INFECrION AND IMMUNITY, Oct. 1972, p. 564-573Cbpyright © 1972 American Society for Microbiology

Vol. 6, No. 4Printed in U.S.A.

Characterization and Comparison of MycobacterialAntigens by Two-Dimensional

ImmunoelectrophoresisDIANNA B. ROBERTS,1 GEORGE L. WRIGHT, JR., LEWIS F. AFFRONTI,

AND MELVIN REICH

Department of Microbiology, The George Washington University School of Medicine, Washington, D.C. 20005

Received for publication 27 April 1972

Two-dimensional immunoelectrophoresis (2D-IEP), in which a complex of anti-gens is subjected to electrophoresis first through an agarose matrix in one directionand secondly through an antiserum-agarose matrix at right angles to the first direc-tion, was evaluated as a tool for analysis of mycobacterial antigens. Cell extractsfrom four species of mycobacteria, Mycobacterium tuberculosis (four strains), M.bovis strain BCG, M. scrofulaceum, and M. phlei, were assayed by 2D-IEP with fouranti-mycobacterial antisera. Besides displaying the precipitin curves in a more easilyinterpreted format than did conventional immunoelectrophoresis (IEP), 2D-IEPoffered greater sensitivity in terms of numbers of precipitin curves when like reac-tions were compared with IEP patterns. As many as 60 immunoprecipitates wereobserved on 2D-IEP slides compared to 18 on comparable IEP plates. Technicalreproducibility of patterns from run to run was excellent. Other parameters, such asthe influence of using different batches of antigen on the pattern, are discussed.Each of the cell extract antigens gave a unique pattern of precipitin peaks whichcould be easily differentiated from the patterns given by the other mycobacterialcell extracts when reacted with any of the antisera in 2D-IEP. Since both the speciesand strains of mycobacteria could be easily and reproducibly differentiated solelyon the basis of two-dimensional immunoelectrophoretic patterns obtained withany of the antisera employed in this study, it may be possible, by using IEP, todifferentiate and identify all species and strains of mycobacteria with one standard,highly sensitive antiserum, rather than a battery of antisera.

Immunoelectrophoresis (7) is a valuable tech-nique for the analysis of mixtures of antigens.Its value, however, is limited in that, first, theprecipitin patterns are not representative ofquantitative variations among the antigens and,second, their interpretation requires consider-able skill and experience. Ressler (18) showedthat antigens could be forced by an electriccurrent into an agar matrix containing antiserum,thus producing a number of narrow curves with acommon base, the relative heights of which areproportional to antigen concentrations. Laurell(13) combined these methods so that the peaksno longer had a common base but were separatedaccording to the electrophoretic mobility of thevarious antigens in agarose. This crossed elec-trophoresis technique is called two-dimensional

1 Part of this report was submitted in partial satisfaction of re-quirements for the M.S. degree, Department of Microbiology, TheGeorge Washington University, Washington, D.C. 20005.

immunoelectrophoresis, quantitative immuno-electrophoresis (4, 11), or two-dimensionalelectroimmunodiffusion (A. J. Crowle, personalcommunication).Two-dimensional immunoelectrophoresis was

developed and has been used primarily for theanalysis and quantitation of normal and ab-normal serum antigens (5, 6) but should be ap-plicable to other mixtures of antigens (2).Although gel diffusion and immunoelectro-

phoresis have been used for the analysis of myco-bacterial antigens (3, 14, 16, 17), they are notsensitive enough for studying all the antigenicmoieties of a crude or multicomponent antigenmixture. For example, acrylamide pore gradientelectrophoresis of a mycobacterial cell extractpreparation results in the detection of between40 and 60 bands which stain with protein-specific dyes (1); yet in immunoelectrophoresisonly 11 to 20 bands appear after reaction with

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homologous antiserum (L. F. Affronti and G. L.Wright, Jr., 1968, Amer. Rev. Resp. Dis. 98:153).Furthei more, differences in the precipitin pro-files of the various species are often difficult todetect.

Since two-dimensional immunoelectrophoresisappeared to be more sensitive and the two-dimensional immunoelectrophorograms wereeasier to interpret than was the case in con-ventional immunoelectrophoresis, it was thoughtworthwhile to assess the value of this procedurefor the analysis of mycobacterial antigens. Thisreport describes the adaptation of the techniqueto the analysis of mycobacterial antigens andits possible use in taxonomic classification ofspecies and strains of mycobacteria.

