INFECTION AND IMMUNITY, Apr. 1972, p. 474-481Copyright ©) 1972 Americ.an Society for Microbiology

Vol. 5, No. 4Printed in U.S.A.

Characterization and Comparison of MycobacterialAntigens by Discontinuous Pore Gradient

Gel ElectrophoresisLEWIS F. AFFRONTI, GEORGE L. WRIGHT, JR., AND MELVIN REICH

The George Washinigtoni Uniiversity Medical Ceniter, Department of Microbiology, Washington, D.C. 20005

Received for publication 15 November 1971

Concentrated culture filtrates and cell extracts were prepared from selectedmycobacteria and analyzed by a multistage polyacrylamide electrophoretic pro-cedure. Various staining procedures were used to detect protein, carbohydrate,nucleic acid, and lipid constituents. Visual examination of the more prominentlystained components indicated differences and similarities in each species of Myco-bacteria. The majority of the individual components were of small molecularweight; however, there appeared in all culture filtrates and extracts examined aslowly moving protein component of relatively large molecular size. Evidencebased on immunodiffusion suggests that it is a common mycobacterial antigen.

It has been demonstrated by a number ofstudies that the mycobacterial cell and its meta-bolic products are of an exceedingly complexnature. The biology of the tubercle bacillus andthe relationship of its complex products withinthe host have been studied for many years tobetter understand the pathogenesis of tubercu-losis. There have been many physicochemicaltechniques which have been applied to the sepa-ration and isolation of these complex cellularproducts. It is also important that their chemicalcomposition be known so that the mechanism oftuberculous disease and, consequently, how tocombat it may be better understood. Moreover,by employing more refined mycobacterial frac-tions of known composition, the similarities anddifferences that exist among the various acid-fastorganisms could be more sharply defined andthus contribute to a more satisfactory under-standing of mycobacterial relationships.The present study is a direct outgrowth of our

long-range investigations concerned with de-veloping and standardizing methods for isolatingand purifying mycobacterial antigens and eluci-dating their possible biological effects. Specifi-cally, it has been one of the major aims of thisstudy to identify and compare mycobacterialantigens by utilizing a modified pore gradientpolyacrylamide electrophoresis method. Thepresent report characterizes these biologicallyactive components from various selected strainsof mycobacteria utilizing this technique. Anti-genic profiles from each of them are obtained

and compared, and the possible interrelation-ships are examined.

MATERIALS AND METHODS

Source of organisms for culture filtrates and bac-terial extracts. Selected mycobacterial species wereobtained from the American Type Culture Collectionand the culture collection of the Microbiology De-partment of The George Washington UniversitySchool of Medicine. These included: Mycobacteriumtuberculosis H37Ra, M. tutberculosis H37Rv, M.bovis, M. bovis BCG, M. aviuim, M. kansasii, M.intracellitlare, M. scrofulaceum, M. phlei, M. for-tuitum, and M. smegmatis. Cells were grown as sur-face pellicles on Proskauer-Beck medium at 37 Cfor 6 to 8 weeks before separation from the culturefluid by Seitz filtration. The culture filtrates wereconcentrated by pervaporation to one-tenth theiroriginal volume. The total volumes obtained wereeach divided into two equal portions. One portionwas lyophilized and stored at -20 C, and the otherwas divided into 2-ml samples and also stored at-20 C. When ready for use, each of the lyophilizedfiltrates was reconstituted with sterile, distilled water.The bacterial extracts were prepared by suspend-

ing the collected cells in isotonic saline and centri-fuging them at 2,010 X g. Centrifugation was re-peated twice, and one volume of the washed cellswas suspended in 1.5 volumes of 0.15 M phosphate-buffered saline (PBS), pH 7.2. This suspension, whichwas maintained in an ice bath, was sonically treatedfor 20 min with a Branson model 575 sonifier. Thissonic extract was centrifuged at 10,000 X g for 1 hr,and the sediment was resuspended in 1.5 volumes ofPBS and subjected to sonic treatment for an additional20 min. The sonic extract was centrifuged as above,

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VOL. 5, 1972 CHARACTERIZATION OF MYCOBACTERIAL ANTIGENS. I

and the two supernatant fluids were pooled andcentrifuged at 144,000 X g for 100 min. The finalcell extract was then stored at -80 C in small vol-umes. Protein was analyzed by the Lowry methodor by the Nessler reaction (6).

