JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 1981, p. 637-6420095-1137/81/040637-06$02.00/0

Vol. 13, No. 4

Detection of Legionella pneumophila Capsular-Like EnvelopeAntigens by Counterimmunoelectrophoresis

RANDALL A. SMITH,* SAMUEL DiGIORGIO,' JAMES DARNER,2 AND ANN WILHELM'Department ofMicrobiology and Immunology, Wright State University School ofMedicine, Dayton, Ohio

45435,' and Bacteriology Service, Veterans Administration Hospital, Dayton, Ohio 454282

Received 17 October 1980/Accepted 19 January 1981

The capsular-like envelope of Legionella pneumophila strains Togus 1 (sero-type 2) and Philadelphia 1 (serotype 1) was isolated and purified by columnchromatography on Sepharose 6B. Antibody raised in rabbits to these twoantigenic materials did not cross-react in gel diffusion. Upon electrophoresisfollowed by gel diffusion, the majority of both envelope materials was found tomigrate towards the cathode. A minor antigenic component of each envelope onlymigrated slightly towards the anode. Using the envelope antigens and the twoanti-envelope sera in a counterimmunoelectrophoresis (CIE) assay, positive re-sults were only obtained when the antigenic materials were placed in the cathodalwell. The Togus 1 and Philadelphia 1 antigens did not cross-react in CIE. Thesensitivity of the CIE assay was poor (15.6 ,ug/ml by carbohydrate content)compared to its sensitivity in other microbial systems. Although CIE may not bea useful diagnostic aid in identifying Legionella species due to its low sensitivity,it may be of value in serotyping the microorganism since we did not see cross-reactivity between the two strains when anti-envelope sera were used.

Legionella pneumophila, a small gram-nega-tive rod, is the causative agent of two distin-guishable clinical syndromes. An acute respira-tory form of legionellosis known as Legionnairesdisease is typified by a severe and sometimesfatal pneumonitis (9, 10, 14, 21). A milder formof legionellosis, Pontiac fever, is characterizedby a lack of pneumonia and fatalities (9, 11).Although this microorganism is frequently dif-ficult to cultivate on laboratory media, it isthought to be a free-living bacterium which hasbeen isolated from environmental sources (9,21).

Presently, six serotypes of L. pneumophilahave been described as determined by a directimmunofluorescence assay using conjugatedrabbit antisera (17). This assay, in conjunctionwith culturing on appropriate laboratory media,is also used in evaluating clinical specimens forthe presence of Legionella (3, 16, 17, 23). Tilton(20) and Berdal et al. (1) have found the enzyme-linked immunosorbent assay to be a sensitivetechnique for detecting Legionella antigens.However, cross-reactivity between Legionellaserotypes, and sometimes with unrelated bacte-ria, occurs when the antisera used are preparedagainst the whole cell (20). Recently, Wilkinsonand Fikes (22) found that L. pneumophila andatypical Legionella-like organisms could bereadily identified by a slide agglutination test.

Their results indicated that the slide agglutina-tion test is a reliable alternative to the directimmunofluorescence assay.Johnson et al. (12) demonstrated that a

loosely bound capsular-like envelope materialcan be readily isolated from Legionella strainsand purified by column chromatography. Thispurified material contains the type-specific Le-gionella antigen, whereas the crude saline washmaterial contains both the group- and type-spe-ciflc antigens. They noted that this capsular-likeenvelope antigen may be important in the in-duction of host protection, since this materialwas found to diminish the indirect immunofluo-rescent and microagglutination antibody titerswith convalescent sera from patients who hadhad legionellosis. Also, the majority of convales-cent sera will form precipitin bands to this an-tigenic material in gel diffusion assays.By chemical analysis, Johnson et al. (12)

found this envelope material to be composed ofcarbohydrate (35%), protein (2.6%), phospho-lipid (1.8%), and 2-keto-3-deoxyoctonate (1%); itexhibits endotoxin-like properties. Carbohy-drate and carbohydrate-containing capsular andenvelope antigens of various bacteria (4, 6, 15,18), fungi (13, 18), viruses (8, 18),and protozoans(18, 19) can be readily detected by counterim-munoelectrophoresis (CIE) assays. The advan-tages of CIE in serodiagnosis of disease are its

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638 SMITH ET AL.

sensitivity (<50 ng of antigen per ml) and rapid-ity; test results can be obtained in 60 min or less(4, 18).Presented here are the results of our experi-

ments in using CIE to identify L. pneumophilastrains Togus 1 (serotype 2) and Philadelphia 1(serotype 1) capsular-like envelope antigens byusing anti-envelope sera prepared in rabbits.

