Vol. 38, No. 1INFECTION AND IMMUNITY, Oct. 1982, p. 35-400019-9567/82/100035-06$02.00/0Copyright 0 1982, American Society for Microbiology

Purification of TR-b, a Reiter Treponeme Protein AntigenPrecipitating with Antibodies in Human Syphilitic Sera

C. SAND PETERSEN,* N. STRANDBERG PEDERSEN, AND N. H. AXELSENDepartment of Treponematoses, Statens Seruminstitut, Copenhagen, Denmark

Received 20 April 1980/Accepted 23 June 1982

TR-b is a Reiter treponeme antigen, cross-reacting with an antigen in Trepone-ma pallidum (Nichols pathogenic strain). Sera from patients with secondarysyphilis contain precipitating antibodies against TR-b. The isolation of TR-b froma bacterial sonic extract is described here. It involved five fractionation steps:anion-exchange chromatography (DE-52 Whatman), gel filtration (Ac-A-22 Ultro-gel), and affinity chromatography on phenyl-Sepharose CL 4B, iminodiaceticacid-Sepharose CL 4B, and lysine-Sepharose 4B, respectively. The purified TR-bwas enriched 199 times compared with the starting material, and the recovery was12%. TR-b was shown to be a protein; it did not bind to a series of lectins, and bygel filtration and polyacrylamide gel electrophoresis, the molecular weight wasdetermined to be 610,000 to 630,000. It was found by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be composed of identical 70,000-daltonsubunits. The isolated TR-b was immunologically pure when tested in crossedimmunoelectrophoresis against polyspecific anti-Reiter immunoglobulin. Thepurified TR-b antigen was used for the production of a monospecific rabbitantiserum, giving strong fluorescence with both the Reiter treponeme and T.pallidum in an indirect immunofluorescence test.

Patients with secondary syphilis have precipi-tating antibodies against 5 of 40 antigens in theapathogenic Reiter treponeme (14). One of thesefive antigens (TR-a) is represented by a weakand not well-reproduced precipitate in crossedimmunoelectrophoresis (IE) (14), but the re-maining antigens (TR-b, TR-c, TR-dl, TR-d2,TR-e) have recently been shown to be related tocorresponding antigens in Treponema pallidum(15). It is, therefore, probable that antibodiesagainst TR-b, TR-c, TR-dl, TR-d2, and TR-e,when present in syphilitic sera, are raised inresponse to the corresponding antigens of T.pallidum (15). TR-e has been isolated and identi-fied as the periplasmatic flagellum or axial fila-ment of the Reiter treponeme (11, 17). TR-dl hasbeen found to consist ofRNA (12), and recentlyTR-c and TR-d2 have been purified and shownto be protein antigens (9; manuscript in prepara-tion). TR-e has been shown to be very useful inenzyme-linked immunosorbent assays for thedetection of immunoglobulin G (IgG) and IgMantibodies in syphilis (16, 18).

In this study, a method for the purification ofTR-b from a sonically treated supematant of theReiter treponeme is presented.

MATERIALS AND METHODSReiter treponeme. The same strain of the Reiter

treponeme as used previously (14) was grown and

harvested as described elsewhere (14). Eight grams(wet weight) of treponemes was suspended in 80 ml of0.05 M Tris-hydrochloride buffer, pH 8.0. This suspen-sion was sonicated in an ice bath with an MSE 150-Wultrasonic disintegrator (Manor Royal, Crawley, En-gland) with output frequency of 20 kHz and an ampli-tude of 20 microns for 6 x 30 s at 30-s intervals. Thesonic extract was centrifuged at 35,000 x g for 15 minat 4°C, and the supernatant was used as the crudeproduct for the purification of antigen TR-b.