Discontinuous pore gradient polyacrylamide gelelectrophoresis. Electrophoresis in polyacrylamidegel was performed as reported by Wright and Af-fronti (8) with tris(hydroxymethyl)aminomethaneglycine buffer, pH 8.3, and 5 ma per tube. Thismethod utilizes a multistage gel for the separation ofmycobacterial cellular products and is a modifica-tion of the discontinuous pore gradient techniquedescribed by Wright et al. (9) for the separation ofserum proteins. The complex ionic tuberculin mix-ture migrates through a four-stage separating gel inglass tubes (6 mm inner diameter by 12 cm). Thesegel columns consist of (total gel concentration fol-lowed by gel height): a 3.50%h/ (1 cm) upper gel fol-lowed by a 4.75%o (1 cm), a 7% (1.5 cm), and finallya 12%0 (5 cm) bottom gel. From 0.2 to 0.5 ml ofsample containing 300 ,ug of protein and 20%7 sucrosewas layered over the 3.75%, gel. Electrophoresis wasallowed to proceed at room temperature until thebromophenol blue tracking dye reached a point 1cm from the bottom of the tube. The differentialstains used for approximate identification of sepa-rated components were amido black and CoomassieBlue for protein, the periodic acid-Schiff reagent(PAS) for mucoproteins and polysaccharides, theFeulgen stain and acridine orange for nucleic acid,gallocyanin for ribonucleic acid (RNA), and theOil Red 0 stain (ORO) for lipids. After the myco-bacterial samples were subjected to electrophoresisand their patterns were well established, the Rpvalues for each of the components which had beendeveloped by the Coomassie Blue stain were calcu-lated.

Conventional disc polyacrylamide electrophoresis.The conventional disc electrophoresis procedurewhich was described by Ornstein and Davis (4) andwhich employs homogeneous gel columns of singleconcentrations was used.

Polyacrylamide gel densitometry. Polyacrylamidegels which had been stained for protein were scannedusing a Gilford densitometer and multirange re-corder (model 2000) used together with a BeckmanDU spectrophotometer. This technique had the ad-vantage of eliminating subjectivity involved in visualobservation of the stained patterns.

Antisera. Twelve male albino guinea pigs weighingfrom 500 to 700 g were sensitized with a mixture ofM. tuberculosis H37Ra culture filtrate and driedcells in oil (Bayol F). Each milliliter contained 10mg of cells, and each guinea pig was given 0.4 mlsubcutaneously in several sites on the back of theneck at weekly intervals for 2 weeks. Sensitizationwas evident since lesions of the Koch phenomenonwere seen 1 week after the last injection. The injectedanimals were bled from the heart, and the sera werecollected in the conventional manner.

Recovery of protein components from acrylamidegel. The protein components studied in precipitintests and by immunodiffusion were obtained by

pooling eluates from 50 gel columns after electro-phoresis. The combined 3.50 and 4.75% gel portionswere removed and pooled, and the componentswere eluted from the gel by grinding in a mortartogether with an amount of distilled water equal to10 times the volume of the gel. They were allowed todiffuse at 4 C for 48 hr, after which time the liquidwas removed and concentrated by means of eitherultrafiltration or pervaporation. The eluates from thepooled 7%o gels and the eluates from the 12%O gelswere similarly prepared.

Precipitin tests. The precipitin tests used were asdescribed in a previous report by Affronti (1). Theconcentrated eluates from the combined 3.50 and4.75%h0 gels and those from each of the pooled 7 and12%o gels were made to the following concentrationson the basis of their protein content: 50, 10, 5, and1 ,ug per ml.

Double immunodiffusion. The concentrated eluatesrecovered from the gels were analyzed by doubleimmunodiffusion in 1% agar and were buffered with

-~~~:.0

FIG. 1. Comparison of proteini-stained profiles ofM. tuberculosis culture filtrate samples as obtainiedby the coniventionial disc and the disconitiinuouis poregradient electrophoretic methods. Samples were sub-jected to electrophoresis in gel columnis as follows(total gel contcenitrationi followed by gel heighit):A, 4.75%l' (6.5 cm); B, 7%l (6.5 cm); C, 4.75%c (8.5cm); D, 7% (8.5 cm); E, four-stage disconitinuouspore gradienit polyacrylamide gel.

