Vol. 29, No. 2JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 1991, p. 302-3090095-1137/91/020302-08$02.00/0Copyright © 1991, American Society for Microbiology

Comparison of Crossed Immunoelectrophoresis, Enzyme-LinkedImmunosorbent Assays, and Tube Agglutination for Serodiagnosis

of Yersinia enterocolitica Serotype 0:3 InfectionANDERS PAERREGAARD,l* GEOFFREY H. SHAND,2 KNUD GAARSLEV,3 AND FRANK ESPERSEN1

Department of Clinical Microbiology, Statens Seruminstitut, Rigshospitalet, Blegdamsvej, DK-2100 Copenhagen 0,'Department of Diagnostic Bacteriology and Antibiotics, Statens Seruminstitut, DK-2300 Copenhagen S,3

and Department of Microbiology, Dakopatts, DK-2600 Glostrup, Denmark

Received 24 August 1990/Accepted 1 November 1990

Antibodies against Yersinia enterocolitica serotype 0:3 were measured by crossed immunoelectrophoresis(XIE) using whole-cell sonic extract as antigen and by enzyme-linked immunosorbent assays (ELISAs) usingeither purified lipopolysaccharide or whole formalinized cells expressing virulence plasmid-encoded surfaceantigens (pYV+ cells). The results were compared with those obtained with the standard tube agglutinationmethod. Sera from three groups of people were examined by using these assays. The first group consisted ofhealthy blood donors, the second consisted of patients with recent infection due to microorganisms other thanY. enterocolitica 0:3, and the third consisted of patients with recent Y. enterocolitica 0:3 infection. Sera fromthe last group were also obtained at regular intervals for 12 months postinfection. Results obtained with XIEand the ELISAs were in good agreement with those obtained with tube agglutination. Variation, diagnosticsensitivity, and diagnostic specificity were satisfactory for all the assays studied. However, the lipopolysaccha-ride ELISA was less laborious than tube agglutination and XIE and carried a somewhat greater diagnosticspecificity than the pYV+ ELISA. XIE and the pYV+ ELISA, on the other hand, also had advantages. XIEenabled simultaneous examination of the individual antibody response against a wide range of chromosome-encoded antigens, and the pYV+ ELISA enabled detection of specific pYV antibodies when sera were adsorbedwith formalinized pYV-cured Y. enterocolitica 0:3 cells prior to the assay.

Yersinia enterocolitica is an enteric pathogen whichcauses acute gastrointestinal disease in humans. The organ-ism probably also plays an important role in the pathogenesisof several immunopathological disorders, the most commonbeing reactive arthritis (10, 12, 16). In Canada, NorthernEurope, and Japan and increasingly in the United States (2),organisms causing human disease are predominantly of the0:3 serotype. The Yersinia virulence plasmid, pYV, encodesa number of proteins considered to be important virulencefactors (5). Expression of these virulence characteristics istemperature dependent, being optimal at 37°C. Immunoblot-ting has revealed that Y. enterocolitica infection gives rise toan antibody response directed against a wide range ofbacterial antigens (15, 33, 34).The ability to quantitate the immunoglobulin class-specific

antibody response to these antigens, both chromosomal andplasmid encoded, is of diagnostic interest and may also leadto a better understanding of the process of pathogenesis.

In this study we first examined the antibody responseagainst a large pool of chromosomally encoded Y. enteroco-litica serotype 0:3 antigens by means of crossed immuno-electrophoresis (XIE). Thereafter, we evaluated and com-pared the diagnostic value of enzyme-linked immunosorbentassays (ELISAs), in which the antigens used were eitherpurified Y. enterocolitica 0:3 lipopolysaccharide (LPS) orwhole formalinized bacterial cells expressing pYV-encodedantigens. We also developed a simple method for quantita-tion of antibodies (designated pYV Ab) against pYV-en-coded antigens. The results were compared with thoseobtained with the traditional tube agglutination method.

* Corresponding author.