T. paUidum. T. pallidum antigen for electrophoresiswas prepared as described earlier (15).Rabbit antibodies. Antibodies against the Reiter

treponeme were obtained by the immunization ofrabbits with a crude bacterial sonic extract as previ-ously described (14). A purified immunoglobulin frac-tion with a protein concentration of 15.8 gfliter wasprepared from a pool of these antisera by the methodof Harboe and Ingild (4). Monospecific antibodiesagainst TR-b were obtained by the immunization oftwo rabbits with the purified TR-b in Freund incom-plete adjuvant by the procedure described by Harboeand Ingild (4), using 10 Fg of TR-b protein per injec-tion. Rabbit anti-T. pallidum immunoglobulin was thesame as described earlier (15).Human sera. Sera were obtained from selected

patients with secondary syphilis with strong reactiv-ities in traditional syphilis serological tests and fromhealthy persons as described earlier (14).Fused rocket IE. The.fractionations were monitored

by means of fused rocket IE (19).Crossed IE with intermediate gel. Crossed IE with

intermediate gel was performed as described by Axel-sen (1).

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36 PETERSEN, PEDERSEN, AND AXELSEN

Tandem crossed IE. The identity between the puri-fied TR-b and the TR-b in the crude Reiter sonicextract was tested by tandem crossed IE (6).Agarose gel electrophoresis. Agarose gel electropho-

resis was performed as described by Johansson (5).Staining methods. Immunoprecipitates of TR-b were

stained for RNA and DNA with "Stains All" {1-ethyl-2 - [3 - (1 - ethyl - naphtho[1 ,2d]thiazolium - z - ylidene) - 2 -

methylpropenyll naphtho[1,2d]thiazolium bromide;Serva Feinbiochemica, Heidelberg, Germany} by themethod of Gould and Matthews (3). Periodic acid-Schiff reagent (24) and Sudan black B (21) were alsoused for the staining of carbohydrates and lipids in theimmunoplates.SDS-PAGE. Sodium dodecyl sulfate-polyacrylamide

gel electrophoresis (SDS-PAGE) was performed on ahomogeneous gel with 10% acrylamide in 0.1 M phos-phate buffer (pH 7.0) and 0.2% SDS by the method ofWeber and Osborn (21). The protein markers werethyroglobulin (molecular weight, 33 x 104), ferritin(molecular weight, 220 x 103 and 18 x 103), albumin(molecular weight, 67 x 103), catalase (molecularweight, 60 x 103), lactate dehydrogenase (molecularweight, 36 x 103), phosphorylase b (molecular weight,94 x 103), ovalbumin (molecular weight, 43 x 103),carbonic anhydrase (molecular weight, 30 x 103),trypsin inhibitor (molecular weight, 20.1 x 103), and a-

lactalbumin (molecular weight, 14.4 x 103), all fromPharmacia Fine Chemicals, Uppsala, Sweden.Continuous gradient PAGE. Electrophoresis in gels

containing 2 to 16% and 4 to 30% continuous gradientsof polyacrylamide (Pharmacia Fine Chemicals) wasused to determine the molecular weight of the nativeform of TR-b. The electrophoresis buffer contained0.09 M Tris, 0.08 M boric acid, and 0.003 M disodiumEDTA. A kit of molecular weight proteins from Phar-macia Fine Chemicals (see above) was used for thecalibration of the linear best-fit relationship betweenRf and the log of the molecular weight.

Gel filtration on Ac-A-22 Ultrogel. The molecularweight of TR-b was also estimated by gel filtration onAc-A-22 Ultrogel. The total gel bed volume (V,) was128 ml, and it was packed in a column with beddimensions of 1.6 x 70 cm (K 16/70 from PharmaciaFine Chemicals). The column void volume (VI,) wasthe elution volume for dextran blue 2000. The elutionvolume (Vi) was measured, and Ka. = Ve - VJV, -

V0 was calculated for each of the proteins. The calibra-tion curve was calculated for the relation between Ka,and the log of the molecular weight of the knownproteins. Molecular weight standards were thyroglob-ulin (molecular weight, 669,000), ferritin (molecularweight, 440,000), aldolase (molecular weight, 158,000),albumin (molecular weight, 67,000), ovalbumin (mo-lecular weight, 43,000), chymotrypsinogen (molecularweight, 25,000), and RNase (molecular weight,13,200), all from Pharmacia Fine Chemicals.Chemical analysis. Protein concentration was deter-

mined by the method of Lowry et al. (7), with bovineserum albumin as the standard. Hexoses and pentoseswere estimated by the anthrone (13) and orcinol (8)methods, respectively.

