INFEcrION AND IMMUNITY, Aug. 1975, p. 387-397Copyright 0 1975 American Society for Microbiology

Vol. 12, No. 2Printed in U.S.A.

Serological Studies of Actinomyces israelii by CrossedImmunoelectrophoresis: Standard Antigen-Antibody System

for A. israeliiKENNETH HOLMBERG,* CARL-ERIK NORD, AND TORKEL WADSTROM

Department of Bacteriology, the National Bacteriological Laboratorv, Stockholm, Sweden

Received for publication 6 February 1975

Standard preparations of crude cytoplasmic and whole cell-associated antigenmixtures of Actinomyces israelii were analyzed by crossed immunoelectrophore-sis (CIE), with a standard polyvalent antiserum comprising purified and concen-trated immunoglobulin G antibodies to formolized whole cells of A. israelii sero-types 1 and 2. The standard antigens provided four antigen-antibody systems forA. israelii. The immunoprecipitation patterns of the system were compared, andthe immunochemical characteristics of individual precipitates were analyzed.Each system contained specific precipitates, but also one or two precipitateswhich were immunochemically identical to precipitates of the other systems. Thestandard system for A. israelii based on cytoplasmic antigens was best reproduci-ble and revealed the highest number of immunoprecipitates. These precipitatespossessed immunochemical properties which made them suitable for CIEstudies. The cytoplasmic antigen mixture of A. israelii was, therefore, adopted asthe most suitable for further development of a crossed immunoelectrophoreticsystem for A. israelii. In subsequent assays the cytoplasmic antigen mixture wasanalyzed by CIE with a standard antiserum (StAbII) prepared from rabbit antiseraraised in rabbit against cell lysates of A. israelii, serotypes 1 and 2. A standardantigen-antibody system for A. israelii was obtained which revealed an immuno-precipitation pattern of 10 distinguishable precipitates. The resolving power andseparation by CIE of this standard system for A. israelii was compared with thatof crossed immunoelectrofocusing. The results suggest that these methodssupplement each other. Crossed immunoelectrofocusing appeared to be a usefultool for separation of specific components of the protein-antigen complex of A.israelii for analytic serology. The CIE in conjunction with a standard referenceantigen-antibody system for A. israelii based on cytoplasmic antigens offers greatpotentialities in diagnostic A. israelii serology.

The cellular antigens of Actinomyces israeliihave been poorly defined. Crude and partlypurified antigen preparations have been com-monly used for serological studies (6, 23, 24, 25,40). An account of the analytical serology of A.israelii was recently given in a review byBowden and Hardie (6). They reported that theantigens in acid-extracts and culture superna-tant fluids could be divided into two groups.Antigens of group 1 mainly consisted of non-precipitating carbohydrates associated withthe cell walls. Antigens belonging to group 2were mainly soluble proteins sensitive to treat-ment with proteases. The antigens of group 1were reported to evoke a poor immunologicalresponse in rabbits, whereas those from group 2elicited antibodies detectable in gel diffusiontests. Both groups of antigens were reported tobe reasonably species specific. However, exten-

sive cross-reactions have been shown by im-munofluorescence (8, 17, 25) and gel diffusiontests between whole cells or crude antigenpreparations of A. israelii and antisera raisedagainst other species of Actinomyces and Pro-pionibacterium acnes (6, 25).

Although immunodiffusion tests have beenused for analysis of A. israelii antigens, thistechnique is probably not sensitive enough forstudying complex antigen mixtures. Crossedimmunoelectrophoresis (CIE) (26, 39) offersgreater possibilities in such studies for thecharacterization of crude cellular antigens of A.israelii without further purification.The aim of this investigation was to analyze

various A. israelii antigen preparations as possi-ble reference antigens for the development of astandardized antigen-antibody system for A.israelii for CIE studies.

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MATERIALS AND METHODS

Bacterial strains. Type strains of A. israelii sero-

type 1, ATCC 12103 and ATCC 10048. and serotype 2,ATCC 27036, were used for the production of'standardantigens (7, 8). In addition, antigens were preparedfrom two strains of A. israelii serotype 2 (WVU 307and Holm 1452/40-41). kindly provided by M. Ge-rencser, West Virginia University, Morgantown, W.Va., and Per Holm, Statens Seruminstitut, Copenha-gen, Denmark. One strain, SBL 225/74, isolated andtyped as A. israelii by criteria devised by K. Holmbergand C.-E. Nord (J. Gen. Microbiol., in press). was

also used for antigen preparation.Antigen preparation. Strains of A. israelii were

grown to cell densities of 0.3 to 0.5 g/liter in a

prereduced, anaerobic sterilized (PRAS) peptone-yeast-glucose broth according to Holdeman andMoore (16) in an atmosphere containing 100% CO2 at37 C, harvested by centrifugation (4,000 x g, 4 C, 20min), washed with saline, and stored at -20 C untilused.

