Comparison of crossed immunoelectrophoresis, enzyme linked immunosorbent assays, and tube agglutination for serodiagnosis of Yersinia enterocolitica serotype O:3 infection

(1)

0095-1137/91/020302-08$02.00/0

Comparison

of Crossed

Immunoelectrophoresis,

Enzyme-Linked

Immunosorbent Assays,

and

Tube

Agglutination

for

Serodiagnosis

of

Yersinia enterocolitica

Serotype

0:3

Infection

ANDERS PAERREGAARD,l* GEOFFREY H. SHAND,2 KNUDGAARSLEV,3 ANDFRANK ESPERSEN1 Department of ClinicalMicrobiology, StatensSeruminstitut, Rigshospitalet, Blegdamsvej, DK-2100Copenhagen0,'

Department of DiagnosticBacteriology and Antibiotics, Statens Seruminstitut, DK-2300Copenhagen S,3 and Department ofMicrobiology, Dakopatts, DK-2600Glostrup, Denmark

Received 24 August 1990/Accepted 1 November 1990

Antibodies against Yersinia enterocolitica serotype 0:3 were measured bycrossed immunoelectrophoresis (XIE) using whole-cell sonicextractas antigenandby enzyme-linked immunosorbentassays (ELISAs) using either purified lipopolysaccharide or wholeformalinized cells expressing virulence plasmid-encoded surface

antigens(pYV+ cells). The resultswerecompared with those obtained with the standard tubeagglutination

method. Sera from threegroupsofpeoplewereexaminedby usingtheseassays. Thefirstgroupconsisted of healthyblooddonors, the secondconsisted of patients with recent infection due tomicroorganismsotherthan Y.enterocolitica0:3,andthe thirdconsisted ofpatients withrecentY. enterocolitica0:3 infection. Sera from thelastgroup werealso obtainedatregularintervals for 12 monthspostinfection.ResultsobtainedwithXIE andthe ELISAswereingoodagreement with those obtained with tubeagglutination. Variation, diagnostic

sensitivity, and diagnostic specificityweresatisfactoryfor all theassaysstudied. However,the lipopolysaccha-ride ELISA was less laborious than tube agglutination and XIE and carried asomewhat greater diagnostic specificity than thepYV+ ELISA. XIE and thepYV+ ELISA,onthe otherhand, alsohadadvantages. XIE enabled simultaneousexamination of the individual antibodyresponse againstawide rangeof chromosome-encodedantigens,and thepYV+ELISA enabled detection ofspecific pYVantibodieswhenserawereadsorbed

with formalinized pYV-curedY. enterocolitica 0:3 cellspriortotheassay.

Yersinia enterocolitica is an enteric pathogen which causesacutegastrointestinal disease in humans. The organ-ismprobablyalsoplaysanimportantrole in thepathogenesis ofseveral

immunopathological disorders,

themost common

being reactive arthritis (10, 12, 16). In Canada, Northern Europe,andJapanandincreasinglyin theUnitedStates(2), organisms causing human disease are

predominantly

ofthe 0:3serotype.The Yersiniavirulence

plasmid,

pYV, encodes

anumber ofproteins considered to be important virulence factors (5). Expression ofthese virulence characteristics is temperaturedependent, being optimalat37°C. Immunoblot-ting hasrevealed that Y.enterocoliticainfection gives riseto an antibody response directed against a wide range of bacterial antigens (15, 33, 34).

Theabilitytoquantitate theimmunoglobulin class-specific antibodyresponse tothese antigens, both chromosomal and plasmid encoded, is ofdiagnostic interest and may also lead to abetterunderstanding ofthe processofpathogenesis.

In this study we first examined the antibody response againstalarge pool of chromosomally encoded Y. enteroco-litica serotype 0:3 antigens by means of crossed immuno-electrophoresis (XIE). Thereafter, we evaluated and com-pared thediagnosticvalue ofenzyme-linked immunosorbent assays (ELISAs), in which the antigens used were either purified Y. enterocolitica 0:3 lipopolysaccharide (LPS) or whole formalinized bacterial cells expressing pYV-encoded antigens. We also developed a simple method for quantita-tion of antibodies (designated pYV Ab) against pYV-en-coded antigens. The results were compared with those obtained with the traditional tube agglutination method.