Quantification of TR-b. The quantification of TR-bwas performed by rocket IE (22), using the monospe-

cific antiserum against the purified TR-b.Lectin binding. The binding of purified TR-b to

lectins was tested in small columns and by crossed

immuno-affino-electrophoresis (2). The following lec-tins were used: concanavalin A-Sepharose, lentil-lec-tin Sepharose 4B, wheat germ lectin Sepharose 6 MB,Helix pomatia lectin Sepharose 6 MB (all from Phar-macia Fine Chemicals), peanut agglutinin (IBF Reac-tifs, Clichy, France), and Ricinus agglutinin (MilesLaboratories, Elkhart, Ind.).Immunofluorescence experiments. T. pallidum

(Nichols pathogenic strain) and the Reiter treponemewere fixed to glass slides (20), and monospecific rabbitanti-TR-b was added to the slides. In some of theexperiments, monospecific rabbit anti-TR-b waspreabsorbed with various concentrations of purifiedflagella (11). After being washed, a fluorescein isothio-cyanate-conjugated swine anti-rabbit immunoglobulin(code F 2190, lot no. 069 B, DAKOPATTS, Copenha-gen, Denmark) was added to the slides, and thetreponemes were microscopically tested for fluores-cence. Pre-immunization serum was used as a controland gave no fluorescence.

Materials. The following gel materials were used inthe isolation procedure: DEAE-cellulose (DE-52,Whatman), Ac-A-22 Ultrogel (Reactifs IBF), phenyl-Sepharose CL 4B, lysine Sepharose 4B (Pharmacia),and iminodiacetic acid Sepharose CL-4B (PierceChemical Co., Rockford, Ill.). The concentration stepwas performed on an Amicon stirred ultrafiltration cellmodel no. 202 with a PM 10 filter (all from AmiconCorp., Lexington, Mass.).

RESULTSPurification procedure. An 80-ml amount of

the Reiter sonically treated supernatant wasadded to a column of DEAE-cellulose with a bedvolume of 180 ml, and elution was performedwith a stepwise salt gradient, including 180 ml ofeach of 0.05, 0.1, 0.15, 0.25, 0.5, and 0.75 MNaCl in 0.05 M Tris-hydrochloride, pH 8.0. Theflow rate was 100 ml/h. A total of 115 fractions involumes of 10 ml each were collected, and everysecond fraction was tested in fused rocket IEagainst rabbit anti-Treponema Reiter immuno-globulin. Fractions nos. 78 to 102 containing TR-b (Fig. 1A) were pooled and concentrated 20times. This suspension was then gel filtered at4°C on a column (2.5 by 100 cm) with Ac-A-22Ultrogel (Pharmacia K 25/100) with a bed vol-ume of 480 ml. The column was calibrated withdextran blue 2,000 (Pharmacia), and the columnconstant (Kay) was estimated to be 52. Thecolumn was eluted with 0.05 M Tris-hydrochlo-ride (pH 8.0) containing 0.2 M NaCI and 15 mMNaN3 at a flow rate of 10 ml/h. A total of 100fractions of 5 ml each were collected. The frac-tions were tested by fused rocket IE. TR-b waspresent in fractions nos. 50 to 62 (Fig. 1B),which were pooled and applied to a column ofphenyl-Sepharose CL-4B with a bed volume of 8ml. The elution of the bound antigens was ob-tained by a stepwise ethylene glycol gradient,using 8 ml each of 10, 20, 50, and 75% ethyleneglycol in 0.05 M Tris-hydrochloride, pH 8.0.Twenty-six fractions of 3 ml each were collect-

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PURIFICATION OF TR-6 37

ed, with a flow rate of 20 ml/h. TR-b was elutedin fractions nos. 2 to 6 with 10% ethylene glycol,as visualized in fused rocket IE (Fig. 1C). Thepooled fractions 2 to 6 were dialyzed for 24 hagainst 1 liter of 0.05 M Tris-hydrochloride to

ADE-52Whatm

B

a -TR

Inn vA

Ac-A -22Ultrogel

L ...