Cytoplasmic and whole cell-associated antigenswere prepared from batches of 5 g (wet weight) ofwashed cells as shown in Fig. 1.

Cytoplasmic antigens: aqueous extract of soniclysate. The washed cells, diluted 1:5 (vol/vol) withdistilled water, were disintegrated for 20 x 45 s at20,000 Hz/s, using a Braun-Sonic '300S (B. Braun,Melsungen, West Germany) 19-mm probe with a

9.5-mm tip, cooled with ice water. This treatmentyielded a cell disruption of' approximately 90'>. Thedegree of cell disruption was confirmed by examina-tion of' Gram-stained smears and electron microscopy.The cell debris was removed by centrif'ugation at10,000 x g for 10 min at 4 C. The cell lysate was

f'urther centrif'uged for 60 min at 105,000 x g at 4 C in

(1)

(2)

(3) Sonication(20,000 Hz/s:20 x 45 s)

(4) Centrifugation(10,000 X g:30 min)

Discard pellet

(5) Ultracentri-fugat ion(10,500 g:l h)

(6) Supernatant(frozen 20 Ccytoplasmicfract ion)

(7)

a Beckman L2-65B preparative ultracentrifuge withSW40 rotor, and the resulting clear fraction of thesupernatant fluid constituted the cytoplasmic anti-gens of' A. israelii.Whole cell-associated antigens. (i) Urea ex-

tract. The washed cell pellet of whole cells wasdiluted 1:3 (vol/vol) with 4 M fresh urea solution. Themixture was homogenized for 1 to 2 min on a Vortexmixer and stirred at 4 C for 16 h. The suspension wascentrifuged (10,000 x g, 4 C, 30 min), the pellet wasdiscarded, and the supernatant fluid was dialyzedagainst several volumes of buffer (0.2 M sodium phos-phate, pH 7.2) for 16 h.

(ii) Hydrochloric acid extract. The washed cellswere suspended in 10 ml of 0.2 M HCl, homogenizedon a Vortex mixer, and heated in boiling water for 10min. The suspension was cooled at room temperature,and the pH was adjusted to 7.2 with 0.2 M NaOH.The cell debris was sedimented by centrifugation(5,000 x g, 4 C, 15 min), and the supernatant fluidwas dialyzed as above.

(iii) Trichloroacetic acid extract. The washedcells were extracted with 20 ml of' 10% (vol/vol)trichloroacetic acid. The extraction was performed at55 C in a water bath for 15 min, followed by cooling inan ice-water bath and centrif'ugation (5.000 x g, 4 C,15 min). The supernatant was then decanted andstored f'rozen ( 20 C). The cell debris was resus-pended in 10 ml of' distilled water, and the centrif'uga-tion was repeated. The supernatant fluids were pooledand shaken with a double volume of acetone for about60 min. The precipitate which formed was removed bycentrif'ugation (5,000 X g, 4 C. 15 min). The superna-tant fluid was decanted, and the acetone was evapo-rated from the precipitate on f'ilter paper. The precipi-tate was then dissolved in distilled water, and the pHwas adjusted to 7.2 with 0.2 M NaOH.

Cultivation (PRAS PYG broth:5 davs:.37 C)

Centrit'ugation (4,000 X g:20 min)

Extraction(0.2 M HCI: 100 C: 10 mini

Centrif'ugat ion(5,000 x g:15 mini

Discard pellet

Dial,sis(0.2 M NaH2PO4:pH 7.2)

Dialvsate(frozen -20 C)

Ext ract ion(10T TCA:55 C: 15 min)

Centrif'ugation(5,000 ( g:15 min)

Discard pellet

Precipitation(acetone:60 min)

Centrif'ugat ion(4,000 x g:30 min)

Extraction(4 M urea:4 C:16 h)

Centrit'ugat ion(10,000 x g:30 min)

Discard pellet

Dialvsis(0.2 M NaH2PO4:pH 7.2)

Dialysate(frozen -20 C)

Precipitatedissolved in distilledwater and frozen: -20 C

StAgSL StAgHCl StAgTCA StAgureaFIG. 1. Flow scheme for preparation of cytoplasmic and whole cell-associated antigens of Actinomvces israelii.