MATERIALS AND METHODS

Patients and normal controls. Sera from three groups ofsubjects were included. Group I was composed of 78 healthyDanish blood donors (mean age, 38 years; range, 18 to 68years); 31 were females and 47 were males. Group II wascomposed of 39 patients with culture-proven acute diarrhealdiseases or bacteremias caused by organisms other than Y.enterocolitica serotype 0:3. The- microorganisms isolatedfrom this group were Y. enterocolitica 0:9 (n = 2), Yersiniapseudotuberculosis serotype O:IV (n = 1), Campylobacterjejuni (n = 1), Clostridium difficile (n = 2), Salmonella typhi(n = 6), Salmonella paratyphi (n = 6), other Salmonellaserotypes (n = 4), Escherichia coli (n = 2), Klebsiellapneumoniae (n = 3), Morganella morganii (n = 1), Staphy-lococcus aureus (n = 2), Streptococcus pneumoniae (n = 2),other Streptococcus species (n = 4), Haemophilus parain-fluenzae (n = 1), and Brucella abortus (n = 2). These serawere included to detect false-positive reactions in the assaysexamined. They were obtained 2 to 6 weeks after the onsetof disease. Group IlIa was composed of patients withculture-proven Y. enterocolitica 0:3 infection. Serum sam-ples were collected 2 to 4 weeks after the start of diarrhea.Nine of these patients were further followed prospectivelyover a 12-month period (group IIlb). Blood was drawn fromthese patients 2, 5, 7, 12, 26, and 52 weeks after the start ofdiarrhea. In order to ensure that the sera were comparablewith respect to this time schedule, all blood samples weredrawn by one of the authors during visits to the patients intheir homes. Presence or absence of joint symptoms wasrecorded at the same time.

Strains and culture conditions. The two Y. enterocoliticaserotype 0:3 strains, YeL and YeWH, used for XIE havebeen described previously (25). Strain YeL was also used for

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tube agglutination. For the other procedures a virulenceplasmid-carrying (pYV+) Y. enterocolitica 0:3 strain,YeO3O1P+, and its isogenic plasmid-cured (pYV-) deriva-tive, YeO301P-, were used (26).Whole bacterial cells to be used in ELISAs, immunoblot-

ting, and adsorption of antibodies were cultured underconditions known to promote expression of pYV-encodedantigens. Strains YeO301P+ and YeO301P- were grown ina rich medium consisting of 1% tryptone, 0.5% yeast extract,and 0.2% glucose supplemented with the salts described byHiguchi et al. (20) plus 20 mM sodium oxalate. Cultivationwas performed in 100 ml of medium, which was incubatedfor 16 h at 20°C in a water bath with shaking. Thereafter,fresh medium was added to give a 20-fold dilution, andincubation was continued for 6 h at 37°C. The bacteria wereharvested by centrifugation and washed twice in saline.

Before it was decided how to cultivate bacteria for extrac-tion of LPS, a pilot study was performed to evaluate theinfluence of the pYV plasmid and variation in growth tem-perature on composition of the LPS. Strains YeO301P+ andYeO301P- were cultivated as described above; however,for each strain the final 6 h of incubation was performed at 20and 37°C in parallel, giving four different bacterial batches.On the basis of the results of this pilot study, LPS to be usedin ELISA was extracted from cells of strain YeO301P- thathad been grown at 20°C for 48 h on Truche agar plates andharvested in distilled water.

Bacteria (strain YeL) to be used for tube agglutinationwere grown for 48 h at 22°C on thick nutrient agar plates andharvested in saline. For immunization of rabbits, strainYeO301P+ was cultured at 20°C for 48 h on Truche agarplates (25).

Preparation of killed whole bacterial cells. (i) ELISAs.Bacteria (strains YeO301P+ and YeO301P-) to be used asantigens for the ELISAs were suspended in saline with 1%formaldehyde at 20°C for 2 h, washed three times, and keptat -20°C until use.

(ii) Tube agglutination. Equal volumes of strain YeL insaline and 96% ethanol were slowly mixed and left at 20°Cfor 24 h. This stock solution was stable for approximately 4weeks at 4°C. For the final use, the stock solution wasdiluted in phosphate-buffered saline (PBS) to a concentrationof approximately 5 x 109 cells per ml. This preparation couldbe used for 3 consecutive days.