MATERIALS AND METHODS

Bacterial strains. The Philadelphia 1 (serotype 1)and Togus 1 (serotype 2) strains of L. pneumophilawere obtained from the Centers for Disease Control,Atlanta, Ga. The bacteria were cultured at 37'C in a90% air-10% C02 incubator on Mueller-Hinton agarsupplemented with 1% IsoVitaleX (BBL MicrobiologySystems, Cockeysville, Md.) and 1% hemoglobin. Tomaintain viability of the respective bacterial strains,they were transferred twice weekly.Harvesting of bacteria and envelope antigen.

The procedures followed for harvesting the bacterialstrains and envelope antigens were essentially thoseof Johnson et al. (12). Briefly, the bacteria were dis-lodged from the culture plates by the addition of 0.02M phosphate-buffered saline (pH 7.0), followed byscraping with a rubber policeman. The plates werethen placed on a clinical rotator (Erbaugh Corp., AnnArbor, Mich.) and rotated at 100 rpm for 30 min. Aftercentrifugation at 34,000 x g for 30 min at 4°C, thesupernatant (wash) containing the crude envelope ma-terial was carefully decanted, filter sterilized by beingpassed through a 0.45-jum pore size filter, and subse-quently lyophilized.The respective bacterial pellets were suspended in

phosphate-buffered saline (pH 6.4) and steamed for 60min at 101°C. The killed cell suspensions were storedat 4°C until used to immunize rabbits and to titrateantisera by the microagglutination assay.

Purification ofenvelope antigens. The envelopeantigens of the two Legionella strains were isolatedby the methods of Johnson et al. (12). Isolation of thecapsular-like material from the respective bacterialwashes was accomplished by column chromatographyon a Sepharose 6B column (2.6 by 40 cm; PharmaciaFine Chemicals, Inc., Piscataway, N.J.), monitored at280 nm. The envelope or capsular-like materials fromboth bacterial strains, which were eluted in the voidvolume, were pooled and lyophilized. A sample of eachwas removed to measure the carbohydrate content bythe phenol-sulfuric acid method (5).

Preparation of antisera. Eight-week-old NewZealand white female rabbits were used to prepareantisera to the respective capsular-like (envelope) andsomatic antigens of Legionella. Rabbits received atotal of three intramuscular injections over a 6-weekperiod, consisting of either 1 mg of envelope material(based on carbohydrate content) per injection or I09killed bacteria per injection. Freund complete adju-vant was used for the first two injections, and theincomplete form was used for the last injection. Anti-body titers were determined by using the heat-killedbacteria and the serum samples in the microagglutin-ation assay described by Farshy et al. (7). Rabbits

were exsanguinated and their sera were collected ifthe titers were 2,048 or greater. The respective anti-sera, envelope, and crude wash materials were reactedin gel diffusion to determine the purity of the chro-matographed envelope materials.CIE procedures. Glass microscope slides (25 by 75