*Correspondingauthor.

MATERIALS ANDMETHODS

Patients and normal controls. Sera from three groups of subjectswereincluded. Group I was composed of 78healthy Danish blood donors (mean age, 38 years; range, 18 to 68 years); 31 were females and 47 were males. Group II was composed of 39 patients withculture-provenacutediarrheal diseases orbacteremias caused by organisms other than Y. enterocolitica serotype 0:3. The- microorganisms isolated from this group were Y. enterocolitica0:9(n = 2), Yersinia pseudotuberculosis serotype O:IV (n = 1),

Campylobacter

jejuni(n = 1), Clostridium difficile (n = 2), Salmonellatyphi (n = 6), Salmonella paratyphi (n = 6), other Salmonella serotypes (n = 4), Escherichia coli (n = 2), Klebsiella pneumoniae (n = 3), Morganella morganii (n = 1), Staphy-lococcusaureus(n= 2), Streptococcus pneumoniae (n =2), other Streptococcus species (n = 4), Haemophilus parain-fluenzae (n = 1), and Brucella abortus (n = 2). These sera wereincludedtodetectfalse-positivereactions inthe assays examined. They were obtained 2 to 6 weeks after theonset of disease. Group IlIa was composed of patients with culture-proven Y. enterocolitica 0:3 infection. Serum sam-pleswere collected 2 to 4 weeks after the start of diarrhea. Nine of these patients were further followed prospectively over a12-monthperiod (group IIlb). Blood was drawn from thesepatients 2, 5, 7, 12, 26, and 52 weeks after the start of diarrhea. In orderto ensurethat the sera were comparable with respect to this time schedule, all blood samples were drawnby oneof the authors during visits to the patients in their homes. Presence or absence of joint symptoms was recorded atthe same time.

Strains and cultureconditions. The two Y. enterocolitica serotype 0:3 strains, YeL and YeWH, used for XIE have been describedpreviously (25). Strain YeL was also used for

302

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tube agglutination. For the other procedures a virulence plasmid-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 under conditions known to promote expression of pYV-encoded antigens. StrainsYeO301P+ andYeO301P- were grownin a rich medium consisting of

1%

tryptone, 0.5% yeast extract, and 0.2% glucose supplemented with the salts describedby Higuchi etal. (20) plus 20 mM sodium oxalate. Cultivation

was performed in 100 ml of medium, which was incubated for 16 h at

20°C

in a water bath with shaking.

Thereafter,

fresh medium was added to give a 20-fold dilution, and incubation was continued for 6 h at37°C. The bacteriawere harvested by centrifugation and washed twice in saline.

Before it was decided how to cultivate bacteriafor extrac-tion of LPS, a pilot study was performed to evaluate the influence of the pYV plasmid and variation in growth tem-perature on composition of the LPS. StrainsYeO301P+and YeO301P- were cultivated as described above; however, for each strain the final 6hof incubationwasperformedat20 and

37°C

inparallel, giving four different bacterial batches. On the basis of the results of this pilot study, LPS to beused in ELISA was extracted from cells ofstrainYeO301P- that had been grown at20°C for 48 h on Truche agarplates and harvested in distilled water.

Bacteria (strain YeL) to be used for tube agglutination were grown for 48 h at 22°Con thick nutrient agar plates and harvested in saline. For immunization of rabbits, strain YeO301P+ was cultured at 20°C for 48 h on Truche agar plates (25).

Preparation of killed whole bacterial cells. (i) ELISAs. Bacteria (strains

YeO301P+

and

YeO301P-)

to be used as antigens for the ELISAs were suspended in saline with 1% formaldehyde at20°C for 2 h, washed three times,and kept at -20°C until use.