(iii) Adsorption of sera. Bacteria (strains YeO301P+ andYeO301P-) to be used for adsorption of sera were washedonce in saline, killed by incubation at 20°C for 2 h in salinewith 1% formaldehyde, washed three times, and kept at 4°Csuspended in saline in quantities of 1 g (wet weight) until use.

Preparation of LPS. LPS was extracted by the hot phenol-water method (38). The LPS separated by ultracentrifugationwas further purified by the method of Hasin et al. (18) andfinally resuspended in endotoxin-free water. Purity wasdetermined by measuring A260 and A280 which confirmed thatLPS preparations were substantially free of contaminationwith nucleic acids and protein. The four batches of LPS wereanalyzed by sodium dodecyl sulfate-polyacrylamide gel elec-trophoresis (SDS-PAGE) with silver stain and by reaction inELISA.XIE. A previously described reference system in which

sonicated preparations of Y. enterocolitica serotype 0:3were run against corresponding rabbit immunoglobulins bymeans of XIE was used (25, 27). This system contains 58regularly visible precipitates. Only chromosomally encodedantigens are detected. The sera to be examined were incor-porated at a concentration of 40 pL1/cm2 in the intermediate

gel on plates (5 by 5 cm). Antibodies present in the interme-diate gel against antigens included in the reference systemwere detected by a reduction in the areas enclosed by thecorresponding precipitates (1). Quantitation of antibodiesagainst Y. enterocolitica antigens in the intermediate gel wasperformed by comparison with a series of plates containingincreasing amounts of the reference rabbit antiserum in theintermediate gel (1, 22). Amounts were 0, 0.1, 0.2, 1, 2, 10,20, and 40 RI1/cm2. For each precipitin in a patient's serum atiter was determined in the range of 0 to 8 (1, 22). These titerswere added to give the total score of this patient's serum.ELISA. Immunoglobulin A (IgA), IgG, and IgM antibodies

in human sera were analyzed by three different indirectELISAs. For the assay of antibodies to LPS (LPS ELISA),96-well polystyrene microtiter plates (Polysorb; Nunc,Roskilde, Denmark) were coated with a 1% solution of LPSin PBS, pH 7.2. Plates were incubated for 2 h and thenovernight at 4°C. Those plates not used immediately werewashed three times with PBS and dried. Dried plates couldbe stored at 4°C for at least 2 months with no significant lossof activity.

Test sera were diluted 1:6,000 and 1:750 in PBS containing0.1% Tween for detection of IgG and IgA/IgM antibodies,respectively, and 100 ,ul was incubated in each coated wellfor 2 h. After being washed with PBS-Tween, wells wereincubated for 1 h with 100 ,ul of peroxidase-conjugated rabbitanti-human IgG (Dakopatts, Glostrup, Denmark) diluted1:5,000 in PBS-Tween or 100 ,ul of peroxidase-conjugatedrabbit anti-human IgA or IgM (Dakopatts) diluted 1:2,000.Plates were then washed again, and color was developed byusing o-phenylenediamine (0.67 mg/ml) in 0.1 M citrate-phosphate buffer, pH 5.0, containing 0.0125% H202. Thecolor reaction was stopped after 10 min by addition of 150 p.lof 1 M sulfuric acid to each well. Optical density at 492 nm(OD492) was measured with a photometer (EIA Reader 2550;BioRad, Richmond, Calif.).For assays of antibodies to whole Y. enterocolitica cells,

microtiter plates (Maxisorb; Nunc) were coated with forma-linized whole cells (strain YeO3O1P+:pYV+ ELISA andYeO3O1P-:pYV- ELISA) diluted to an OD540 of 0.2 incarbonate buffer, pH 8.6. After overnight incubation asdescribed above, plates were washed three times with PBScontaining 5% lactose and 0.5% bovine serum albumin.Thereafter, the assay procedure was carried out as describedabove, except that serum samples were diluted 1:6,000 and1:3,000 for assays of IgG and IgA/IgM antibodies, respec-tively. In all cases, the dilutions of serum samples weredesigned to bring the final OD of the majority of sera withinthe 0 to 2.0 range of the photometer. Optimal concentrationof reagents was determined by checkerboard titration.