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AFFRONTI, WRIGHT, AND REICH

sodium diethylbarbiturate, pH 8.2. Plastic templateswith wells 0.2 cm in diameter and 0.5 cm equidistantfrom each other were placed on glass plates. When thegel had hardened, the concentrated eluates and theantisera were placed into the wells. The units werethen placed in a humidified chamber at room tem-perature until the precipitin pattern had developed.The plates were stained with amido black for pro-teins.

RESULTS AND DISCUSSIONWith the introduction of a polyacrylamide

electrophoretic multistage system for the sepa-ration and isolation of mycobacterial antigens, asimple method is available for the improvedseparation and resolution of mycobacterialproducts. Also, by utilizing various differentialstaining procedures, profiles of mycobacterialstrains can be established for comparison, thusallowing a more refined characterization oftheir interspecific relationships.A comparison with the conventional disc

electrophoresis procedure of Ornstein andDavis (4) which uses gel columns of singleconcentrations is given in Fig. 1. A 300-Agamount of protein was applied and after elec-trophoresis was stained for protein with 0.05%Coomassie Blue (Colab, Chicago, Ill.). It can benoted that increased resolution and separationof the mycobacterial cell extract constituentsare achieved by electrophoresis in a pore gradientpolyacrylamide gel system. Even a modificationof the conventional disc electrophoresis pro-cedure, wherein the length of the gel column wasincreased to the same gel length as utilized in the

l 2 3 4

pore gradient gel system, resulted in neitherbetter separation nor better resolution of themycobacterial samples. Because of these find-ings, the multistage method was selected as theprocedure of choice for this study.

Electropherograms of culture filtrates andcell extracts from representative mycobacterialstrains (M. tuberculosis H37Ra, M. tuberculosisH37Rv, M. phlei, and M. fortuitwn) stainedfor protein by Coomassie Blue are given in Fig. 2.An increased number of protein-stained com-ponents was generally evident in cell extractswhen they were compared with their respectiveculture filtrate samples containing an equivalentamount of protein (300 ,Ag), as can be noted inTable 1. It could be demonstrated, however,that the protein-stained components of theoriginally electrophoresed culture filtrate couldbe made comparable in number and positionwith those of its respective cell extract samples.This was done by simply effecting a 100-foldgreater concentration of the culture filtratesamples and adjusting the protein concentrationoptimally so that comparable profiles resulted.This indicated that there are several componentsin the originally electrophoresed culture filtratesamples which were below the sensitivity levelof the protein stain; but when a greater con-centration was effected, detectable levels wereattainable. No increase in the number of stainedbands could be detected when the cell extractsamples were similarly concentrated and sub-jected to electrophoresis. In fact, some com-ponents coalesced to form broader bands, as a

5 6 7 8FIG. 2. Representative electropherogram of nonlyophilized culture filtrate and cell extracts from various myco-

bacterial strains. (1 and 2) M. tuberculosis H37Ra culture filtrate and cell extract, respectively; (3 and 4) M.tuberculosis H37Rv culturefiltrate and cell extract, respectively; (5 and 6) M. phlei culturefiltrate and cell extract,respectively; (7 and 8) M. fortuitum culture filtrate and cell extract, respectively.

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VOL. 5, 1972 CHARACTERIZATION OF MYCOBACTERIAL ANTIGENS. I

result of column overloading, and thus yielded aprotein profile with a decreased number of totalbands.

It can also be seen from Fig. 2 that the ma-jority of the culture filtrate and cell extractcomponents are of low molecular weight sincethey migrate and separate in the small-pore gels(12%). The possibility that the fast-movingcomponents, i.e., those found in the small-poregel, might represent highly charged substances oflarge molecular weight can be discounted, par-ticularly in view of the report of Tombs (7)which convincingly demonstrated that the mo-bility of a protein in a gel is related to the pro-portion of pores larger than the limiting size.

Therefore, the position of the components withinthe gel can be accounted for mainly on the basisof a pore filtration mechanism instead of chargedifferences.The number of protein, PAS, nucleic acid, and

ORO-positive staining components demon-strated by the differential staining technique foreach of the cell extracts studied is summnarizedin Table 2. These data reflect the finding thatonly a small number of protein-staining com-ponents migrated into the 3.50, 4.75, and 7%gel sections. This small number of components(from 1 to 13) probably represents molecules oflarge size since they are retarded by gels of largeporosities.