MATERIALS AND METHODS

Preparation of mycobacterial antigens. Selectedstrains and species of mycobacteria were obtainedfrom the American Type Culture Collection, theTrudeau Culture Collection, and the culture collec-tion of the Microbiology Department of The GeorgeWashington University School of Medicine. Theseincluded Mycobacterium tuberculosis strain H37Ra(H37Ra), M. tuberculosis strain H37Rv (H37Rv),M. bovis strain BCG (BCG), M. scrofuilaceum, andM. phlei. The cells were grown as surface pellicles onProskauer-Beck medium (Difco Laboratories, De-troit, Mich.) at 37 C for 6 to 8 weeks before they wereseparated from the culture fluid by filtration. Theculture filtrates were concentrated by pervaporationto one-tenth their original volume. The concentratedfiltrates were then dialyzed against 10 changes of 20times their volume of cold distilled water. The proteinconcentration of the dialyzed culture filtrate (CF)was determined by using the method of Lowry et al.(15). The CF was used immediately or stored in 5-to 10-ml quantities at -80 C.

Bacterial cell extracts were prepared by dilutingthe washed cell mass with two volumes (w/v) of0.88% saline containing 0.02%7 Merthiolate (PS).This mixture was placed in an ice bath and subjectedto ultrasonic vibrations provided by a Biosonic soni-fier for 10 min. The partially broken cells were al-lowed to cool for 5 min, and then sonically treatedfor an additional 10 min. The sonic extract was cen-trifuged at 15,000 X g for 45 min at 3 C. The super-natant fluid was drawn off and saved, and the pelletwas resuspended in three volumes (w/v) of PS. Thesonic treatment and centrifugation steps were repeatedtwo more times. The pellet from the third 15,000 X gcentrifugation was reserved for use in making anti-serum as described below. The pooled supematantfluids were centrifuged at 100,000 X g for 2.5 hr at3 C. The pellet from this centrifugation was alsoreserved for use in making antiserum. The super-natant fluid was termed cell extract (CX) and waspassed through a sterilizing membrane filter (0.45mm pore size, Millipore Corp., Bedford, Mass.). Theprotein concentration was ascertained as described

above for CF. The cell extract was divided into smallquantities and stored at -80 C.

Preparation of antisera. With the exception of theantiserum directed against M. tuberculosis strainH37Rv, the antisera were prepared, with slight modi-fications, in accordance with the procedure describedby Hirschfield (9). At least three rabbits were usedin making antiserum to each organism. That volumeof CX which contained 5 mg of protein was mixedwith that volume of CF which contained 5 mg ofprotein. One-half milliliter of a 10% aqueous solu-tion of AlCl3 was added to the CX-CF mixture, thepH was adjusted to 7.2 with 10% aqueous NaOH,and the now turbid suspension was centrifuged at2,000 X g for 20 min. The supematant fluid wasdrawn off, reprecipitated, and centrifuged as above.These steps were repeated three more times. Thepellets from the five centrifugations were suspendedin three volumes of PS and centrifuged at 2,000 X gfor 20 min. The supernatant fluid was discarded andthe pellet was washed six more times.

With this pelleted precipitate were mixed 0.5 g ofthe 15,000 X g pellet, 0.5 g of the 100,000 X g pellet,and sufficient (approximately 2 ml) unprecipitated CXand CF (the protein contributions from each prepa-ration being equal) to make a suspension which couldpass through an 18-gauge hypodermic needle. Thetotal volume of the suspension did not exceed 6 mlper rabbit.The suspension, which contained 10 mg of alum-

precipitated protein, about 6 mg of unprecipitatedprotein, and 1 g of centrifugation pellet material, wasinjected intramuscularly (im) into both hind quartersof the rabbit, half on each side. Forty days later, 2mg of unprecipitated protein, half from CX and halffrom CF, was injected subcutaneously into the flanksof the rabbit. Seven to 10 days later, a small sample ofblood was drawn from the ear and the serum wastested against CF and CX in the conventional agargel double-diffusion method. The development ofeight or more precipitin bands in 48 hr indicated theserum was ready to be harvested. If fewer than eightbands appeared, the rabbit received a second booster.The second booster was seldom necessary. Once theantiserum was deemed satisfactory, the rabbit wasexsanguinated by cardiac puncture and the serum wasdrawn off. The antisera of the same specificity whichgave about (+ 1) the same number of precipitin bandswere pooled, divided into 10- to 20-ml quantitiesand stored at -80 C.