All samples were tested in triplicate, and the mean valuewas calculated. Positive and negative control sera wereincluded in every plate. To minimize intraplate and day-to-day variations, samples of control sera with low (n = 1),intermediate (n = 2), and high (n = 1) OD values wereincluded on every plate for construction of a standard curve.The OD of every sample was adjusted to the standard curve,and the results are given in arbitrary ELISA units. Back-ground was the OD of wells incubated with buffer alone.Results are given without subtraction of the backgroundvalues.Tube agglutination. Samples (0.3 ml) of the final prepara-

tion of ethanol-killed cells were added to 0.2 ml of twofolddilutions of serum in PBS, starting with a dilution of 1 in 10.The results were read as the highest final dilution in the

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304 PAERREGAARD ET AL.

0.5-ml sample with granular agglutination after incubationfor 20 h at 52°C.

Adsorption of sera. Fifty serum samples from group I, 27from group II, and all sera from groups Illa and IlIb wereadsorbed with pYV- Y. enterocolitica serotype 0:3 cells toremove chromosomally encoded antigens. Adsorption wasperformed by incubating 2 ml of a 1:100 dilution of the serumin question (in PBS containing 0.1% Tween) with 1 g (wetweight) of pelleted formalinized YeO301P- cells grown at37°C. Incubation was performed for 4 h at 4°C in vials slowlytumbling end over end. Thereafter, the bacteria were pel-leted by centrifugation, and the supernatant was reincubatedunder identical conditions with another 1 g of bacteriaovernight. Finally, the bacteria were discarded after centrif-ugation, and the adsorbed sera were passed through a0.45-Rm filter (Sartorius, Gottingen, Federal Republic ofGermany) before storage at -20°C until use. The adsorbedsera were assayed in a final dilution of 1:500 in the ELISAs,since that dilution gave OD values within the 0 to 2.0measuring range of the photometer.As controls, sera were also adsorbed with a mixture (1:1)

of pYV+ and pYV- Y. enterocolitica cells to remove anti-bodies to chromosome as well as plasmid-encoded antigens.The adsorption was carried out according to the scheduledescribed above.SDS-PAGE and immunoblotting. Whole-cell bacterial sam-

ples were dissolved in denaturation buffer by boiling for 10min, separated by SDS-PAGE, and electroblotted ontonitrocellulose paper as described previously (29). Rabbitserum for immunoblotting was obtained after subcutaneousvaccination of three rabbits with strain YeO301P+ accordingto the schedule used by Skurnik (30). Incubations of verti-cally cut nitrocellulose paper strips with sera and withhorseradish peroxidase-conjugated swine antibody to rabbitimmunoglobulins (Dakopatts, Glostrup, Denmark) wereboth performed for 2 h at room temperature with threefollowing washes. Finally, the strips were visualized withtetramethylbenzidine (Merck, Darmstadt, Federal Republicof Germany) (21).

Statistics. The specificity and sensitivity of the methodswere determined as described by Wulff (40) by using theresults obtained with sera from groups II and IlIa. Thediagnostic sensitivity is the predictive value of a negativetest (PVneg). The diagnostic specificity is the predictive valueof a positive test (PVP,S). The nosographic sensitivity is thepercentage of patients with the disease who have a positivetest. The nosographic specificity is the percentage of patientswithout the disease who have a negative test.On the basis of repeated determinations of 10 serum

samples with low (n = 3), medium (n = 4), and high (n = 4)test results, the intraday and day-to-day coefficients ofvariation for the methods (CVid and CVdd, respectively)were determined. The standard deviation was calculated byusing the formula \ S/27, where d2 is the sum of squareddifferences between double determinations of the samesample and n is the number of observations.For each method the cutoff level (to discriminate between

sera that were positive and negative for recent Y. enteroco-litica infection) that gave the best PVneg and PVpOS waschosen. Discrimination between sera that were positive andnegative for pYV Ab in the pYV+ ELISA was, however,handled differently. Antibodies to pYV as well as chromo-some-encoded antigens were removed from sera by means ofadsorption with formalinized pYV+ and pYV- Y. enteroco-litica cells. The upper limit obtained with these preadsorbedsera was chosen as the cutoff.