TABLE 1. Number of mycobacterial components separated by electrophoresis on polyacrylamide gel

Mycobacterium M. tuber- .tuberculosis culosis M. avium M. bovis hf. |. scrofu- M. ntra- Mp.i

Component H37Ra H37Rv kansasii laceumn cellulare fortuitum M.liistained for

CEa CF CE CF CE CF CE CF CE(CF CE CF CE CF CE CF CE CF

Protein... 50 47 47 42 47 42 55 44 52 38 56 39 51 48 58 48 61 52Carbohy-I

drate 8 12 8 8 8 6 5 7 16 20 8 3 10 10 5 9 7 9Lipid.. 8 10 10 11 6 2 9 12 15 6 15 7 9 8 6 7 12 10NucleicI

acid 2 2 3 1 1 1 3 4 2 3 2 2 22 1 2 1

a CE, cell extract; CF, culture filtrate.

TABLE 2. Distributioni of the ntumber of componienzts inl mycobacterial cell extracts developed by variousstainis in polyacrylamide gel segmentts of differenit porosities

No. of components

Component :BMycobacterium M.tuberculosis tuberculosis M. bovis At. avium Mf. kansasii M. M ieH37Ra H37Rv intracellulare M. phi

Protein3.5%/'a Segment 3 1 1 2 2 2 34.75% Segment 10 10 12 4 7 10 137.00% Segment 11 11 13 11 13 13 1012.00% Segment 26 25 29 30 30 31 35

Carbohydrate3.5% Segment 1 1 1 2 2 2 34.75% Segment 2 1 1 1 5 2 17.00% Segment 1 2 2 0 6 0 312.00% Segment 4 4 1 5 3 1 1

Lipid3.5% Segment 1 1 1 2 2 2 34.75% Segment 2 1 1 1 5 1 17.00% Segment 1 2 4 0 5 2 412.00% Segment 4 6 3 3 3 1 4

Nucleic acid3.5% Segment 0 0 0 0 0 0 04.75% Segment 0 0 0 0 0 0 07.00% Segment 0 0 0 0 0 0 012.00%O Segment 2 3 3 1 4 2 2

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AFFRONTI, WRIGHT, AND REICH

PAS-positive components and those whichstained for lipid were detected in each of thefour polyacrylamide gel sections (3.50, 4.75,7, and 12%,). Most of the PAS-positive andORO-positive components could be correlatedwith the protein-staining components, indicatingthat they were probably glycoprotein or muco-protein lipid complexes. All mycobacterial cul-ture filtrates and cell extracts analyzed containedPAS-positive and ORO-positive substances.The fact that the PAS-positive components weredistributed in gels of four different pore sizeswould suggest that these carbohydrates are ex-tremely heterogeneous, ranging from complexesof large molecular weight to those of smallershape and size. Moreover, as demonstrated bythe specific gallocyanin RNA stain, RNA wasonly found in the 12% gel stage (Table 2).These data support the view, therefore, thatboth culture filtrates and cell extracts containvarious chemical components of widely differingmolecular sizes. Allowing molecules to be driventhrough gels of progressively decreasing poresize, such as occurs in the system described above,not only greatly enhances the resolution of thesecomponents but also suggests that this methodmight be a useful tool for molecular size determi-nations. In other words, it would be expectedthat large molecules would be retarded whilesmaller ones would migrate relatively unim-peded, depending upon the pore size of the gel.The relative distance that the components wouldmigrate through the gels thus would serve asanother criterion for further characterization ofprotein mixtures. In ordei to test this possibility,bovine serum albumin standards of known mo-lecular weight varying from 67,000 to 268,000(Table 3) were selected and subjected to elec-trophoresis in a single gel system containing 7, 9,and 12% polyacrylamide to compare theirmobilities in these gel concentrations. For thistool to be valid, a linear function relationshipbetween relative mobility and gel concentrationis required. As shown in Fig 3, an approximatelinear relationship exists in the region of theacrylamide concentration between 7 and 12%.

TABLE 3. Stanidards uised jfr polyacrylamide gelelectrophoresis

Alolec-Protein and symbol ular

weight

Bovine serum albumin BSA-1. 67,000(BSA)

Dimer BSA-2........ 134,000

Trimer BSA-3 210,000

Tetramer BSA-4 ........ 268,000

RELATION BETWEEN GEL CONCENTRATIONAND RELATIVE MOBILITY

1.0 BSA-1

0.9

>-0 BSA-2t- 0.8 A-JO 0.70*

0.6 BSA-3>-0.5\

QABSA-40.3

0.2

0.1Gel conc. gm/100mI

7 8 9 10 11 12PERCENTAGES

FIG. 3. Relationt betweeni gel conicenitrationt anidrelative mobility.