Three procedures and six rabbits were employedin making antiserum to M. tuberculosis strain H37Rv.Two rabbits were injected im with a mixture con-taining 10 mg of alum-precipitated protein (halffrom CF and half from CX), 30 mg of dried wholecells, and 3.5 mg of unprecipitated protein from CX.Two rabbits each received im an emulsion madefrom 3.3 ml of Freund's incomplete adjuvant, 10mg of protein equally from unprecipitated CF andCX, and 30 mg of dried cells. A third pair of rabbitseach received im half the dose of the alum suspensiongiven each of the first pair of rabbits plus half thedose of the adjuvant emulsion given each of thesecond pair of rabbits. A booster injection of the

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type described above was given to each of the sixrabbits 40 days after the initial inoculation. Ten dayslater the rabbits were exsanguinated and their serawere pooled, divided into 10-ml quantities, and storedat -80 C.

Conventional immunoelectrophoresis. A modifica-tion of the "macro" method described by Grabarand Williams (8) was used in performing immuno-electrophoresis (IEP). Fifteen milliliters of a hot(90 C) 1% agarose (Sea-Kem, Bausch & Lomb,Rochester, N.Y.) solution in IEP gel buffer (sodiumbarbital-barbital, I = 0.093, pH 8.6) was pouredonto a glass lantern slide (8.2 by 10.1 cm). The anti-gen wells (0.7 cm in diameter) were cut with theircenters 2.9 cm from the cathodic edge of the slideand 0.5 cm from the antiserum troughs, which were0.2 by 9 cm and held 0.3 ml of antiserum. The antigenwells were charged with 70 ,liters of CX containing200 i 35,ug of protein. Electrophoresis was conductedusing IEP well buffer (sodium barbital-barbital, I =0.06, pH 8.6) at 12 ma per slide at 5 C until thebromophenol blue tracking dye included in theantigen well was 0.5 cm from the anodic edge of theslide. The troughs were charged with antiserum, andthe plates were incubated at room temperature in ahumid chamber for 72 hr.

Two-dimensional immunoelectrophoresis. Two-di-mensional immunoelectrophoresis (2D-LEP) wasemployed, with slight modifications, as described byLaurell (13). The first electrophoresis was carriedout as described above for conventional IEP. Thecenter of the antigen well was 1.2 cm from the bottomedge and 2.9 cm from the cathodic side of the slide.After the first electrophoresis, the agarose above aline parallel to and 2.2 cm from the bottom edge ofthe slide was removed and replaced with 12 ml of awarm (50 C) mixture of one part 2% agarose in IEPgel buffer and one part antiserum, either undiluted(final dilution 1:2) or diluted 1 to 1 with IEP gelbuffer (final dilution 1: 4). After the agarose-anti-serum mixture had solidified, the slide was returnedto the electrophoretic chamber and electrophoresiswas conducted at right angles to the first direction ofseparation at 10 ma per slide for 20 hr at 5 C. Thesteps required to perform 2D-IEP are illustrated inFig. 1.

After the second electrophoresis, the slides wereplaced in a humid chamber and incubated for 24 hrat room temperature to increase the intensity of theprecipitin curves. Once the curves had developed, theslides were washed with several changes of PS, driedunder bibulous paper, stained with Ponceau S (1%in 3% trichloroacetic acid), destained in 5% aceticacid, again dried, and finally photographed.The diagrammatic representations of the precipitin

patterns were made by tracing the images of the topsof the peaks produced when the stained slide wasprojected through a photographic enlarger ontowhite paper.

RESULTSTypical two-dimensional immunoelectropho-

retic patterns and the effect of dilution of anti-serum on these patterns are presented in Fig. 2.

2.

:

0 4=:' '

4

FIG. 1. Diagrammatic illustration of the procedurefor perjorming two-dimensional immunoelectrophoresis.1. A hot (90 C) solution of 1% agarose in barbitalbuffer is poured onto an 8 by 10 cm glass slide. 2.Electrophoresis in the first direction is carried out at12 ma per slide. The agarose in the region of the di-agonal lines is then cut away. 3. A warm (50 C) mix-ture of equal parts 2% agarose in barbital buffer andantiserum is poured onto the slide to cover the regionabove the dashed line. 4. The second electrophoresisis then carried out at righlt angles to the first at 10 maper slide for 20 hr.