StAg: Standard antigen designation; see text. TCA: Trichloracetic acid; PRAS PYG broth: prereducedanaerobic-sterilized peptone-yeast-glucose broth.

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Characterization of antigens. The protein con-centration of the antigen preparations was deter-mined by the method of' Lowry et al. (27), or bymeasurements at 280 nm in a Zeiss PMQ spectropho-tometer using bovine serum albumin as a standard.The carbohydrate concentration of the antigen mix-tures was determined by the phenol-sulf'uric acidreaction with glucose as standard (28). For quantita-tive determination of hexoses, the cysteine-sulf'uricacid method was used modif'ied as described byWright and Rebers (41).Enzvmic digestion of' antigen preparations was

performed with trypsin (trypsin 2x crystallized,Sigma Chemical Co., St. Louis, Mo.) and Pronase(Grade B, Sigma) in 0.05 M sodium phosphate buffer(pH 7.2) at 37 C for 18 h.

For chemical characterization of' the immuno-precipitates. duplicate gels were stained with Coo-massie brilliant blue R (ICI, Manchester. England)for protein (14) and periodic acid-Schiff reagent forglycoproteins and polysaccharides (29).

Standardization of antigens. Equal volumes fromdifferent batches of antigen preparations were pooledand concentrated by dialysis against polyethvleneglycol (molecular weight 20.000) (Kebo AB, Stock-holm, Sweden). These mixtures represented the poly-valent standard A. israelii antigens and were codedStAgSL (sonic lysate). StAgurea. StAgHCl. andStAgTCA. All standard antigens were stored at 20 Cor lyophilized. Lyophilized antigens were reconsti-tuted in phosphate-buffered saline (pH 7.2).

Immunogens. For immunization, both formalin-treated cells (10%7c vol/vol, 1 h) and sonic cell lysates of'A. israelii, serotype 1 (ATCC 12103) and serotvpe 2(WVU 307), were used. Suspensions were prepared of'(i) formolized whole cells, containing approximately 1x 106 cells/ml and of (ii) crude cell lysates to an opticaldensity of 0.2 at 540 nm.Preparation of standard antisera. Antisera to A.

israelii were prepared as f'ollows. Rabbits were immu-nized by intravenous injections of the immunogens.The rabbits received 1 ml of the immunogens twice aweek for 5 weeks and were bled 1 week after the lastinjection. Test bleedings were made prior to theimmunization to check the absence of' preimmuneantibodies against the immunogen and during theimmunization. Two pools of antisera, one againstformolized whole cells (StAbI) and one against celllysate (StAbIl). were produced. Each pool containedantisera from 9 to 10 rabbits raised against ATCC12103 and antisera from the same number of rabbitsraised against WVU 307. The immunoglobulin frac-tion of the pooled antisera was obtained by precipita-tion with ammonium sulfate (37% saturation, 4 C).The precipitate was redissolved in and dialyzedagainst 0.017 M sodium phosphate (pH 6.3). Furtherpurification was achieved by DEAE-cellulose columnchromatography. The qualitative demonstration ofimmunoglobulins was done by immunoelectrophoresisagainst goat anti-rabbit plasma protein serum (Beh-ringwerke AG, Marburg-Lahn, Germany). Purifiedimmunoglobulin G was concentrated by ultrafiltrationon a Sartorius membrane filter (SM 13200; Sartoriusmembrane filter, BmbH Gottingen, Germany) to afinal concentration of 20 mg/ml. The two standard

immunoglobulin G pools were stored frozen with 15mM NaN3 as preservative.

Experimental procedures. CIE was performed asdescribed bv Weeke (38), and in the modificationstandem-crossed immunoelect rophoresis and crossed-line immunoelectrophoresis as devised by Kroll (20,21) were used. Tris(hydroxymethyl)aminomethane-barbital buffer (P. J. Svendsen, personal communica-tion) was used: 5.5-diethylbarbituric acid (Barbital)(Merck). 22.4 L: tris(hvdroxvmethvl)aminomethane(Sigma), 44.3 g; calcium lactate, 0.533 g; NaN3, 0.65g; distilled water, 1,000 ml; pH 8.6, diluted 1:4 for use(ionic strength, 0.02). A 1% (wt/vol) agarose solution(Miles Seravac, Berks, England) dissolved in tris-(hydroxymethyl)aminomethane-barbital buffer waspoured onto glass plates to give a gel of 1 mm. Electro-phoresis was performed at 12 C in the first dimension,applying 8 to 9 V per cm for 60 min, followed bv elec-trophoresis in the second dimension with the samefield strength for 90 min or 1 to 2 per cm overnight.From preliminary experiments with varying quantitiesof antigen preparations and antisera, 5 ul of antigenand 5 gl of antisera per cm2 of' gel were used in the ex-perimental system. The gels were washed with 0.1 MNaCl and distilled water (38). After drying, the plateswere stained for proteins in a solution of 0.5% (wt/vol)Coomassie brilliant blue R in ethanol-acetic acid-water. Excess dye was removed by washing in dye-freesolvent (14). Carbamylated human transferrin (39) orhuman albumin (2) was used as a reference standardto study the reproducibility of the CIE experiments.Human serum albumin was purchased from Kabi AB,Stockholm, Sweden, and human transferrin was ob-tained from Behringwerke AG, Germany. Rabbitantisera against human transferrin and albumin werepurchased from Dakopatts A/S, Copenhagen, Den-mark.