mm) or lantern slides (84 by 94 mm) were coveredwith 0.75% agarose (type III; Sigma Chemical Co., St.Louis, Mo.) prepared in 0.05 M barbital buffer (pH8.6). Opposing wells of 2.5-mm diameter were cut 3mm apart by using a gel punch. Electrophoresis (con-tinuous) was performed at 7 V/cm for 60 min at roomtemperature. In initial experiments, the direction ofmigration (anodal or cathodal) of the respective Le-gionella envelope antigens was determined. After elec-trophoresis of the envelope materials (5 til at 250 /ug/ml), adjacent 2.5-mm wells were cut on the anodal andcathodal sides of the antigen well. Rabbit anti-enve-lope specific antserum was added to these wells in 5-pl amounts. The slides were then placed in a moistchamber for 18 h at 37°C to allow the development ofprecipitin bands. The direction of migration of therabbit immunoglobulin G (IgG) was determined byusing immunoelectrophoresis. A 2-til sample of rabbitanti-envelope serum was electrophoresed for 60 minas described. The center trough was then filled withgoat anti-rabbit IgG (H-chain specific; Cappel Labo-ratories, Inc., Cochranville, Pa.), and the slides wereincubated in a moist chamber overnight. On this sameslide, CIE was performed concurrently with the elec-trophoresis of the rabbit antisera. The antisera wellsfor both CIE (Fig. 5, no. 2) and immunoelectrophoresis(Fig. 5, no. 3) were cut the same distance from theends of the slide.

RESULTSThe envelope material from both Legionella

strains was eluted from the Sepharose 6B col-umn in the void volume (molecular weightgreater than 4 x 106) and formed a single precip-itin band in a gel diffusion assay with the ho-mologous whole-cell antiserum. Multiple precip-itin bands formed when the homologous whole-cell antiserum was reacted with the crude washmaterial. These results are shown in Fig. 1; thePhiladelphia antigens and antisera were used.

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FIG. 1. Gel diffusion reaction of Philadelphia Istrain crude wash material with homologous rabbitanti-envelope and anti-whole cell antisera. Wells: 1and 3, anti-whole cell; 2 and 4, anti-envelope; 5, crudewash material.

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DETECTION OF LEGIONELLA BY CIE 639

No gel diffusion cross-reactivity was evident be-tween the Philadelphia 1 and Togus 1 strainswhen we used the envelope antigenic materialsand their envelope-specific antisera as shown inFig. 2.CIE is applied to antigen-antibody systems in

which the antigen migrates towards the anodeduring electrophoresis, forming a precipitin bandwith the homologous antiserum (IgG) which isback-migrating due to electroendosmosis. To de-termine the feasibility of utilizing CIE to detectthe Legionella capsular-like antigenic material,it was first necessary to determine the directionof migration of the column-purified envelopematerials. After electrophoresis of the respectiveantigens for 60 min, adjacent wells were cut onthe anodal and cathodal sides of the antigenwells. These were filled with 5 pld of homologousrabbit anti-envelope serum, and the slides wereincubated for 18 h in a moist chamber. Theresults of this experiment are shown in Fig. 3.Although the envelope materials eluted off theSepharose 6B column as a single peak, two dis-tinct precipitin bands resulted from electropho-

FIG. 2. Gel diffusion reaction of Philadelphia Iand Togus I strain envelope antigens with homolo-gous and heterologous rabbit anti-envelope sera.Wells: I and 2, Togus anti-envelope; 3 and 4, Phila-delphia anti-envelope; 5, Togus envelope material; 6,Philadelphia envelope material.

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FIG. 3. Electrophoresis of Togus I and Philadel-phia I envelope antigens followed by gel diffusionwith homologous anti-envelope sera. Wells: 2, Togusantigen electrophoresis for 60 min at 7 V/cm; 4,Philadelphia antigen electrophoresed as above; 1,anti-Togus envelope serum added after electropho-resis; 3, anti-Philadelphia envelope serum added as

above.

resis and gel diffusion. The major precipitatingband of both the Togus 1 and Philadelphia 1antigens migrated toward the cathode, whereasa minor component diffused or slightly migratedtowards the anode.