(ii) Tube agglutination. Equal volumes of strain YeL in saline and 96% ethanol were slowly mixed and left at 20°C for 24 h. This stock solution was stable forapproximately4 weeks at 4°C. For the final use, the stock solution was dilutedinphosphate-buffered saline (PBS)to aconcentration of approximately5 x 109cellsper ml. Thispreparationcould be used for 3 consecutive days.

(iii) Adsorption of sera. Bacteria (strains YeO301P+ and YeO301P-)to be used foradsorption of serawere washed once in saline, killed by incubationat20°C for 2 h in saline with 1%formaldehyde, washedthree times, and kept at 4°C suspended insaline inquantitiesof 1 g(wet weight) until use. Preparation of LPS. LPSwasextractedby the hot phenol-watermethod(38).TheLPSseparatedbyultracentrifugation wasfurtherpurified bythe method of Hasin et al. (18) and finally resuspended in endotoxin-free water. Purity was determined bymeasuring

A260

andA280whichconfirmedthat LPS preparations were substantially free of contamination withnucleic acids and protein. The four batchesof LPSwere analyzed bysodiumdodecylsulfate-polyacrylamide gel elec-trophoresis(SDS-PAGE) withsilver stainandbyreaction in ELISA.

XIE. A previously described reference system in which sonicated preparations of Y. enterocolitica serotype 0:3 were run againstcorresponding rabbitimmunoglobulins

by

means of XIE was used (25, 27). This system contains 58 regularly visibleprecipitates. Onlychromosomally encoded antigensaredetected. The sera to be examined were incor-porated at aconcentration of 40 pL1/cm2in the intermediate

gelonplates (5 by 5cm). Antibodiespresentin the interme-diate gel against antigens included in the reference system were detected by a reduction in the areas enclosed bythe corresponding precipitates (1). Quantitation of antibodies against Y.enterocolitica antigensinthe intermediategelwas

performedby comparison witha series ofplates

containing

increasingamounts of the reference rabbit antiseruminthe intermediategel (1, 22). Amounts were0, 0.1, 0.2,

1, 2,

10,

20, and 40

RI1/cm2.

Foreachprecipitinin apatient's serum a

titerwasdetermined intherangeof0to8

(1, 22).

These titers were added togive the total score of thispatient's serum.

ELISA.

Immunoglobulin

A

(IgA),

IgG,

and

IgM

antibodies in human sera were analyzed

by

three different indirect ELISAs. Fortheassayof antibodies toLPS

(LPS

ELISA),

96-well polystyrene microtiter

plates

(Polysorb;

Nunc,

Roskilde,Denmark)werecoated witha1% solution of LPS in PBS, pH 7.2. Plates were incubated for 2 h and then overnight at 4°C. Those

plates

not used

immediately

were washed three times withPBS and dried. Dried

plates

could be storedat4°C foratleast 2 monthswithno

significant

loss ofactivity.

Test sera werediluted1:6,000 and 1:750inPBS

containing

0.1% Tweenfor detection of

IgG

and

IgA/IgM

antibodies,

respectively, and 100 ,ulwas incubated in eachcoated well for 2 h. After

being

washed with

PBS-Tween,

wells were incubatedfor1 hwith100,ul of

peroxidase-conjugated

rabbit anti-human

IgG

(Dakopatts,

Glostrup,

Denmark)

diluted 1:5,000 in PBS-Tween or 100 ,ul of

peroxidase-conjugated

rabbit anti-human

IgA

or IgM

(Dakopatts)

diluted

1:2,000.

Plateswere then washed

again,

andcolorwas

developed

by

using

o-phenylenediamine (0.67

mg/ml)

in 0.1 M citrate-phosphate buffer, pH 5.0,

containing 0.0125%

H202.

The color reactionwas

stopped

after 10min

by

addition of150

p.l

of1M sulfuric acidtoeach well.

Optical

density

at 492nm (OD492)wasmeasured witha

photometer

(EIA

Reader

2550;

BioRad,

Richmond,

Calif.).