The higher the dilution of antiserum, the fainterand taller the peaks are for a given duration ofelectrophoresis in the second direction. At adilution of 1:8 the peaks were too faint to beseen clearly. At a 1:2 dilution of the antiserumthe peaks were quite opaque and easily seen, buttended to be grouped together near the linemarking the beginning of the antigen-antiserummatrix above the line of the first direction ofseparation of the antigen. At the 1:4 dilution,the peaks were still easily seen, tall enough to bewell separated from each other, and less anti-serum was required. Because only half as muchantiserum was required (3 ml per slide) at the1:4 dilution and discrete patterns were obtain-able, the 1:4 dilution was adopted for the com-parison of mycobacterial cellular antigens fromorganisms homologous and heterologous to thespecificity of the antiserum.By comparing the 2D-IEP patterns obtained

(Fig. 2) with either dilution (1:2 or 1:4) ofantiserum with classical IEP patterns (Fig. 3)one may observe the ability of the 2D-IEPsystem to accentuate differences between antigenpreparations from two mycobacterial speciesreacted against homologous and heterologousantisera. The classical IEP patterns (Fig. 3)

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A B

C

E F

FIG. 2. Effect of dilution of antiserum on two-dimensional immunoelectrophoretic patterns. The anti-serum dilution used in each of the patterns in the leftcolumn (A, C, E) was 1:2; a 1:4 dilution was used ineach of the patterns in the right column (B, D, F).The antigen:antiserum reactions pictured are: A andB, Mycobacterium tuberculosis strain H37Ra cellextract versus rabbit anti-M. tuberculosis strain H37Ra;C and D, M. bovis strain BCG versus rabbit anti-M.tuberculosis strain H37Ra; E and F, M. bovis strainBCG versus rabbit anti-M. bovis strain BCG.

representing reactions of H37Ra cell extract(CX) and BCG CX with antisera directed againstH37Ra and BCG are dissimilar. Yet the 2D-IEPpatterns (Fig. 2) which represent some of thesesame reactions make the antigenic differencesbetween the two species more apparent, and thusallow easier differentiation of the cell extractsfrom the two species. Besides displaying theprecipitin curves in a more easily interpretedformat, the 2D-IEP technique offers greatersensitivity than classical IEP. Ten to 18 immuno-precipitin arcs were detectable in each classicalIEP pattern (Fig. 3), whereas 27 (Fig. 2B) to48 (Fig. 2E) precipitin curves were detected inthe 2D-IEP electrophorograms representingreactions of H37Ra CX and BCG CX, respec-tively, with their homologous antisera. Un-fortunately, not all peaks which are visible on

the stained slides themselves are visible in thereproduced photographs.The 2D-IEP electrophorograms presented in

Fig. 4 were obtained with homologous myco-bacterial antigen-antiserum systems. The patternswere distinct and unique for each species tested.Sixty immunoprecipitin curves were detectedin the H37Rv CX-anti-H37Rv homologoussystem (Fig. 4B) which exemplifies the highdegree of sensitivity obtainable with the 2D-IEPsystem.By projecting the stained image of the two-

dimensional pattern onto white paper and tracingjust the peaks of the precipitin curves, a "finger-print" profile was obtained. This permitted aneven clearer interpretation and comparison ofthe two-dimensional patterns obtained withdifferent species and strains of mycobacteria.Figure 5 is a representative example of some ofthe two-dimensional "fingerprints" obtainedwhen the different mycobacterial CX antigenpreparations were reacted against homologousand heterologous antisera. Regardless of thetotal number of precipitin curves detected, each"fingerprint" was unique for the species or strainCX antigens tested against any single antiserum.Somewhat similar patterns were obtained whenthe CX from a given strain was reacted withthe homologous and the two heterologous anti-sera employed.

In an attempt to state quantitatively the ob-servation that a pattern given by one strain orspecies could be differentiated from the patterngiven by another strain or species of mycobac-teria, the electrophorograms were divided arbi-trarily into eight vertical zones as indicated inFig. 5. The number of precipitin curves in eachzone was counted and the results are illustratedin Fig. 6. Each mycobacterial species and strainused in this study could be differentiated bycomparing the graphic profiles obtained witheach antiserum.When M. phlei CX was analyzed by the three

antisera, precipitin peaks were not found inzones 1, 2, 6, 7, or 8. This pattern was easilydifferentiated from the 2D-IEP pattern of M.scrofulaceum in that the peaks in the latter weresometimes found in zone 1 or 2 and always inzone 6 or 7. The majority of peaks in the M.bovis pattern were centered around zone 4,whereas the majority of peaks in the M. tubercu-losis strain H37Rv pattern were found in zones4 through 6 and for the M. tuberculosis strainH37Ra pattern in zones 1 through 4.No single zone was characteristic of the species

or strain tested if the antigen preparations werereacted against either anti-BCG or anti-H37Ra

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A

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e,,

Ra

FIG. 3. Conventional immunoelectrophoretic patterns of reactions of Mycobacterium bovis strain BCG (BCG)cell extract (CX) and M. tuberculosis strain H37Ra (Ra) CX with rabbit anti-BCG (troughs A and C) and rabbitanti-Ra (trough B).