It can be seen that as the pore size decreases, thelarge-molecular-weight proteins exhibit a slowermobility than the smaller proteins. Relativemobility versus very low (<7%r) or very high(> 12%) gel concentrations did not result in astraight line relationship, thus invalidating theuse of gel concentrations in these extreme ranges.Therefore, within certain ranges, this demon-strates a relationship between molecular size andmobility in gels of different concentrations andpermits the use of migration distance, or theconventional concept of RF, as one possiblecriterion for characterizing protein componentsof a mixture separated by gel electrophoresis.Moreover, as was previously reported by Marcha-lonis (3), this method also provides a simplemeans of estimating molecular weights of pro-tein in a mixture.The applicability of using mobilities or R,

values in polyacrylamide gel electrophoresis asan aid in identifying similar or identical proteincomponents which possibly would result incharacteristic band patterns for each species andthus serve to complement the more conventionalmethods of bacterial classification appeared tobe of limited value in this study. In an attemptto eliminate the subjectivity involved in visuallyreading these stained patterns, densitometrictracings were performed using a Gilford densi-tometer. Figure 4 gives representative tracingsfrom three mycobacterial species including M.tuberculosis H 37Ra, the reference strain. Fiftypeaks corresponding to the total number of com-ponents separated by electrophoresis were easilynoted. Although differences in strains were dis-cernable with this technique, they were subtle

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VOL. 5, 1972 CHARACTERIZATION OF MYCOBACTERIAL ANTIGENS. I

M. tuberculosis H37RaCel I - Extract

3 f 4% 7% 12%

FIG. 4. Representative densitometric tracintg ofM. tuberculosis H37Ra, H37Rv, anid M. bovis Percenit-ages inzdicate gel conicenitratioli.

and their reproducibility was less than ideal.As will be described in the following paper, how-ever, a method is now available which we believehas considerable merit in permitting one toestablish definitive characteristic patterns formycobacterial species and which could possiblybe used as an aid in their classification.

Figure 5 is a schematic drawing which repre-sents the electropherograms and RF values offour strains of mycobacterial culture filtratesthat were previously lyophilized and the proteincomponents stained with Coomassie Blue. Ascan be noted, lyophilization apparently altered

the electrophoretic protein patterns since thetotal number of stained components was reducedsignificantly, especially in the 3 and 4%i, gelregions, from that observed in the nonlyophilizedculture filtrate samples (compare with Fig. 2).however, lyophilization did not reduce the majorprotein-stained components since these remainedvisually prominent in both nonlyophilized andlyophilized samples. Although differences inprotein profiles are present among these myco-bacterial species either due to a direct deleteriouseffect of the lyophilization process on susceptibleprotein components or due, in fact, to the exist-ence of true protein compositional differences, itis clear that striking similarities are evident whenthe major components selected on the basis ofvisual prominence in corresponding gel segmentsare compared. 1 his similarity in certain cor-responding gel segments among culture filtratesfrom different mycobacterial species is also re-flected by the presen.ce in these filtrates of com-ponents with identical or nearly identical RFvalues.

However, without a means of estimating thenormal variation in RF values from trial to trial,this approach would only have validity providing,as Rouatt et al. (5) have recently suggested, thathomologous bands could be identified withcertainty. Thus, if the migration of a protein in agel derives essentially from a pore filtrationmechanism, as Tombs proposes (7), then pro-teins with the same RF values would be expectedto be identical. This expectation was proven inthe course of attempting to characterize theprotein component in culture filtrates from fourmycobacterial species which migrated into thelower portion of the 4.75% gel segment and hadan average RF value of 0.30. This band wasconsidered the ideal candidate for selection fortwo reasons: first, it appeared as a single com-ponent devoid of any neighboring bands andthus, from a practical viewpoint, was relativelyeasily sectioned out; and secondly, it was presentin all culture filtrates and extracts analyzed, thatis, it can be considered as a very likely candidatefor a possible "common antigen." Precipitintests were performed on corresponding pooledgel segments to establish the presence or absenceof antigenicity for the total components withineach of the major gel portions, i.e., the 4.75,7, and 12%o gel segments. Precipitin tests werepositive with the highest concentration (50 ,g)of the eluates of electrophoresed culture filtratesamples from pooled 4.75c% gel segments of M.tuberculosis H37Ra as test antigen against itshomologous antiserum. Eluates from the pooled7 and 12%C gel segments also gave positive reac-

tions, thus confirming the antigenicity of all of

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AFFRONTI, WRIGHT, AND REICH INFECT. IMMUNITY

SCHEMATIC DIAGRAM AND Rf VALUESOF FOUR STRAINS OF MYCOBACTERIA

5056

58

60

6467 70727579

809496

Myco. Myco.kansasii scrofulaceum

42

-- 6-656

62

67

gi74

82

84

94 9297

Myco. Nintracelluare tuber

FIG. 5. Schematic diagram antd RF values offour straiis of nmycohacleria.