Double immunodiffusion was carried out accordingto Ouchterlony (30) and conventional immunoelectro-phoresis according to Grabar and Williams (13).Rocket electrophoresis into agarose containing A.israelii antiserum was performed as described byLaurell (26). Thin-layer isoelectric focusing on poly-acrvlamide gel (IFPAG) (36. 37) was conducted atconstant wattage (60 W) according to Soderholm etal. (32) and Soderholm and Wadstrom (34). Carrierampholytes (Ampholine, LKB-Produkter, Bromma,Stockholm, Sweden) were used in a f'inal concentra-tion of 2.0w (wt/vol) to establish the pH gradients.The antigen preparations were subjected to focusingin the pH range 3.5 to 10.0 and 3.5 to 5.0. Gels werefixed and stained as previously described (34). Isoe-lectric focusing in the first dimension in combinationwith electrophoresis into an antibody-containing aga-rose gel in the second dimension (crossed immuno-electrofocusing) was performed as described by Soder-holm and Smvth (33). After separation by IFPAG thegel was sliced, and the strip containing the antigenpreparation was turned upside down and applied onthe top of an agarose antibody-containing gel. Elec-trophoresis was then performed for 3 h with a fieldstrength in the antibody-containing gel of' 10 V/cm.

Chemicals. All chemicals were of analytical gradeunless otherwise stated. Peptone and yeast extractwere purchased from Difco. The salts and constitu-

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390 HOLMBERG, NORD, AND WADSTROM

ents for buffers were obtained from Merk AG, Darm-stadt, Germany.

RESULTSAntisera raised in rabbits after intravenous

immunization with formolized whole cells of A.israelii formed precipitates in CIE experimentsagainst cytoplasmic and whole cell-associatedantigens of A. israelii. As considerable varia-tions in the immunoprecipitation patternagainst the same antigen preparation were ob-served with one antiserum to the other, a stand-ard antibody pool against A. israelii was pre-pared. This polyvalent antibody pool, codedStAbI, provided the reference antibody stand-ard employed in primary attempts to develop astandard antigen-antibody system for A. israeliiby means of CIE.

Antigen preparations made by the same pro-cedure from the same strain of A. israeliiyielded different immunoprecipitate patternswith the standard antiserum. Polyvalent stan-dard antigens designated StAgSL, StAgHCl,StAgTCA, and StAgurea of A. israelii weretherefore prepared. Each standard antigen con-tained two different batches of antigen prepara-tions by each extraction procedure from sixstrains of A. israelii (ATCC 12103, ATCC 10048,WVU 307, Holm 1452/40-41, SBL 225/74,ATCC 27036). Each standard antigen was con-centrated to contain 4 to 6 mg of protein per ml.These standard antigens gave four differentantigen-antibody systems for A. israelii with thereference standard antiserum. These systemscontained a spectrum of immunoprecipitationarcs which varied in resolution when the anti-gen-antibody ratio was altered. In a series ofCIE the concentrations of the reactants werevaried over 10-fold ranges to yield the bestresolution of the complex antigen-antibody sys-tems. As a result of these chess-board titrationsof the reactants it was found that an Ag/Abratio of 5-Al sample of undiluted StAg to 5 ul ofStAb/cm2 of gel or 5-Al sample of StAg diluted1:2 to 2.5 ul of StAb/cm2 of gel revealed themaximal number of precipitates for all the sys-tems. When the immunoelectrophoreses wererun with these ratios between the antigen andthe antibody amount, with purified human al-bumin or carbamylated transferrin as an inter-nal reference marker, the system based onStAgSL contained six precipitation arcs, codedSL1 to SL6 (Fig. 2A), and the systems based onStAgHCl and StAgurea each contained five pre-cipitation arcs, coded HCll to HCl5 and urealto urea5, respectively (Fig. 2B, C). The systembased on StAgTCA consisted of only one pre-cipitate, coded TCA1. The antigen-antibody