A

C

B

D

E

FIG. 4. Two-dimensional immunoelectrophoretic

patterns obtained with homologous mycobacterialantigen-antiserum reactions at an antiserum dilution

of 1:2. A, Mycobacterium tuberculosis strain HS7Ra

cell extract (CX) versus rabbit anti-M. tuberculosis

strain H37Pa. B, M. tuberculosis strain HS7Rv CX

versus rabbit anti-M. tuberculosis strain H37Pv.

C, M. bovis strain BCG CX versus rabbit anti-M.

bovis strain BCG. D, M. scrofulaceum CX versus

rabbit anti-M. scrofulaceum. F, M. phlei versus rabbit

anti-M. phlei.

antiserum, the overall pattern being required todifferentiate between the various mycobacterialcell extracts. However, when the cell extractantigens were reacted with the antiserum pro-duced to H37Rv, the mycobacteria could bedifferentiated by the overall antigen profile aswell as by the total number of precipitin curvesand number of immunoprecipitins in zone 3.

It is interesting to note, as shown in Fig. 6, thatat a dilution of 1:4, the three antisera testedgave more precipitin curves with at least oneheterologous strain or species of mycobacteriathan with the homologous strain or species. Onepossible explanation for this is that there maybe certain antigens in the homologous CX whichstimulate the synthesis of antibodies but, whenanalyzed with antiserum diluted 1: 4, are ingreat antigen excess. Lesser amounts of thesesame antigens in heterologous CX might thenbe precipitable. If this accounts for the observa-tion, a higher concentration of homologousantiserum would be expected to precipitate theseantigens in the homologous cell extract, yieldinga greater number of precipitin peaks. Sixtyprecipitin curves were detected in 2D-IEPelectrophorograms of the homologous H37Rv-anti-H37Rv system when the antiserum wasdiluted 1:2 compared with 41 at a 1:4 dilutionof antiserum. Forty-two were detected at 1 :2and 27 at 1:4 with the H37Ra homologoussystems; and 48 at 1:2 and 34 at 1:4 with theBCG homologous system. Twofold concentrationof the H37Ra CX resulted in a decreased numberof precipitin curves at a 1:2 dilution of anti-serum, and a fivefold concentration yielded only

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B ~~~~~C

D ---E

FIG. 5. Typical 'fingerprint" profiles of two-dimensional immunoelectrophoretic patternis obtained with myco-bacterial antigens tested with antiseruim diluted 1:4. The numbers at the bottom of each profile are zone nwnbers;the vertical lines are the boundaries of the zonies (see text). The anitiserum employed was directed against Myco-bacterium tutberculosis strain H37Rv. The antigens are: A, M. tuberculosis strain 1137Rv cell extract (CX); B, M.tuberculosis strain H37Ra CX; C, Mv. bovis straini BCG CX; D, M. scrofulaceum CX; anid F, M. phlei CX. (Thecontinuous lines between two peaks indicate reactions of identity.)

H437Rv BCG

41 33 219

40334 I1I S

.J A.

1A

ZONE NUMBER

FiG. 6. Anztigen profiles of mycobacterial cell extracts obtained by counting the number of precipitin peaks ineach of the eight zones (illustrated by Fig. 5). The organiism from which the antigen was made is written at thetop of the column in which the profile appears. The antiserum used (all at 1:4 dilution) is written across the rowin which the profile appears. The niumber in the upper right corner of each profile indicates the total number ofprecipitin curves obtained in that antigen-anitiserum initeraction. Note that the mycobacterial species and strainscould easily be differentiated with aniy one of the three anitisera.

A-- -P~ R

10 curves at the 1:2 diution of antiserum. Intwo heterologous systems, BCG-anti-H37Raand BCG-anti-H37Rv, the same numbers ofcurves were detected with either a 1:2 or 1:4

dilution of antiserum. These results appear tosupport the hypothesis mentioned above.To establish the reproducibility of 2D-IEP

with respect to day-to-day or run-to-run re-

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peatability, the effects of different batchesof cell extracts from cells grown under the sameconditions, different batches of cells grown fordifferent lengths of time, different batches ofantiserum, and resemblances of closely relatedstrains of one species, several experiments wereconducted. Typical examples of the results ob-tained are shown in Fig. 7 and 8. Identical 2D-IEP patterns of the reaction of H37Ra CX andanti-H37Ra (diluted 1:4) obtained from threedifferent runs are shown in Fig. 7. Both theH37Ra CX and the antiserum used to obtainthe patterns in Fig. 7 were from different batchesthan those used to obtain the pattern shownelsewhere (Fig. 2B). Although the antiserumused to produce the patterns in Fig. 7 was moresensitive than that used to obtain the patternFig. 2B (37 peaks compared to 27), the grosspatterns are quite similar. Moreover, if thefingerprint profile of the patterns in Fig. 7 isdetermined (zone 1, 2 peaks; zone 2, 1 peak;zone 3, 10 peaks; zone 4, 20 peaks; zone 4, 3peaks; zone 6, 1 peak; and zones 7 and 8, nopeaks), it is found that the peaks are allocatedto the zones in nearly the same proportions asin Fig. 2B (fingerprint profile in Fig. 6). Themajority of antigen peaks detected if the CX isderived from H37Ra are in zones I through 4.Examples of the effects on the reproducibility