interspecific relationships for this "common-= band" having almost identical RF values. It can

be seen from Fig. 6 that this component gavereactions of identity for each of these myco-bacterial species, thus confirming immunobio-logically the results obtained physicochemically.The component was found in the 4.75%cc gelsegments of all the mycobacterial and culturefiltrates and extracts analyzed, and therefore itcan be considered a common antigen. It shouldbe pointed out that re-electrophoresis of eachof the common bands again yielded single com-ponents. The characterization of this antigenand its possible relationship with the commonantigen which was present in all mycobacterialspecies which Castelnuovo (2) examined andreported will be the subject of a later communi-cation.

ACKNOWLEDG MENTS

FIG. 6. Gel diffusioni resuilts of culture filtrate We gratefully acknowledge the technical assistance of Lindauates h1avinlg nlearly identical RF values, from tour Grow, Dianna Rober-ts, Karen Far-rell, and Mary Gaffney.rames of mycobacteria.Thde elucltes gave reaction1 of This research is supported by Public Health Service gr-antsrai*sofycbatera.Theltaes av ractoto A108529 and A107099 from the National Institute of Allergy and'entity indicaiting a "comimoni anitigen." Well nto. ' Infectious Diseases, and by a grant from The John A. Hartfordf. tuiberculosis H37Ra; well nio. 2. M. kansasii; Foundation, Inc.ell no. 3, M. scrofiilaceum; well no. 4, M. initra-lllare; center well, guin?ea pig anti-H37Ra serum. LITERATURE CITED

these fractions. After proof of their antigenicity,immunodiffusion studies were done with culturefiltrate eluates from the pooled 4.75c%, gel seg-

ments from each of the four different myco-

bacterial strains, M. tuberculosis H37Ra, M.kansasii, M. scrofulaceum, and M. intracellulare.This was done in an attempt to establish possible

1. Affronti, L. F. 1959. Purified protein derivatives (PPD) andother antigens prepared from atypical acid fast bacilli andNocardia asteroides. Amer. Rev. Resp. Dis. 79:284-294.

2. Castelnuovo, G., S. Yamanaka, M. Zoppis, and E. Dotti.1969. Protein components of Mycobacterium phlei: frac-tionation procedures. Tubercle 50:194-202.

3. Marchalonis, J. J. 1969. Isolation and characterization of im-munoglobulin-like proteins of the Australian lungfish.Aust. J. Exp. Biol. Med. Sci. 47:405-419.

480

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-98

/lyco.rculosis37Ra

elizstiidiMwcce

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VOL. 5, 1972 CHARACTERIZATION OF MYCOBACTERIAL ANTIGENS. I

4. Ornstein, L., and B. J. Davis. 1961. Disc electrophoresis.Distillation Products Industries, Rochester, N.Y.

5. Rouatt, J. W., G. W. Skyring, V. Pukayastha, and C. Quadling.1970. Soil bacteria: numerical analysis of electrophoreticprotein patterns developed in acrylamide gels. Can. J. Micro-biol. 16:202-205.

6. Seibert, F. B., and L. F. Affronti. 1963. Analytical chemicalmethods, p. 1-33. Iii R. Parlett (ed.), Methodology manualfor investigation of mycobacterial and fungal antigens.American Thoracic Society, New York.

7. Tombs, M. P. 1965. The interpretation of gel electrophoresis.Anal. Biochem. 13:121-132.

8. Wright, G. L., Jr., and L. F. Affronti. 1968. Gradient poly-acrylamide electrophoresis of mycobacterial cell products.Amer. Rev. Resp. Dis. 98:153.

9. Wright, G. L., Jr., K. B. Farrell, and D. B. Roberts. 1971.Gradient polyacrylamide gel electrophoresis of human serum

proteins: improved discontinuous gel electrophoretic tech-nique and identification of individual serum components.Clin. Chim. Acta 32:285-296.

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