80 -

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Gr.I Gr. II Gr. Illa 3 5 7 12 26 52(weeks after start of symptoms)

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FIG. 1. Levels of precipitating antibodies against Y. enteroco-litica serotype 0:3 in human sera as measured by XIE. Results aregiven as total precipitin scores (see text). Group I (Gr. I), Healthyblood donors; group II (Gr. II), patients with recent diarrhea orbacteremia due to microorganisms other than Y. enterocolitica 0:3;group Illa (Gr. IIIa), patients with recent Y. enterocolitica 0:3infection; group IlIb (Gr. IlIb), longitudinally observed patientswith Y. enterocolitica 0:3 infection. Dashed line marks the diagnos-tic cutoff level.

For comparison of results obtained with different groupsof sera, the Mann-Whitney rank sum test for unpaired datawas used. The Spearman rank correlation test was used forcomparison of results obtained with different methods; P <0.05 was chosen as the level of significance.

RESULTS

XIE. Antibodies against 19 antigens were detected in serafrom patients with recent Y. enterocolitica serotype 0:3infection. Antibodies against a small number of additionalantigens were also detected in some sera, but not all platescould be evaluated with respect to changes in the corre-sponding precipitates because of their localization in thecentral part of the reference system.

Sera from blood donors (group I) had a median number of10 precipitins (range, 8 to 13), giving a median score of 27(range, 23 to 43), while sera from patients with othergastrointestinal or invasive infectious disease (group II) hada median number of 16 precipitins (range, 14 to 18), giving amedian score of 41 (range, 34 to 68). In the group consistingof patients with recent Y. enterocolitica serotype 0:3 infec-tions (group Illa), the median number of precipitins was 17(range, 15 to 19) and the median score was 62 (range, 43 to79). The scores obtained with sera from the different patientgroups are illustrated in Fig. 1. Within the three groups, thevariations in precipitin titers and scores were much morepronounced than the variation in the number of precipitins.The scores of groups I and IlIa both overlapped with thescores of group II. The sera in group II that gave the highestscores originated from patients with infections caused byother yersineae and other members of the family Enterobac-teriaceae. PVpos, PVneg' CVdd, and CVdd for the method aregiven in Table 1. None of the individual antigens provedspecific for Y. enterocolitica serotype 0:3 infection.The antibody levels in sera from patients with Y. entero-

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SERODIAGNOSIS OF YERSINIA ENTEROCOLITICA 0:3 INFECTION

TABLE 1. Clinical applicability of and variation and correlation among results of the tube agglutination, XIE, and ELISAs for detectionof recent Y. enterocolitica serotype 0:3 infectiona

Assay Nosographic Nosographic PV PV * CVid CVdd Correlation with tubesensitivity specificity neg po agglutination (rho)b

Tube agglutination 100 (86-100) 100 (94-100) 100 (91-100) 100 (86-100) 5 (12) 10 (36)

XIE 92 (74-99) 85 (68-95) 82 (63-94) 93 (78-99) 4 (12) 10 (25) 0.62*

LPS ELISAIgA 100 (86-100) 97 (87-100) 100 (91-100) 96 (80-100) 15 (40) 17 (43) 0.67*IgG 100 (86-100) 97 (87-100) 100 (91-100) 96 (80-100) 7 (18) 17 (45) 0.71*IgM 100 (86-100) 100 (91-100) 100 (91-100) 100 (86-100) 14 (36) 12 (32) 0.70*

pYV+ ELISAIgA 88 (69-98) 85 (70-94) 94 (75-97) 79 (60-92) 6 (17) 11 (29) 0.65*IgG 92 (74-99) 87 (73-96) 92 (78-98) 79 (59-92) 5 (13) 15 (38) 0.64*lgM 100 (86-100) 100 (91-100) 100 (91-100) 100 (86-100) 6 (16) 11 (27) 0.70*

a Nosographic sensitivity and specificity, PVneg, PVpos, CVid, and CVdd are given as percentages with 95% confidence limits. For CV values, only the upperlimit is given.

b Spearman rank correlation test; asterisks indicate P < 0.05.

colitica infection, as evaluated by the scores, decreased withtime but exceeded the scores obtained by most blood donorsera for the whole 12-month observation period (Fig. 1).ELISA. The backgrounds (median and range) in the

ELISA systems, as evaluated by the OD values of wellscontaining buffer only, were 0.087 and 0.038 to 0.160 (LPSELISA), 0.164 and 0.082 to 0.287 (pYV+ ELISA), and 0.090and 0.079 to 0.105 (pYV- ELISA).