78 90 6102

a -TR

_: .' ... '.

54 58 6256 60 64

C Phenyl SepharoseCL 4B

a-TR

.& W

2 4 6

3 5

DIminodiacetic acidSepharose CL 4B

a -TR

20 22 24

21 23

E Lysine Sepharose4B

a -TR

3 5

4 6

FIG. 1. Fused rocket IE profiles of the Reitertreponemal antigens eluted from the anion-exchangechromatographic gel (DE-52 Whatman) (A), Ac-A-22Ultrogel (B), phenyl-Sepharose CL-4B (C), iminodia-cetic acid-Sepharose CL-4B (D), and lysine-Sepharose4B (E). Samples (10 pL1) from the fractions were

analyzed against polyspecific rabbit anti-Reiterimmunoglobulin (a-TR) in the upper gels. Arrowspoint to the TR-b precipitate. The fraction numbersare indicated. The plates were stained with Coomassiebrilliant blue R-250.

remove the ethylene glycol and, thereafter, add-ed to a column of iminodiacetic acid SepharoseCL-4B with a bed volume of 8 ml. The gel wasfirst washed with 30 ml of 0.05 M Tris-hydro-chloride to remove unbound antigens followedby specific elution with 20 ml of 10 mM EDTA in0.05 M Tris-hydrochloride. A total of 25 frac-tions of 25 ml each were collected. The flow ratewas 10 ml/h. TR-b was eluted with EDTA anddetected in fractions nos. 20 to 25 (Fig. 1D),whereas several other contaminating treponemalantigens passed unbound through the matrix.The final purification of TR-b was obtained byaffinity chromatography on lysine-Sepharose4B. The pooled fractions nos. 20 to 25 from theiminodiacetic acid Sepharose column was addedto a column of 8 ml of lysine-Sepharose 4B gel,and elution was performed with a stepwise saltgradient with 8 ml each of 0.05, 0.1, 0.25, and 0.5M NaCl in 0.05 M Tris-hydrochloride, pH 8.0;20 fractions of 4 ml each were collected. TR-bwas eluted with 0.05 M NaCl. Fractions nos; 3 to6 containing TR-b free of contaminating antigenswere pooled, and this was the end product of thepurification procedure. The yield of pure TR-bin one procedure was 2 mg of protein. Recover-ies and enrichments of TR-b after the variousfractionation steps are given in Table 1. Thepurification of TR-b was performed five timeswith nearly identical results.

Several results indicated that the purified TR-b is a protein. It gave an absorption maximum at280 nm upon spectral analysis, and it reactedwith the Folin reagent of the Lowry reaction.Staining for polysaccharide, lipid, RNA, andDNA was negative, and the binding experimentswith six lectins all gave negative results. Nodetectable hexoses and pentoses were estimatedby the anthrone and orcinol methods, respec-tively.The purified antigen TR-b showed a reaction

of identity in tandem crossed IE with the TR-bantigen in the reference pattern. Only one pre-cipitate was seen when the isolated antigen wastested against a polyspecific anti-Reiterimmunoglobulin detecting more than 40 trepone-mal antigens (Fig. 2A). Purified TR-b reactedwith human syphilitic serum as visualized incrossed IE (Fig. 2B).

In Fig. 3, polypeptide analysis of the purifiedTR-b protein by SDS-PAGE shows one poly-peptide with an apparent molecular weight of70,000. In agarose gel electrophoresis, TR-bonly produced one band. The molecular weightof the native isolated TR-b was estimated to be630,000 and 610,000 by PAGE and gel filtrationon Ac-A-22 Ultrogel, respectively. Thus, thenative TR-b probably consists of nine identicalsubunits.The immunization of rabbits with the purified

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38 PETERSEN, PEDERSEN, AND AXELSEN

TABLE 1. Recovery and enrichment of TR-b in the different purification steps% Recovery of TR-b in Enrichment of TR-b