system based on StAgSL appeared to be thebest reproducible system. All precipitates weredemonstrable on the anodic side of the applica-tion well. The precipitation arcs in the differentsystems were numbered from the anodic side ofthe first-dimensional electrophoresis.The immunochemical relationship between

the antigenic components of the standard anti-gens was analyzed by tandem-crossed immuno-electrophoresis and cross-line immunoelectro-phoresis in the modification called absorption ofantibodies in situ (3-5). Several immunochemi-cally identical antigenic components were de-tected in the standard antigens, indicated byfusion of the precipitates, identical electropho-retic mobility, and identical precipitate mor-phology in tandem-crossed immunoelectro-phoresis experiments (Fig. 3A to F).Two of the antigenic components of StAgSL

showed total or partial identity to componentsof the other standard antigens. Thus, compo-nent SL3 showed identity to HC14 and urea3,whereas component SL6 showed identity toHC15, urea5, and TCA1.The identity of these antigenic components

was confirmed in crossed-line immunoelectro-phoretic experiments. In separate experiments 5,ul each of the standard antigens StAgHCl,StAgurea, and StAgTCA per cm2 were includedin a gel strip, placed along the first-dimensionalelectrophoresis gel bearing 5 ,ul of separatedStAgSL, and subjected to electrophoresis to-gether into an agarose gel containing the refer-ence antiserum. The test plates were comparedwith a control plate with no antigen in the gelstrip. In test plates with StAgHCl and StAgureain the gel strip, the precipitates representing thecomponents of StAgSL, coded SL3 and SL6,were lacking, whereas the remaining precipi-tates were identified as antigenic componentscoded SL1, SL2, SL4, and SL5. Thus, thesecomponents appeared to be specific for StAgSL.In similar experiments, StAgHCl and StAgureawere found each to possess three specific anti-gens. These were identified as the antigeniccomponents coded HCl1, HCl2, HCl3 andureal, urea2, urea4. It appeared as a result ofthe above experiments that a total number of 12immunochemically different antigenic compo-nents could be detected by CIE in the standardantigens of A. israelii. The number of antigeniccomponents (precipitates) of the individualstandard antigens and their immunochemicalrelationship are shown in Table 1.The possible presence of cathodically migrat-

ing antigenic components of the standard anti-gens was controlled by an experimental tech-nique similar to that of Ganrot (12). The

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A. ISRAELII ANTIGENS IN IMMUNOELECTROPHORESIS

A+

SU.

SL2t SL

B

+1cm

C ++

HCI3 4 urea2./,H12 ~~urea4 / ureel4HCIIjurea5j /

-IN

+1c - ure a37-1 M §

TCAI

0 t

FIG. 2. Crossed immunoelectrophoresis of 5 lI of the standard antigens (A) StAgSL, (B) StAgHCl, (C)StAgurea, and (D) StAgTCA of A. israelii. The second-dimension gel contained 5 gl of a rabbit standardantiserum to A. israelii, StAbI, per cm2. The first-dimension electrophoretic run was done at 9 V/cm for 50 min,and the second run was done at 2 V/cm overnight.

first-dimension gel slab was placed in the mid-dle of the antibody-containing gel. No suchantigens were demonstrable.The precipitating activities of the antigens ex-

cept for SL3 and SL6 were completely de-stroyed after 18 h of incubation with Pronasebut all were resistant to trypsin treatment. Heatstability tests yielded complete loss of precipi-tating activity upon heating at 80 C for 15 minat pH 7.2.Chemical analysis of the standard antigens

revealed differences in their content of proteinand carbohydrate. The carbohydrate-to-proteinratios, calculated on the basis of carbohydratesin hydrolysates of the standard antigens, rangedfrom 10.0 to 7.0. When these ratios were calcu-lated on the basis of the content of hexosepolysaccharides, the ratio for StAgSL was 20.0,whereas the ratios for the other standard anti-gens were almost the same as in the firstestimation.