(i) LPS ELISA. LPS extracted from bacteria grown withand without the virulence plasmid and at different tempera-tures appeared to be identical in SDS-PAGE after silverstaining and gave very close comparable results when usedin the ELISA to assay a number of sera from blood donorsand patients with recent Y. enterocolitica serotype 0:3infections (data not shown). Therefore, LPS extracted fromplasmid-cured cells grown at 20°C was used for the rest ofthe study, as such cells give the highest yield of serogroup-specific sugars (36).

Figure 2A shows the results obtained with the differentgroups of sera in the LPS ELISA. The PVneg and PVpos ofthe methods were high, and the variations were satisfactory(Table 1). Persistence of IgA, IgG, and IgM antibodies inlongitudinally observed patients is also illustrated in Fig. 2A.Almost no sera were positive with respect to IgA or IgMantibodies for more than 3 months postinfection. IgG anti-bodies, on the other hand, persisted for longer periods in allthe patients, still giving positive results by 12 months inseveral cases.

(ii) pYV+ ELISA. Y. enterocolitica serotype 0:3 cells usedas antigen for the pYV+ ELISA expressed several of thepYV-encoded antigens (Fig. 3). Figure 2B gives the resultsobtained with the pYV+ ELISA. PVneg and PVpOS fordetection of IgA and IgG antibodies were somewhat lowerthan in the LPS ELISA. However, this was not the case forIgM antibodies (Table 1). Variation was low (Table 1). IgAand IgM antibody levels declined faster than IgG levels (Fig.2B), as was the case for the LPS ELISA. On the other hand,the pYV+ ELISA revealed several patients with sera thatwere still positive in all three immunoglobulin classes after12 months.

(iii) Detection of pYV-specific antibodies. After adsorptionwith formalinized pYV- cells, the sera contained only smallamounts of LPS antibodies and antibodies against chromo-somally encoded surface antigens (both IgG class), as eval-

uated by LPS ELISA (ranges for groups I, II, and IlIa andIlIb were 0.041 to 0.074, 0.043 to 0.066, and 0.027 to 0.075,respectively, with no significant differences between thegroups) and by the pYV- ELISA (ranges for groups I, II,and Illa and ITlb were 0.151 to 0.320, 0.052 to 0.380, and0.077 to 0.268, respectively, with no significant differencesbetween the groups).When tested for IgG class antibodies in the pYV+ ELISA

(Fig. 4), sera from group IIIa adsorbed with pYV- cells gavesignificantly higher values of ELISA units than sera fromgroups I and II. As a control, the sera were also adsorbedwith a mixture (1:1) of pYV+ and pYV- cells (to removeantibodies to pYV as well as chromosome-encoded Y. en-terocolitica serotype 0:3 antigens) and were thereafter as-sayed in the pYV+ ELISA. The results thus obtained did notdiffer between the four groups (total range, 0.112 to 0.283).Results obtained with sera adsorbed with pYV- cells wereconsidered to be positive for pYV Ab if the test resultexceeded the upper limit of this range, i.e., 0.283. pYV-specific IgG antibodies reached a maximum at 5 weekspostinfection. Thereafter, the antibody levels gradually de-clined, but pYV Ab were still detectable in most patientsafter 12 months.Tube agglutination. The results obtained with traditional

tube agglutination are shown in Fig. 5. PVpOS PVneg, CVid,and CVdd were very satisfactory (Table 1). The correlationsbetween results obtained with XIE and the ELISAs andthose obtained with tube agglutination are also included inTable 1.

Reactive arthritis. Within group Illa, the levels of IgA andIgG antibodies did not differ among patients with arthritis (n= 5) and arthralgia (n = 7) and those without joint com-plaints (n = 13). Neither was persistence of such antibodiesfound in group IIlb when patients with arthralgia (n = 3)were compared with those without (n = 6) (no patientsdeveloped arthritis in this group).