For assays of antibodies to whole Y. enterocolitica

cells,

microtiter plates

(Maxisorb; Nunc)

werecoated with forma-linized whole cells

(strain

YeO3O1P+:pYV+

ELISA and

YeO3O1P-:pYV- ELISA)

diluted to an

OD540

of 0.2 in carbonate

buffer,

pH 8.6. After

overnight

incubation as described above,

plates

were washed three times with PBS containing

5%

lactose and 0.5% bovine serum albumin. Thereafter,theassay

procedure

wascarriedout asdescribed above, except that serum

samples

were diluted

1:6,000

and 1:3,000 for assays of

IgG

and

IgA/IgM antibodies,

respec-tively. In all cases, the dilutions of serum

samples

were

designedto

bring

thefinal OD of the

majority

ofserawithin the 0 to 2.0range of the

photometer.

Optimal

concentration ofreagentswas determined

by

checkerboard titration.

All samples were tested in

triplicate,

andthe mean value was calculated. Positive and

negative

control sera were

included inevery

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 were

included oneveryplateforconstructionofastandardcurve. TheODofevery

sample

was

adjusted

tothestandard 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

background

values.

Tube agglutination.

Samples

(0.3

ml)

of the final prepara-tion ofethanol-killed cells were added to 0.2 ml of twofold dilutionsofserumin

PBS,

starting

withadilution of 1 in 10.

The results were read as the

highest

final dilution in the

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0.5-ml

sample

with

granular agglutination

after incubation for 20 hat

52°C.

Adsorption

ofsera.

Fifty

serum

samples

from group I, 27 from group

II,

and all serafrom groups Illaand IlIb were

adsorbed with

pYV-

Y. enterocolitica serotype 0:3 cellsto remove

chromosomally

encoded

antigens. Adsorption

was

performed

by

incubating

2mlofa1:100dilution oftheserum in

question (in

PBS

containing

0.1%

Tween)

with 1 g

(wet

weight)

of

pelleted

formalinized YeO301P- cells grownat

37°C.

Incubationwas

performed

for 4 hat

4°C

in vials

slowly

tumbling

end over end.

Thereafter,

the bacteria were

pel-leted

by centrifugation,

and the supernatantwasreincubated under identical conditions with another 1 g of bacteria

overnight. Finally,

thebacteriawerediscarded after

centrif-ugation,

and the adsorbed sera were

passed through

a

0.45-Rm

filter

(Sartorius, Gottingen,

Federal

Republic

of

Germany)

before storage at

-20°C

untiluse. The adsorbed sera were

assayed

inafinal dilution of1:500 inthe

ELISAs,

since that dilution gave OD values within the 0 to 2.0

measuring

rangeofthe

photometer.

As

controls,

sera were also adsorbedwithamixture

(1:1)

of

pYV+

and

pYV-

Y. enterocolitica cells to remove anti-bodiesto chromosomeaswellas

plasmid-encoded antigens.

The

adsorption

was carried out

according

to the schedule described above.

SDS-PAGE and

immunoblotting.

Whole-cell bacterial

sam-ples

were dissolvedin denaturation buffer

by boiling

for 10 min,

separated by SDS-PAGE,

and electroblotted onto nitrocellulose paper as described

previously

(29).

Rabbit serumfor

immunoblotting

was obtained aftersubcutaneous vaccination of three rabbitswithstrainYeO301P+

according

to the schedule used

by

Skurnik

(30).

Incubations of

verti-cally

cut nitrocellulose paper

strips

with sera and with horseradish

peroxidase-conjugated

swine

antibody

torabbit

immunoglobulins (Dakopatts, Glostrup, Denmark)

were

both

performed

for 2 h at room temperature with three

following

washes.

Finally,

the

strips

were visualized with

tetramethylbenzidine (Merck, Darmstadt,

Federal

Republic

of

Germany) (21).

Statistics. The

specificity

and

sensitivity

ofthe methods were determined as described

by Wulff (40) by using

the results obtained with sera from groups II and

IlIa.

The

diagnostic sensitivity

is the

predictive

value of a

negative

test

(PVneg).

The

diagnostic specificity

isthe

predictive

value ofa

positive

test

(PVP,S).