Staining of gels containing immunoprecipi-tates of StAgSL with periodic acid-Schiff rea-

gent revealed two weak periodic acid-Schiff-

positive components, SL3 and SL6. Correspond-ing periodic acid-Schiff-positive componentswere detectable in gels containing immunopre-cipitates of the whole cell-associated standardantigens. These periodic acid-Schiff-positivecomponents also stained with Coomassie bril-liant blue, indicating that they were probablyglycoprotein complexes.On the basis of the immunochemical exami-

nation of the standard antigens, the standardpreparation of cytoplasmic antigens, StAgSL,was adopted as the most suitable for furtherdevelopment of the crossed immunoelectropho-retic system for A. israetii.A standard antiserum, StAblI, prepared from

a pool of rabbit antisera against cell lysates ofATCC 12103 and WVU 307, was in subsequentCIE experiments analyzed for antibodies withspecificity for antigenic components of StAgSL.After determination of the optimal ratio of thereactants for CIE, this antigen-antibody systemfor A. israelii revealed 10 antigenic componentsof StAgSL. The immunoprecipitation pattern ofthe system is shown in Fig. 4A. The precipita-

.

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392 HOLMBERG, NORD, AND WADSTROM

A B

Llrea4r.C 65 HC14 S6

A ,urea3HCI5.~~~ irea21$L6 H~~~~~CI2 SLG4\

J/

SL 7/ HCII SLS

SL5 SL2 SL

x Yx

.=

D E

-HCI 4HC15K HCI3

,A HCI2Lirea5 11C011urea6b2 /// /

\ 1f/// reai

ro Lire/

TCAl HC 14~~~/HC13

- /

11)0x Y

Li (.\312AI! cilTCA1 ,,/7;ea2

// irEi

r.

X Y

Fic(. ,3. Tandem-crossed immunoelectrophoretic comparison of four standard antigens of A. israelii. StAgSL,StAgurea, StAgHCI, and StAgTCA, using 5 pI of the standard antiserum to A. israelii, StAbII, per cm2 as

reference. Five microliters of the antigen preparations was placed in the application holes X and Y as follows:(A) X, StAgSL; Y, StAgHCI; (B) X, StAgSL; Y, StAgurea; (C) X, StAgSL; Y, StAgTCA; (D) X, StAgHCI; Y,StAgurea; (E) X. StAgHCI; Y. StAgTCA; (F) X, StAgurea; Y, StAgTCA.

TABLE 1. Number of antigenic components of the standard antigens of A. israelii and theirimmunochemical relationship

Standard antigens (StAg)aDetermination

StAgSL StAgHCI StAgurea StAgTCA

Number of ait igenic components 6 5 5 1

Component identical to: SLISL2

HC1lHC12HC13

urea 1urea2urea3

SL3 HC14 urea4SL4SL5SL6 HC15 ureaS TCA1

a Designations: see text.

tion arcs are counted from the anodic side anddesignated 1 to 10. The six antigenic compo-

nents of StAgSL detected in the system basedon StAbl. coded SLI to SL6. were also revealedin this system as indicated by intermediate gelCIE experiments (Fig. 4B). In descending orderthe antigenic components coded SLI to SL6corresponded to the antigenic components 1, 2,7, 8, 9, and 10 in the standard system for A.israelii based on StAbIl.

CIE was performed to investigate stain-to-stain variation in cvtoplasmic antigen prepara-

tions of' A. israelii. The number of antigeniccomponents varied from 1 to 10 for differentstrains of' A. israelii. All antigen preparations of'A. israelii. biochemically and in immunofluo-rescence designated as type 1, contained atleast f'ive antigenic components. Up to 10 dif'-f'erent components were detected in some anti-gen preparations from type 1 strains. No anti-

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A. ISRAELII ANTIGENS IN IMMUNOELECTROPHORESIS 393

gen preparation from type 2 strains containedmore than six antigenic components.The immunoprecipitation patterns by CIE for

different strains of A. israelii were comparedwith the precipitation profiles obtained in gelimmunodiffusion and in conventional immuno-electrophoresis, using the same antigen-anti-body system for A. israelii based on StAgSL andStAbIl (Fig. 5A and B). These techniquesreproduced some of the antigenic differencesbetween the strains of A. israelii. Cytoplasmicantigens of type 2 strains gave a single precipi-tation line which showed a reaction of identitywith precipitates to type 1 strains. These strainsgave two or three precipitation lines.To determine whether the components of

StAgSL could be further separated on the basis

StAbiI

5A 4

I(

O'StASL

of their net charge, experiments were carriedout by means of IFPAG in the pH range 3.5 to10.0. Nine stained protein components wereseparated. The components had isoelectricpoints (pl's) in the range 3.5 to 5.0 as seen fromthe pH reference scale in Fig. 6.