Since each envelope material exhibited anti-genic components migrating or diffusing in bothanodal and cathodal directions, the followingCIE assay was performed. A 5-,l volume ofantigen was placed in the center well of anagarose-coated slide. A 5-,ul sample of homolo-gous antiserum was added to adjacent anodaland cathodal wells. Electrophoresis was per-formed as described. The results of this CIEassay are shown in Fig. 4. A precipitin band onlyformed between the antigen well and the cath-odal antiserum well. This result was somewhatunexpected, since a portion of the envelope ma-terial formed a precipitin band on the anodalside of the antigen well after electrophoresis andgel diffusion as noted in Fig. 3. To determinewhether this negative reaction was due to aprozone effect, the following CIE assay was per-formed. In a series of CIE assays, various serumdilutions (1:2 to 1:100) were placed in the anodalwell, and antigen was placed in the cathodalwell. Again, no precipitin bands were apparentafter electrophoresis. This positive CIE reactionoccurred because a portion of the rabbit IgGmigrated toward the anode, although much of itback-migrated due to electroendosmosis. Thepicture presented in Fig. 5 demonstrates themigration of rabbit IgG in relation to a CIE runusing the Philadelphia 1 antigen and homolo-gous rabbit antiserum. Note that the Legion-ella-specific CIE bands (between wells 1 and 2)form under the center and slightly to the anodalside of the precipitin arc identifying IgG (devel-oped with goat anti-rabbit IgG; trough 4).

FIG. 4. CIE of Togus I and Philadelphia I enve-lope antigens with homologous anti-envelope sera.Wells: 2, Togus antigen; 1, anti-Togus envelope se-rum; 4, Philadelphia antigen; 3, anti-Philadelphiaenvelope serum.

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FIG. 5. Comparison of migration of anti-Philadel-phia envelope sera (IgG) in CIE and immunoelectro-phoresis. Wells: 1, Philadelphia antigen; 2 and 3,anti-Philadelphia serum. Electrophoresis was for 60min at 7 V/cm, followed by addition of goat anti-rabbit IgG (H-chain specific) to trough (4).

CIE has been found to be both a rapid andsensitive diagnostic aid for detecting microbialantigens (4, 6, 8, 13, 15, 18, 19). In several bac-terial systems, as little as 50 ng of capsular orenvelope materials per ml could be detected byCIE (4, 17, 18). The results of titrating therespective Legionella envelope antigens withhomologous antisera by CIE are shown in Fig. 6and 7. The least amount of capsular-like enve-lope antigen detectable by CIE was found to be15.6 ,ug/ml (well 6, Fig. 5 and 6) for both theTogus 1 and Philadelphia 1 strains. Attempts atincreasing the sensitivity of the CIE assay byaltering the pH of the buffer, diluting the anti-serum, increasing or decreasing the agarose con-centration, increasing or decreasing the volts percentimeter, or using agarose of a lower endos-motic index were unsuccessful.

DISCUSSIONJohnson et al. (12) have demonstrated that

the capsular-like envelope materials of L. pneu-mophila can easily be extracted by shaking thebacteria in buffered saline. Upon column chro-matography, the envelope materials are elutedin the void volume as a single homologous frac-tion. This chromatographed antigenic materialcontains the type-specific antigen, and the crudewash material contains both the type- andgroup-specific antigens (12). Antisera producedin rabbits to the envelope antigens of the Togus1 and Philadelphia 1 strains did not cross-reactin gel diffusion with the heterologous antisera(Fig. 2). All rabbit antisera directed against thesomatic antigens or the column-purified enve-lope material reacted in CIE. Cross-reactivity in

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6;

FIG. 6. Titration of Togus 1 envelope antigen withhomologous anti-envelope serum by CIE. Row A,wells 1 to 6: Togus envelope antigen, twofold dilu-tions, from (1) 500 ,ug/ml to (6) 15.6 fg/ml. Row B:Anti-Togus envelope serum (undiluted).

CIE was only evident when antisera directedagainst the somatic antigens were reacted withthe heterologous crude wash material beforecolumn chromatography; column-purified enve-lope antigen exhibited no cross-reactivity withheterologous antisera.During electrophoresis, the major precipitat-

ing band of both envelope antigens migratedtoward the cathode and was the only componentdetected in CIE. A minor precipitating compo-nent of both strains migrated or diffused towardthe anode (Fig. 3). This minor antigenic com-ponent did not react in CIE when serum, undi-luted or at various dilutions, was placed in theanodal side of the antigen well, indicating thatthis lack of reactivity was not due to a prozoneeffect. Considering the position of the precipitinarc relative to the antigen well after electropho-resis and gel diffusion (see Fig. 3), it is probablethat this component of the envelope antigenonly slightly diffuses from the well and is non-migrating in an electrical field. This migrationpattern of the L. pneumophila capsular-like en-velope antigens is the opposite of that seen inCIE analysis of other bacterial capsular andenvelope antigens (4, 6, 15, 18).