of 2D-IEP patterns of different batches of thesame organisms cultured for the same time,

A B

C

FIG. 7. Technical reproducibility of two-dimen-sional immunoelectrophoresis patterns. Plates were

produced on different days. Reactions are of H37Racell extract and rabbit anti-H37Ra diluted 1:4. Bothcell extract anid antiserum are from different batchesthan those used to produce patterns described in othersections of the paper.

B

:...

FIG. 8. Two-dimensional immunoelectrophoretic pat-terns of three strains of virulent M. tuberculosis re-acting with the same anti-H37Ra antiserum usedto obtain patterns in Fig. 7. Cell extracts are fromstrains: A, H37Rv, Trudeau Mycobacteria Collection(TMC) n1o. 103 (used elsewhere in these experimenttsand designated "H37Rv"); B, DT, TMC nio. 119;C and D, H37Rv, TMC no. 102. Patterns A and Bwere obtained with cell extracts of 8-week-old cultures.Patterns C and D were obtained with cell extractsfrom two different batches ofcells harvested at 4 weeks.

different closely related strains cultured for thesame and different times, and different batchesof antiserum used with CX from the same batchare shown in Fig. 8. The bottom two photographs(Fig. 8C and D) represent reactiono of twobatches of the same organism, each grown for4 weeks with the same anti-H37Ra antiserumused to produce the patterns in Fig. 7. It is ap-parent that the two batches of the organismgave nearly identical patterns. The upper twophotographs (Fig. 8A and B) are reactions oftwo virulent strains of M. tuberculosis relatedto the organisms in Fig. 8C and D but grownfor 8 weeks with the same anti-H37Ra antiserum.The necessity for adoption of standard cultureconditions for organisms to be analyzed isemphasized by the differences between the twopairs of slides. In spite of strain differences, batchdifferences, and age of culture differences, thereseem to be overall resemblances among thefour slides which reflect their close taxonomicrelationship. In addition, their fingerprint pro-files approximate those obtained when H37RvCX reacted with a different batch of anti-H37Raantiserum (Fig. 6). The majority of peaks fallinto zones 4 through 6. It seems reasonable toconclude that, with standard culture conditionsand sensitive antiserum, 2D-IEP patterns arereproducible from run to run and with different

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batches of antigen. In addition, although thepower of 2D-IEP as a tool for identifying myco-

bacteria is exemplified by its ability to differ-entiate some strains within species of myco-

bacteria, strain resemblances within species are

also apparent, making assignment of organismsto their proper species relatively easy without theuse of batteries of monospecific antisera. Bestresults are obtained if the same, highly sensitiveantisera are used throughout the analysis of allthe cell extracts.

DISCUSSION

The two-dimensional immunoelectrophoretictechnique as described in this report resulted inthe detection of approximately four times thenumber of antigens detectable by a classical IEPprocedure. The principal reason for the markedincrease in sensitivity of the 2D-IEP system isthat a large proportion if not all of the antigensin the CX are forced by the electric field intocontact with the antibodies as illustrated inFig. 7. Since in conventional IEP the antigensand antibodies make contact by means of simplediffusion which occurs in all directions, only a

portion of the immunologic reagents meet andprecipitate, others being lost in the surroundingagarose gel matrix (Fig. 9).

Because of this high degree of sensitivity the2D-IEP system offers several possibilities forthe qualitative and quantitative identificationand characterization of the highly complexmycobacterial cell extract antigen mixtures.The great sensitivity and technical reproduci-bility (Fig. 7) of the technique plus the ease

with which the two-dimensional immunoelectro-phorograms could be interpreted made possiblethe differentiation of the mycobacterial speciesand strains employed in this study. This was

especially true for the differentiation of the closelyrelated species M. tuberculosis and M. bovis anddifferentiation between two strains of M. tubercu-losis, H37Ra and H37Rv. It was difficult if notimpossible to differentiate clearly the myco-bacteria by classical IEP when only a singleantiserum was used.Although a greater number of antigens could