In other experiments the StAgSL was sub-jected to separation by IFPAG in the pH range3.5 to 5.0 followed by electrophoresis into anantibody (StAbII)-containing gel (crossed im-munoelectofocusing) (Fig. 6). Eight rocket-likeimmunoprecipitates were obtained in the sec-ond-dimension electrophoresis. One of the pre-cipitates with pl 4.70 was further separated intothree rockets. One precipitate with pl 4.63 wasseparated into two rockets. Two protein compo-nents in the IFPAG gel in the pH range 4.10 to

A StAbI B

A2 StAb0l

lc'm 0

FIG. 4. (A) CIE of 5 gIl of StAgSL of A. israelii as antigen and standard anti-A. israelii serum StAbII asreference. The precipitates are enumerated from the anodic side and coded 1 to 10. (B) CIE with intermediategel of 5 gI of StAgSL. The intermediate gel contained 5gIl of StA bII per cm2. The reference gel contained 5ml ofStAbIper cm2. All antigenic components of StAgSL precipitated by StAbI are retained in the intermediate gel.

A

_s

BF..[ r-"'

FIG. 5. (A) Immunodiffusion of the standard antigen StAgSL of A. israelii and cytoplasmic antigen prepara-tions of different strains of A. israelii. Center well contained the standard antibody StAbII to A. israelii. Outerwell contained the antigen preparations as follows: (1) StAgSL, (2) ATCC 12103, (3) WVU 307, (4) Holm1452/40-41, (5) SBL 225/74, and (6) ATCC 27036. (B) Immunoelectrophoresis of the standard antigen, StAgSL,of A. israelii to the standard antiserum, StAbII, to A. israelii.

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4.15 showed reaction of identity in the seconddimension. One protein component in theIFPAG gel with pl 3.91 formed no precipitate inthe antibody-containing gel. The immuno-precipitation pattern showed differences instaining intensity, but the pattern was repro-ducible in repeated experiments.

DISCUSSION

Different methods were employed in the pres-ent study to prepare crude cellular extracts ofA. israelii in attempts to identify those solubleprotein antigens of A. israelii which were mostsuitable for the development of a standardantigen-antibody system for A. israelii for CIEstudies. The chemical extraction methods, em-ployed for the preparation of whole cell-associ-ated antigens, were used because similar meth-ods, had been previously applied (6, 9, 11, 22-25). Cytoplasmic antigens, obtained by prepar-ative ultracentifugation of water extracts of

cell lysates were examined because of theirsuccessful usage as antigens with mycobacteria(42, 43) and Candida albicans (1, 2) for CIEstudies. Reported advantages of plasma anti-gens of Actinomycetales for serological taxo-nomic studies of these bacteria (24) also con-tributed to the choice of the cytoplasmic frac-tion of cell lysates.The chemical extracts from unbroken cells

likely included solubilized substances from thecell walls but also components leached from thecytoplasm. The cytoplasmic ultracentrifugate,used as cytoplasmic antigens, likely included awider spectrum of solubilized proteins by add-ing intracellular substances to those cell wall-bound substances solubilized as a result ofdisintegration.The antigen preparation procedures, per se,

might have involved denaturation of the solubil-ized proteins which disturb the formation ofimmunoprecipitates in CIE (35). This appliesparticularly to the trichloroacetic acid extracts,which may explain why only one antigenic com-

-4--

4&r

@.1#*. Z. .

tpH 3.78 3.95 4

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FIG. 6. Crossed immunoelectrofocusing of 100 Al of the standard antigen, StAgSI, of A. israelii. Isoelectricfocusing was performed in a pH 3.5 to 5.0 gradient. The protein bands were subjected to electrophoresis from astrip of polyacrylamide gel bearing the separated StAgSL into an agarose gel containing 5 ,l of the standardantiserum, StAbIl, to A. israelii per cm2. The fixed and Coomassie brilliant blue-stained proteins of thefirst-dimension electrofocusing in polyacrylamide gel are inserted by photomontage.

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A. ISRAELII ANTIGENS IN IMMUNOELECTROPHORESIS 395

ponent was detected in the standard antigenprepared by this procedure.Much of the evidence gained in the immuno-

chemical characterization of the standard anti-gens of A. israelii suggested that the cytoplas-mic fraction provided the most promising re-

sults as a standard antigen for A. israelii in CIE.These antigenic materials were most easilyaccessible and contained proteins or degrada-tion products of proteins with pl 3.5 to 5.0.Consequently, they were well suited for analysisin CIE at pH 8.6 by means of precipitatingantibodies.The immunoglobulins were purified from

antisera raised in rabbits against formalin-treated whole cells and cell lysates of A. israelii.The standard antibody pools caused by CIEprecipitation contained six and ten antigeniccomponents, respectively, of the cytoplasmicfraction of A. israelii. Among these precipitatesit was possible to demonstrate those which are