Since most of the IgG back-migrates towardthe cathode due to electroendosmosis, CIE has

640 SMITH ET AL.

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DETECTION OF LEGIONELLA BY CIE 641

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FIG. 7. Titration of Philadelphia I envelope anti-gen with homologous anti-envelope serum by CIE.Row A, wells I to 6: Philadelphia envelope antigen,twofold dilutions, from (1) 500 pg/ml to (6) 15.6 g/

ml. Row B: Anti-Philadelphia envelope serum (un-diluted).

been used in microbial systems in which theantigen exhibits anodal migration at an aLkalinepH (8.0 to 8.8). Not all of the rabbit IgG back-migrates. A portion of the IgG migrates towardthe anode as shown in Fig. 5, validating the use

of "reversed CIE" in detecting Legionella-spe-cific envelope antigens. By reversing the classicalCIE procedure (i.e., antigen is added to theanodal well rather than the cathodal well,whereas the antisera are added to the cathodalweil rather than the anodal well) for CIE, we

could successfully detect the Legionella anti-gens by this modified procedure within 60 minof the onset of the test. The major shortcomingof this CIE system was its poor sensitivity indetecting the envelope materials (15.6 ,ug of car-

bohydrate antigen per ml for both the Togus 1and Philadelphia 1 strains). Attempts at modify-ing this assay system to increase the sensitivityproved to be unsuccessful. We conclude that theonly realistic means of altering the sensitivity ofthe test is to alter the charge on the IgG bycarbamylation (2) so it will migrate toward theanode. Preliminary test results indicate that thismay be a feasible approach to increasing the

sensitivity of the CIE assay.Because of its relative insensitivity, the CIE

assay is not a practical diagnostic test to use inscreening for Legionella in specimens such assputum and pleural fluids, which may containonly nanogram amounts of envelope materialsor limited numbers of bacteria. However, CIEmay be useful in determining the serotype ofisolated cultures, since we have not found anycross-reactivity between the Togus 1 and Phil-adelphia 1 strains by CIE when the antiseraused were directed solely against the column-purified capsular-like envelope antigens. Itshould be noted that Wilkinson and Fikes (22)have described a slide agglutination test whichhas been shown to be very effective in serotypingthe various Legionella isolates.

ACKNOWLEDGMENTSWe thank William Johnson, University of Iowa, Iowa City,

for his gracious assistance, and Diane Wuebben for typing thismanuscript.

LITERATURE CITED

1. Berdal, B. P., C. E. Farshy, and J. C. Feeley. 1979.Detection of Legionella pneumophila antigen in urineby enzyme-linked immunospecific assay. J. Clin. Micro-biol. 9:575-578.

2. Bjerrum, D. J., A. Ingild, H. Lowenstein, and B.Weeke. 1973. Carbamylated antibodies used for quan-titation of human IgG. A routine method. Scand. J.Immunol. 2(Suppl. 1):145-148.

3. Cherry, W. B., B. Pittman, P. P. Harris, G. A. Hebert,B. M. Thomason, L. Thacker, and R. E. Weaver.1978. Detection of Legionnaires disease bacteria bydirect immunofluorescent staining. J. Clin. Microbiol.8:329-338.

4. Coonrod, J. D. 1979. Use CIE for antigen detection inbacterial infections. Lab. Management 17:33-36.

5. Dubois, M., K. A. Gilles, J. F. Hamilton, P. A. Reber,and F. Smith. 1956. Colorimetrie method for determi-nation of sugars and related substances. Anal. Chem.28:350-356.

6. El Refaie, M., and C. Dulake. 1975. Countercurrentimmunoelectrophoresis for the diagnosis of pneumococ-cal chest infection. J. Clin. Pathol. 28:801-806.