be demonstrated by increasing the antiserumconcentration, an antiserum dilution of 1:4 (3ml) was adopted because reliable and repro-

ducible two-dimensional patterns could be ob-tained which permitted easy identification ofthe mycobacterial species and strains without ex-

pending a large volume of antiserum. At thisantiserum concentration, however, the number ofantigens detected in the homologous system was

always less than the number detected in at least

+CDo= CD

tfl r

1Z

Conventional Immunoelectrophoresis

+

0

I 1 - +

Two-Dimensional Immunoelectrophoresis

FiG. 9. Diagrammatic explanzation for the greatersenisitivity of two-dimensionial immunoelectrophoresis(2D-IEP) compared to conventional immunoelec-trophoresis (IEP). Because the antigens and anitibodiesdiffuse in all directions (arrows) in IEP, there can beinsufficietnt amounts of certaini anitigens and anitibodiesto allow the formationi of a visible precipitate, or therecan be sufficient loss of either ani antigen or antibodyspecies into the surrounding matrix so that ani effectiveexcess of the other reagenit results in a prozone effect.But in 2D-IEP the antigens are forced electrically intothe anztibody-agarose matrix (arrows). Unider theseelectrodynamic conditions, excess antigen or anitibodyeffects are overcome, and small quantities of an2tigenare precipitated.

one closely related species or strain of myco-

bacteria. This was shown to be a result of con-

siderable antigen excess in the homologous cellextract preparation, while lesser amounts of thesame antigens in the heterologous cell extractswere in appropriate concentrations to produce a

visible precipitin curve. As indicated above, thisproblem could be circumvented by increasingthe concentration of antiserum (to 6 ml), butthen the amount of antiserum consumed pertest is no longer practical for screening or surveypurposes. Even 3 ml (1:4 dilution) of antiserumis approximately 10 times greater than theamounts usually employed in classical IEP andmay be considered by some investigators a

quantity still too large for routine studies. Itmay be possible, however, to develop a "micro"2D-IEP procedure for the characterization ofmycobacterial antigens similar to that describedby Kr0ll (11) and Crowle (personal communica-tion) for the study of serum protein antigensin which the same amount of antiserum used in

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572 ROBERT

classical IEP is employed. Experiments are beingconducted in this laboratory to determine theapplicability of such a procedure for analysisof mycobacterial antigens.Because of the problem of antigen excess in

homologous cell extracts when the 1:4 dilutionof antiserum is used, a direct count of the totalnumber of precipitin curves obtained with CXfrom an unknown species of mycobacterialantiserum or a battery of them may not serveto identify the unknown species. The usefulparameter at this dilution of antiserum is thepattern of the precipitin curves. Both the speciesand strains of mycobacteria could be easilydifferentiated solely on the two-dimensionalimmunoelectrophoretic patterns (Fig. 4 and 5)obtained with any one of the antisera preparedagainst H37Ra, BCG, or H37Rv. This differ-entiation was also successful with different batchesof antigen prepared from cells harvested at 4, 6,and 8 weeks cultured under the same conditions,although optimal ease of identification is ob-tained when the cells are grown for the samelength of time and tested against the samebatch of highly sensitive antiserum. The identi-fication, therefore, of an unknown species orstrain of mycobacteria on the basis of the 2D-lEP pattern would not require a battery of anti-sera One standard, highly sensitive antiserum,such as the anti-H37Rv antiserum produced inthe study, could possibly differentiate all thespecies and strains of mycobacteria. Of course,the analysis of cell extracts from a large numberof mycobacterial species and strains would benecessary to determine if this were possible. Theneed for a suitable reference system to char-acterize mycobacterial antigens and a proposedreference system using classical IEP has recentlybeen described (10; B. W. Janicki, S. D. Chaparas,T. M. Daniel, G. P. Kubica, G. L. Wright, Jr.,and G. S. Yee., Amer. Rev. Resp. Dis., in press).The discussion thus far has centered on the

suitability of the 2D-IEP procedure for theclassification of mycobacteria. Although thevarious species and strains of mycobacteriacould be simply identified by the 2D-IEP tech-nique, it was impossible to determine whichantigens were common and which were specificfor the species or strain by the method as pre-sented. However, Kr0ll (12) has recently de-scribed a modification of a 2D-IEP system inwhich two antigen mixtures are assayed against asingle antiserum. In his technique, precipitincurves which fuse together between the twoantigen mixtures are considered representativeof identical antigen determinants, and thosewhich intersect are considered nonidentical as is

Fs ET AL. INFECT. IMMUNITY

usually described for reactions of identity andnonidentity in gel double-diffusion tests. Itmight, therefore, be possible to determine theantigenic relationships and differences betweentwo species or strains of mycobacteria on asingle 2D-IEP slide, once the patterns have beendetermined individually. Once individual myco-bacterial antigens can be definitively identified,then it should be possible to quantitate themycobacterial antigens in the same way as re-ported for serum protein antigens by using 2D-IEP (4-6, 11-13).The 2D-IEP technique appears to have great

potential for the qualitative and quantitativecharacterization of mycobacterial antigens aswell as any complex mixture of antigens (2).

