reagent concentrationswere also varied with no change seen in precipitinpatterns. However, there was ample evidence thatseparation did occur. (i) Within the same run, onefraction contained only one active componentwhereas another fraction contained two or threecomponents. (ii) Only one fraction contained asensitin. (iii) Two fractions contained only a singlecomponent. The most encouraging results werefound in the observation that two peaks (I-EFII-1 and II-EF III-1) were seen to contain singleprecipitating antigens.Four peaks were not active immunologically.

This finding may be explained in several ways:(i) there may be an antigenically inactive proteinor nucleic acid present; (ii) the activity may nothave been detected by the techniques used; (iii)the peak may be an artifact since, when the elec-trofocusing column is operated without a sample,some peaks do appear. This last explanation isopen to question on the basis that the nonspecificpeaks are usually lower in amplitude and morespread out than the electrofocusing peaks ob-tained in these experiments with active material.The complete chemical characteristics of the

fractionated components are not known. All wereseen to react positively to the Lowry proteindetermination with a protein concentration rang-ing from 32.0 to 362.0 ,ug/ml. Surprisingly, thepeaks with the highest protein concentration wereimmunologically inactive. Ideally the concentra-tions of carbohydrate and nucleic acids wouldhave been determined on all of the peaks. It isproposed that in the future greater volumes ofculture filtrate will be applied to gel filtration andion-exchange columns so that larger samples willbe available for electrofocusing.Most recent investigators who have attempted

to isolate mycobacterial antigens have obtainedonly one or two purified components (7, 13, 20).Such has been the case with this work. However,only the first peak, presumably containing high-molecular-weight substances, from SephadexG-200 was investigated. It is anticipated that,when a similar process is applied to the materialspresent in the second peak from Sephadex G-200,more components will be isolated.

This work has shown that isoelectric focusingcan be useful in ioslating and purifying mycobac-terial antigens However, two problems have beenencountered which should be further discussed.The ampholytes present in the electrofocusingfractions react in protein determination tests.These ampholytes are not easily removed fromthe samples. It has been reported (18) that aSephadex G-25 column will remove the ampho-lytes from the sample provided that the sampleproteins are larger than the ampholytes. But if the

protein concentration is low, this procedure willfurther dilute the protein, making it difficult todetect as it comes from the column. Since theseampholytes have a molecular weight of less than1,000, ultrafiltration through a membrane whichwill pass the ampholytes and retain the proteincould be used.Another problem with isoelectric focusing is

the frequent appearance of a precipitate as abroad band within the column. It is not knownwhether this precipitate is a specific protein thatprecipitated at its isoelectric point or whether it isnonspecific in nature. The precipitate appears atthe top whether the anode is up or down, and itslowly sinks toward the bottom of the column.It seems less likely to appear when the proteinconcentration of the sample is low. When 70 mgof culture filtrate was focused, a heavy precipi-tate appeared. However, a DEAE peak contain-ing 0.65 mg of protein did not form a precipitate.Assuming that protein concentration is the criticalvariable, then the optimal load should be some-where between 0.65 and 70 mg. This may be trueonly for this system. The electrofocusing instruc-tion manual states that 25 mg of each separateprotein can be present in the sample put on thecolumn; i.e., if a sample contained five separateproteins, then the total amount of protein put onthe column could be as high as 125 mg. This is amaximum load, however, and is not recom-mended for high-resolution separation. Addingurea to the sample decreases the tendency for theprecipitate to form, but high concentrations ofurea may alter the sample proteins.Although certain problems remain to be re-

solved, isoelectric focusing appears to be a usefuland sensitive method for the separation ofmycobacterial antigens.

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