Purification step percentage of TR-b in when compared withTreponema Reiter sonic Treponema Reiterextracta sonic extractb

Treponema Reiter sonic extract 100 1.0DE-52 Whatman eluate 76 13Ac-A-22 Ultrogel eluate 57 32Phenyl-Sepharose CL-4B eluate 35 100Iminodiacetic acid Sepharose eluate 20 157Lysine-Sepharose 4B eluate 12 199

a Recovery was estimated by rocket IE.b Enrichments were calculated relative to total protein, setting the amount of TR-b per gram of protein in

Treponema Reiter sonically treated supernatant to 1.0.

TR-b resulted in the production of monospecificantisera. When Reiter sonic extract was submit-ted to crossed IE, employing the specific antise-rum in the second-dimensional gel, only TR-bwas precipitated (Fig. 4B). However, the precip-itate representing the flagellum disappearedwhen the monospecific anti-TR-b was incorpo-rated in the intermediate gel (Fig. 4B). Monospe-cific rabbit anti-TR-b showed strong fluores-cence with both the Reiter treponeme and T.pallidum in the indirect immunofluorescenceassay. Strong fluorescence was also seen whenthe monospecific rabbit anti-TR-b was preab-sorbed with purified Reiter flagella. Rabbit anti-T. pallidum immunoglobulin contained weakantibodies against the purified TR-b antigenwhen analyzed in crossed IE. No precipitationlines were observed when T. pallidum antigenswere analyzed against monospecific rabbit anti-TR-b immunoglobulin in crossed IE.

A

A:a-TR B

0

a-TR

DISCUSSIONMany commercially available affinity chroma-

tography gels were tested in a pilot study fortheir capability of separating the treponemalantigens in experiments using small columns.The three affinity gels used in this study wereselected due to their good separating properties.By the combination of anion-exchange chroma-tography and gel filtration, together with thedescribed affinity chromatographic steps, TR-bwas isolated and purified efficiently. The methoddescribed is technically easy to perform. Oneisolation procedure took 1 week, and all of thegel material could be reused. The method istherefore cheap for the production of TR-b on alarger scale. The reproducibility was good, i.e.,five separate experiments gave similar results.The isolated TR-b antigen was immunologicallypure when tested in crossed IE against polyspe-cific anti-Reiter immunoglobulin, detecting morethan 40 treponemal antigens. The purity of TR-bwas confirmed by the finding that only one bandwith a molecular weight of 70,000 was produced

ss-serum

70000;purified TR -b purified TR-b

FIG. 2. Crossed IE of purified TR-b antigen againstpolyspecific rabbit anti-Reiter immunoglobulin (a-TR)in the upper gels. The intermediate gels containedserum from healthy persons (0) (A) and human syphi-litic serum (SS-serum) (B). The figure shows that TR-bis immunologically pure (A) and that human syphiliticserum reacts with the purified antigen (B).

FIG. 3. SDS-PAGE of isolated TR-b showing oneband with an apparent molecular weight of 70,000.

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PURIFICATION OF TR-6 39

TA-a

~-T' R -

a

a-rR script in preparation), TR-e (11), TR-o (10), anda lipopolysaccharide (C. Sand Petersen, N.Strandberg Pedersen, and N. H. Axelsen, Anal.Biochem., in press). Except for the lipopolysac-charide, all of the isolated antigens cross-react

.b with antigens of T. pallidum, and these antigensare, therefore, valuable tools in the study of the

3nti- immune response in syphilis.TR-b

TR

FIG. 4. Crossed IE of Reiter sonically treated su-

pernatant (TR) against polyspecific rabbit anti-Reiterimmunoglobulin (a-TR) in the upper gels. The interme-diate gels contained normal rabbit serum (0) (A) andmonospecific rabbit anti-TR-b immunoglobulin (anti-TR-b) (B). Monospecific rabbit anti-TR-b immuno-globulin only precipitates with the TR-b antigen, butTR-e (the flagellum) is absent in (B).