DISCUSSION

In this study we have compared results obtained withdifferent methods to study antibody response after infectionwith Y. enterocolitica serotype 0:3, and we have compared

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description of patient groups, see the legend to Fig. 1. Dashed lines mark the diagnostic cutoff levels.

the applicability of the methods for clinical diagnostic pur-poses. XIE enables simultaneous detection of antibodyresponse against the individual antigens in a complex mix-ture without the need for prior purification and therefore

may be used to pinpoint antigens useful for serology. Furthermore, XIE enables a semiquantitation of the antibody-response detected (1, 22).The antibody response in patients with Y. enterocolitica

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SERODIAGNOSIS OF YERSINIA ENTEROCOLITICA 0:3 INFECTION

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FIG. 3. Immunoblotting of rabbit antiserum against whole bacte-

rial cells. Antisera raised against Y. enterocolitica YeO3O1P+ sero-

type 0:3 (not adsorbed, lanes A and B; preadsorbed with cells of

strain YeO3O1P-, lanes C and D) were tested against cells of strain

YeO3O1P+ (+) or strain YeO3O1P- (-). Arrowheads mark the most

prominent pYV-encoded antigens, and their sizes are given in kilo-

daltons. The double arrowheads mark the pYV-encoded outer mem-

brane protein, YadA, and its subunit as visualized by a monoclonal

YadA antibody (31) tested against cells of strain YeO3O1P+ (lane E).

serotype 0:3 infections detected by means of XIE was

directed against a large number of antigens, as previouslydemonstrated in immunoblotting (15, 33, 34). In accordance

with our previous observations that many antigens of Y.

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FIG. 4. Levels of pYV-specific IgG antibodies against Y. entero-colitica serotype 0:3 as measured in pYV+ ELISA. Sera were

preadsorbed with formalinized cells of strain YeO301P- (see text)to remove antibodies against chromosomally encoded antigens. Fordescription of patient groups, see the legend to Fig. 1. The dashedline indicates the upper limit of the range obtained with sera thatwere preadsorbed with a mixture of strains YeO301P+ andYeO301P- to remove antibodies against plasmid-encoded as well as

chromosomally encoded antigens.

FIG. 5. Levels of antibodies against Y. enterocolitica serotype0:3 as evaluated by tube agglutination by using ethanol-killedbacterial cells as antigen. For description of patient groups, see thelegend to Fig. 1. The dashed line indicates the diagnostic cutoff limit.

enterocolitica 0:3 serologically cross-reacted with otherbacterial species, in particular members of the family En-terobacteriaceae (27), patients with Y. enterocolitica 0:3infections differed from patients with other acute infectiousdiseases mainly in total titer scores and not in the numbers ofprecipitins. Consequently, simple counting of precipitins in agiven serum sample was not a suitable diagnostic method.For diagnostic purposes we had to use the intermediate geltechnique, which enables calculation of individual titers andthe total score. This technique is time-consuming and re-quires an experienced laboratory. Therefore, we would notrecommend XIE for routine diagnostic purposes.

Identification of single precipitins specific for infectionwith Y. enterocolitica serotype 0:3 was not successful.Precipitates that in our previous cross-reaction studies werespecific for Y. enterocolitica 0:3, Y. enterocolitica sero-groups, or Yersinia species (27) were not suitable for diag-nostic purposes in this study, either because of an inexpedi-ent position in the central part of the reference pattern orbecause of unexpected precipitins in some sera from groupII.

Several ELISAs for detection of antibodies against Y.enterocolitica have been described. LPS (3, 5, 6, 13, 14),whole pYV- cells (3, 9), SDS extracts of pYV+ and pYV-cells (11), and Yops (23, 28) were used as antigens. How-ever, these methods have not been systematically evaluatedand compared with respect to (i) variation, (ii) specificityagainst controls with other diarrheal and infectious diseases,(iii) sensitivity, and (iv) correlation of results with thoseobtained with the standard agglutination test.Our ELISAs were all easy to handle, permitting the

examination of a large number of samples within a shortperiod of time. The PVneg and PVPOS for detection of recentinfection were in general high, and the variation was satis-factory. We found the PVneg and PVP.S to slightly favor theLPS ELISA compared with the pYV+ ELISA. On the otherhand, the whole-cell antigen was easier to prepare than theLPS antigen, which requires a rather troublesome prepara-tion that most laboratories would like to avoid.The ELISAs enabled the detection of antibodies of spe-