ACKNOWLEDGMENTS

We gratefully acknowledge the technical assistance of KarenFarrell, Linda Grow, and Mary Gaffney.

This investigation was supported in part by a grant from theJohn A. Hartford Foundation, Inc. and a grant from the UnitedStates-Japan Cooperative Medical Science Program administeredby the National Institute of Allergy and Infectious Diseases.

LITERATURE CITED

1. Affronti, L. F., G. L. Wright, Jr., and M. Reich. 1972. Myco-bacterial antigens: characterization and comparison by dis-continuous pore gradient electrophoresis. Infect. Immunity5:474-481.

2. Axelson, N. H. 1971. Antigen-antibody crossed-electrophore-sis (Laurell) applied to the study of the antigenic structureof Candida albicans. Infect. Immunity 4:525-527.

3. Castelnuovo, G., M. E. Duncan, and G. Bellezza. 1964. Myco-bacterial antigens: a study of antigens in bacterial extractsand in culture filtrates of different ages. Tubercle 45:246-254.

4. Clarke, H. G. M., and T. Freeman. 1966. A quantitative im-munoelectrophoretic method (Laurell electrophoresis). Pro-tides Biol. Fluids Proc. Colloq. Bruges 14:503-509.

5. Clarke, H. G. M., and T. Freeman. 1968. Quantitative immu-noelectrophoresis of human serum proteins. Clin. Sci. 35:403-413.

6. Clarke, H. G. M., T. Freeman, and W. Pryce-Phillips. 1970.Quantitative immunoelectrophoresis of serum from hospit-alized chronic schizophrenic and epileptic patients. J.Neurol. Neurosurg. Psychiat. 33:694-697.

7. Grabar, P. 1959. Immunoelectrophoretic analysis. MethodsBiochem. Anal. 7:1-10.

8. Grabar, P., and C. A. Williams. 1953. Methode permettantl'etude conjugee des proprietes electrophoretiques et immu-nochimiques dun melange de proteins. Application auserum sanguin. Biochim. Biophys. Acta 10:193-194.

9. Hirschfield, J. 1960. Immunoelectrophoresis procedure andapplication of the study of group specific variations in sera.Sci. Tools 7:18-25.

10. Janicki, B. W., S. D. Chaparas, T. M. Daniel, G. P. Kubicka,G. L. Wright, Jr., and G. S. Yee. 1971. A reference systemfor antigens of Mycobacterium tuberculosis. Amer. Rev.Resp. Dis. 104:602-604.

11. Kroll, J. 1968. On the immunoelectrophoretic identificationand quantitation of serum proteins. Scand. J. Clin. Lab.Invest. 22:79-81.

12. Kroll, J. 1969. Immunochemical identification of specific pro-tein lines in quantitative immunoelec.rophoresis patterns.Scand. J. Clin. Lab. Invest. 24:55-60.

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COMPARISON OF MYCOBACTERIAL ANTIGENS

13. Laurell, C. B. 1965. Antigen-antibody crossed electrophoresis.Anal. Biochem. 10:358-361.

14. Lind, A. 1961. Serological studies of mycobacteria by means

of diffusion-in-gel techniques. Arstadius Boktrycheri AB,Gothenburg.

15. Lowry, 0. H., N. J. Rosenbrough, A. L. Farr, and R. J.-Ran-dall. 1951. Protein measurement with the Folin phenol rea-

gent. J. Biol. Chem. 193:265-275.16. Parlett, R. C., and G. P. Youmans. 1959. Antigenic relation-

ships between ninety-eight strains of mycobacteria usinggel-diffusion precipitation technique. Amer. Rev. Tuberc.77:450-461.

17. Pickett, M. J., M. M. Pedersen, D. I. Goldstein, and S. Fro-man. 1968. The antigens of Mycobacterium tuberculosis.Protein fractions from cytoplasmic and culture filtrates.Amer. Rev. Resp. Dis. 17:415-422.

18. Ressler, N. 1960. Electrophoresis of serum protein antigens inan antibody-containing buffer. Clin. Chim. Acta 5:359-365.

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