7. Farshy, C. E., G. C. Klein, and J. C. Feeley. 1978.Detection of antibodies to Legionnaires disease orga-nism by microagglutination and micro-enzyme-linkedimmunosorbent assay tests. J. Clin. Microbiol. 7:327-331.

8. Fortunato, J., B. Goldschmidt, J. Menonna, P. Dowl-ing, and S. Cook. 1977. Rapid detection of antibodiesto cytomegalovirus by counterimmunoelectrophoresis.J. Infect. Dis. 135:432-437.

9. Fraser, D. W., and J. E. McDade. 1979. Legionellosis.Sci. Am. 241:82-99.

10. Fraser, D. W., T. R. Tsai, W. Orenstein, W. E. Parkin,H. J. Beecham, R. C. Sharrar, J. Harris, G. F.Mallison, S. M. Martin, J. E. McDade, C. C. Shep-ard, P. S. Brachman, and Field InvestigationTeam. 1977. Legionnaires disease: description of anepidemic of pneumonia. N. Engl. J. Med. 297:1189-1197.

11. Glick, T. H., M. B. Gregg, B. Berman, G. Mallison, W.W. Rhodes, Jr., and I. Kassanoff. 1978. Pontiac fever:an epidemic of unknown etiology in a health depart-

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12. Johnson, W., J. A. Elliott, C. M. Helms, and E. D.Renner. 1979. High molecular weight antigen in Le-gionnaires' disease bacterium: isolation and partialcharacterization. Ann. Intern. Med. 90:638-641.

13. Kerkering, T. M., A. Espinel-Ingroff, and S. Sha-domy. 1979. Detection of Candida antigenemia bycounterimmunoelectrophoresis in patients with inva-sive candidiasis. J. Infect. Dis. 140:659-664.

14. MeDade, J. E., C. C. Shepard, D. W. Fraser, and T. T.R. Redus. 1977. Legionnaires' disease: isolation of abacterium and demonstration of its role in other respi-ratory disease. N. Engl. J. Med. 297:1197-1203.

15. MeIntyre, M. 1978. Detection of capsulated Hemophilusinfluenza in chest infections by counter current im-munoelectrophoresis. J. Clin. Pathol. 31:31-34.

16. McKinney, R. M., L. Thacker, P. P. Harris, K. R.Lewallen, G. A. Hebert, P. H. Edelstein, and B. M.Thomason. 1979. Four serogroups of Legionnaires' dis-ease bacteria defined by direct immunofluorescence.Ann. Intern. Med. 90:621-624.

17. McKinney, R. M., H. W. Wilkinson, H. M. Sommers,B. J. Fikes, K. R. Sasseville, M. M. Yungbluth, and

J. S. Wolf. 1980. Legionella pneumophila serogroupsix: isolation from cases of legionellosis, identificationby immunofluorescence staining, and immunologicalresponse to infection. J. Clin. Microbiol. 12:395-401.

18. Murillo, J. L., and J. A. Titelbaum. 1979. A review ofcountercurrent immunoelectrophoresis. J. Med. Soc. N.Jersey 76:371-375.

19. Pifer, L. L., W. T. Hughes, S. Stagno, and D. Woods.1978. Pneumocystis carnii infection: evidence for highprevalence in normal immunosuppressed children. Pe-diatrics 61:35-41.

20. Tilton, R. C. 1979. Legionnaires' disease antigen detectedby enzyme-linked immunosorbent assay. Ann. Intern.Med. 90:697-698.

21. Vella, E. E. 1978. Legionnaires' disease: a review. J. R.Soc. Med. 71:361-368.

22. Wilkinson, H. W., and B. J. Fikes. 1980. Slide aggluti-nation test for serogrouping Legionella pneumophilaand atypical Legionella-like organisms. J. Clin. Micro-biol. 11:99-101.

23. Winn, W. C., Jr., W. H. Cherry, R. O. Frank, C. A.Casey, and C. V. Broome. 1980. Direct immunofluo-rescent detection of Legionella pneumophila in respi-ratory secretions. J. Clin. Microbiol. 11:59-64.

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