by SDS-gel electrophoresis. In addition, onlyone band was detected in agarose gel electro-phoresis.The molecular weight of TR-b was found to be

in the range of 610,000 to 630,000, which issimilar to that of the previously purified TR-cantigen (9). Also, the chromatographic columnsused in the purification ofTR-b were the same asthose used for the purification of TR-c. TR-b andTR-c were eluted together in the anion-exchangechromatography and in the gel filtration step,but different elution patterns were found for TR-b and TR-c in the hydrophobic interaction chro-matography and in the two affinity chromato-graphic gels, iminodiacetic acid Sepharose CL-4B and lysine-Sepharose 4B. Thus, TR-b waseluted before TR-c on the phenyl-Sepharose andlysine-Sepharose columns but later than TR-c onthe iminodiacetic acid Sepharose column.The immunofluorescence experiments

showed that antibodies against TR-b gave strongfluorescence with both the Reiter treponeme andT. pallidum, indicating the existence of a TR-bhomologous antigen in T. pallidum. We cannotexplain why TR-e (the flagellum) was absentfrom the precipitate pattern of Fig. 4B, but thiscannot be due to the presence of precipitatingantibodies against TR-e in the anti-TR-b prepa-ration. The preabsorption of the anti-TR-b anti-body with purified flagella did not influence thefluorescence observed with unabsorbed anti-TR-b antibody. No T. pallidum antigen corre-

sponding to TR-b could be demonstrated in T.pallidum sonically treated material with the elec-troimmunoprecipitation techniques used. Thus,the evidence for an antigen in T. pallidum cross-reacting with TR-b is still indirect.We have now isolated seven different antigen-

ic components from the Reiter treponeme la-beled TR-b, TR-c (9), TR-dl (12), TR-d2 (manu-

ACKNOWLEDGMENTSMette Paulli Andersen and Tove Kirk are thanked for

perfect technical assistance.This study was supported by grants from the University of

Copenhagen (N. Strandberg Pedersen) and from "Welanderh-jemmenes fond".

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8. Meybaum, W. 1939. Uber die Bestimmung kleiner Pen-tosemengen insbesondere in Derivaten der Adenylsaure.Z. Physiol. Chem. 258:117-120.

9. Sand Petersen, C., N. Strandberg Pedersen, and N. H.Axelsen. 1982. Isolation and characterization of TR-c, anantigen of the Reiter treponeme precipitating with anti-bodies in syphilis. Scand. J. Immunol. 15:459-465.

10. Sand Petersen, C., N. Strandberg Pedersen, and N. H.Axelsen. 1982. Purification of a Reiter treponemal proteinantigen, immunologically related to an antigen in Trepone-ma pallidum. Infect. Immun. 35:974-978.

11. Sand Petersen, C., N. Strandberg Pedersen, and N. H.Axelsen. 1981. A simple method for the isolation of flagellafrom Treponema Reiter. Acta Pathol. Microbiol. Scand.89:379-385.

12. Sand Petersen, C., N. Strandberg Pedersen, and N. H.Axelsen. 1981. Hydrophobic interaction chromatographyused for purification of a Treponema Reiter ribonucleicacid antigen precipitating with antibodies in human syphi-litic sera. Anal. Biochem. 117:231-237.

13. Seifert, S., S. Bayton, B. Novic, and E. Muntwyler. 1950.The estimation of glycogen with the anthrone reagent.Arch. Biochem. 25:191-200.

14. Strandberg Pedersen, N., N. H. Axelsen, B. B. Jorgensen,and C. Sand Petersen. 1980. Antibodies in secondarysyphilis against five of forty Reiter treponeme antigens.Scand. J. Immunol. 11:629-633.

15. Strandberg Pedersen, N., N. H. Axelsen, and C. SandPetersen. 1981. Antigenic analysis of Treponema palli-dum: Cross-reactions between individual antigens of T.pallidum and T. Reiter. Scand. J. Immunol. 13:143-150.

16. Strandberg Pedersen, N., C. Sand Petersen, and N. H.Axelsen. 1982. Enzyme-linked immunosorbent assay fordetection of immunoglobulin M antibody in syphilis

A a-TR B

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