specific for A. israelii and to which the primaryproduction of antibodies is directed in thecourse of natural infections (18). In comparisonwith the complexity identified in relation to thereference antigen-antibody systems for C.albicans (2) and Pseudomonas (19) using a simi-lar type of antigen preparation, 10 referenceprecipitates may seem rather few. However,these systems were based on standard anti-bodies, prepared from polyvalent antiseraraised in rabbits, selected as good producers ofantisera, after prolonged immunization for up

to 1 year with standard antigens. It is possiblethat additional precipitates could have beenincluded in the standard system for A. israelii ifthe system had been based on a standardantibody prepared from antisera obtained fromantisera directed to antigenic components of A.israelii other than just those associated withformalin-treated whole cells and crude celllysates, or after a prolonged immunization pe-

riod. However, it may be argued that sacrificinganimals soon after immunization would yieldantisera with fewer cross-reactions against anti-genic components in common with closely re-

lated species. Analysis of a precipitation systemfor A. israelii by CIE containing 10 precipitatesseems convenient. There is no reason to in-crease the number of precipitates before thepossibilities of this system have been explored.

In attempting to develop a standard antigen-antibody system for A. israelii, the necessity forthe adoption of polyvalent standard antigenand antibody preparations became increasinglyevident. Standard polyvalent antigen had to be

prepared by combining a number of differentbatches of antigen preparations to take accountof strain and batch differences. Individual vari-ation in the ability of rabbits to produce anti-bodies to whole cells of A. israelii made the useof pooled antisera necessary. These findings arein accordance with similar standardized refer-ence antigen-antibody systems for C. albicans(2) and P. aeruginosa (19) in CIE.Applying conventional immunodiffusion and

immunoelectrophoresis, the standard systemfor A. israelii revealed precipitation patterns ofthree precipitates. Up to 10 precipitation arcswere detected by CIE, which exemplifies thehigher sensitivity and the resolving power ofthis technique. The possibility that some of theprecipitates for A. israelii were artifacts of theelectrophoretic separation was considered in aseries of experiments. It is quite possible thatdifferences in charge and size of the antigeniccomponents may compensate in such a way thattwo or more proteins have the same mobilityunder a given set of conditions (15, 31). IFPAG(37) separates proteins differing in charge pro-vided the separation procedure is not influencedby differences in molecular size resulting from-molecular sieving due to the gel porosity. Theuse of a nonrestrictive pore size for IFPAGfacilitated migration of the cytoplasmic pro-teins by their net charge (31). However, differ-ent protein components can have the same pland can therefore be seen as a single band in theIFPAG fractionation. This problem was furtherstudied by combining of IFPAG with electro-phoresis into an antibody-containing agarosegel (crossed immunoelectrofocusing) (33). Fromthe bands in IFPAG, two or three rocket-likeimmunoprecipitations could be detected. Thus,for the standard antigen-antibody system for A.israelii under investigation, the resolving powerof CIE was similar to that of crossed immuno-electrofocusing, the former method giving 10precipitates for the system in comparison with11 precipitation peaks when using the latter.However, the separation obtained with the twomethods differed. Thus, the methods supple-ment each other.

Therefore, crossed immunoelectrofocusingwould be a useful tool for the preparativeisolation of a specific antigenic component ofthe complex antigen mixture of A. israelii foranalytic serology. The CIE in conjunction withthe standardized reference antigen-antibodysystem, based on cytoplasmic antigens of A.israelii, may offer great potential for diagnosticA. israelii serology.

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396 HOLMBERG, NORD, AND WADSTROM

ACKNOWLEDGMENTS

We are indebted to Nils H. Axelsen (Protein Laboratory,Copenhagen, Denmark) and Cyril J. Smyth (KarolinskaInstitutet, Stockholm, Sweden) for their great interest andvaluable advice. We are grateful for the able technicalassistance of Evy Lindstrom and Margareta Hellgren.

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2. Axelsen, N. H. 1973. Quantitative immunoelectrophore-tic methods as tool for a polyvalent approach tostandardization in the immunochemistry of Candidaalbicans. Infect. Immun. 7:949-960.

3. Axelsen, N. H., and E. Bock. 1972. Identification andquantitation of antigens and antibodies by means ofquantitative immunoelectrophoresis. A survey ofmethods. J. Immunol. Methods 1:109-121.

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15. Hedrich, J. L., and A. J. Smith. 1968. Size and chargeisomer separation and estimation of molecular weightsof proteins by disc gel electrophoresis. Arch. Biochem.Biophys. 126:155-164.

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