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cific immunoglobulin classes. For detection of recent infec-tion we found that IgA and IgM antibodies did not addvaluable information to that obtained by IgG antibodiesalone. However, PVneg and PVPOS of the pYV+ ELISA fordetection of recent Y. enterocolitica serotype 0:3 infectionwere somewhat higher for IgM than for IgA and IgG anti-bodies. Furthermore, it is possible that measurement of IgAand IgM antibodies in the LPS ELISA may help to discrim-inate between recent and past infection in patients without a

clinical history of recent disease.Most studies on pYV Ab have been carried out by using

immunoblotting with whole cells or Yops as antigen (e.g.,references 15, 19, 24, and 34). The use of whole bacterialcells expressing pYV-encoded antigens for the ELISA hasnot previously been studied, though Granfors et al. reportedthat the use of an SDS extract of such cells failed to provideinformation additional to that obtained with pYV- cells (11).We found that the pYV+ ELISA enabled specific detectionof pYV Ab when the sera to be examined were preadsorbedwith pYV- Y. enterocolitica serotype 0:3 cells. The level ofpYV Ab in our longitudinally observed Y. enterocolitica 0:3patients was still declining at 12 months postinfection, indi-cating that such antibody response is long lasting. Forty-eight percent of sera from groups I + II revealed positivelevels of pYV Ab. We therefore consider the possibility thatthese sera represent subjects with previous Y. enterocolitica0:3 infection because pYV-encoded antigens have notshown serological cross-reactions with other microbial anti-gens (19, 24). Recent reports have revealed a very highprevalence of pYV Ab in sera from normal subjects (FederalRepublic of Germany, 35% [37] and 43% [28]; Belgium, 46%[32]; and Finland, 22% [24]).

Several of the pYV-encoded antigens have been demon-strated to be important virulence factors (7); furthermore,corresponding antibodies have been reported to providepartial protection against infection in an animal model (35).Therefore, we feel that the establishment of new methods tostudy such antibodies is important. A recent study hassuggested that detection of IgA and IgG pYV Ab may also beuseful to identify patients with chronic infection due to Y.enterocolitica (8).The agglutination test, as traditionally performed in tubes

or microtiter plates by using 0 or OH antigens or untreatedcells, has been the reference standard for measurement ofYersinia antibodies since the late 1960s (e.g., references 3, 4,and 39). We found this method to give very satisfactoryresults. Surprisingly, agglutinating antibodies were still de-tectable after 12 months in sera from most of the longitudi-nally observed patients. This method was, however, more

laborious than the ELISAs. The results obtained with theother methods correlated well with those obtained with thetube agglutination method.None of the methods revealed differences in level or

duration of antibody response among Y. enterocolitica sero-

type 0:3 patients without complications or with reactivearthritis or arthralgia. However, the number of patients wassmall, and the existence of a strong and prolonged IgA (andto a lesser degree also IgG) antibody response in Y. entero-

colitica 0:3 patients with reactive arthritis has been welldocumented in other studies (12, 17).

All the assays studied could be applied for detection ofrecent infection with Y. enterocolitica serotype 0:3. Wefound the LPS ELISA to be preferable because it was lesslaborious than the agglutination test and XIE and carried a

somewhat greater diagnostic specificity than the whole-cellpYV+ ELISA. However, XIE and the pYV+ ELISA were

useful for other reasons. XIE enabled simultaneous exami-nation of the antibody response against a wide range ofchromosomally encoded antigens, and the pYV+ ELISAenabled detection of specific pYV Ab.

ACKNOWLEDGMENTS

We thank Lisbeth Heiden, Bente Larsen, and Kate T0rn0 forskillful assistance.

This work was supported by grants from Lwgeforeningens Forsk-ningsfond, The Ville Heise Foundation, and Erik H0rslev og hustruBirgit H0rslevs Fond. A.P. is a research fellow supported by theDanish Medical Research Council.

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