The

nosographic sensitivity

is the percentage of

patients

with the disease who have a

positive

test.The

nosographic

specificity

is the percentageof

patients

withoutthe 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 of variation for the methods

(CVid

and CVdd, respectively)

weredetermined. The standard deviationwascalculated by

using

theformula\

S/27,

where d2is the sumof squared differences between double determinations of the same

sample

and n is the number ofobservations.

For eachmethod the cutoff level(to discriminate between sera thatwere

positive

and negative forrecent Y. enteroco-litica

infection)

that gave the best PVneg and

PVpOS

was chosen. Discriminationbetween serathat were positive and

negative

for

pYV

Ab in the pYV+ ELISA was, however, handled

differently.

Antibodiesto pYV as well as chromo-some-encoded

antigens

wereremovedfromseraby means of

adsorption

withformalinizedpYV+andpYV- Y. enteroco-litica cells. The upperlimitobtained with these preadsorbed

serawas chosenas the cutoff.

80

- 60-a

40

20.

0

I

Gr.I Gr.II Gr. Illa 3 5 7 12 26 52 (weeksafterstart ofsymptoms)

Gr. llb

FIG. 1. Levels of precipitating antibodies against Y. enteroco-litica serotype0:3 in humansera asmeasuredby XIE. Results are givenas totalprecipitinscores(see text).Group I(Gr. I), Healthy blood donors; group II (Gr. II), patients with recent diarrhea or bacteremiaduetomicroorganisms other than Y.enterocolitica 0:3; group Illa (Gr. IIIa), patients with recent Y. enterocolitica 0:3 infection; group IlIb (Gr. IlIb), longitudinally observed patients with Y.enterocolitica0:3infection. Dashedline marks the diagnos-tic cutofflevel.

Forcomparisonof results obtained with different groups of sera, theMann-Whitney rank sumtestforunpaired data wasused. TheSpearman rankcorrelationtest wasused for comparison of results obtained with differentmethods;P < 0.05was chosenasthe level of significance.

RESULTS

XIE.Antibodiesagainst19antigensweredetected in sera from patients with recent Y. enterocolitica serotype 0:3 infection. Antibodies against a small number of additional antigens werealso detected in some sera, butnotall plates could be evaluated with respect to changes in the

corre-sponding

precipitates because of their localization in the central part of the reference system.

Sera from blood donors(group I) hadamedian number of 10

precipitins

(range, 8 to 13), givinga median score of27 (range, 23 to 43), while sera from patients with other

gastrointestinal

orinvasiveinfectious disease(group II) had amediannumber of 16precipitins (range,14to18),givinga median scoreof 41 (range,34to68). Inthe group consisting ofpatients withrecent Y. enterocolitica serotype 0:3 infec-tions (group Illa), themedian numberof precipitinswas 17 (range, 15to 19)and themedian score was 62 (range, 43 to 79).Thescoresobtainedwith serafrom the different patient groupsareillustratedinFig. 1.Within the three groups, the variations in precipitin titers and scores were much more pronouncedthan thevariationin the number of precipitins. The scores of groups I and IlIa both overlapped with the scoresof group II. The serain group II that gave the highest scores originated from patients with infections caused by other

yersineae

and othermembersof thefamily Enterobac-teriaceae.

PVpos,

PVneg'

CVdd,andCVdd for the method are

given

in Table 1. None of the individual antigens proved

specific

for Y. enterocolitica serotype 0:3infection. The

antibody

levels inserafrompatients with Y.

entero-XIE

0

A

0 04o S

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e- 3

S

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TABLE 1. Clinicalapplicabilityofand variation andcorrelation among resultsofthetubeagglutination, XIE,andELISAsfor detection ofrecent Y.enterocolitica serotype0:3 infectiona

Assay Nosographic Nosographic PV PV *

CVid

CVdd Correlation with tube

sensitivity specificity neg po agglutination(rho)b

Tubeagglutination 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*

LPSELISA

IgA

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+

ELISA

IgA 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*

aNosographic sensitivity and specificity,PVneg,

PVpos,

CVid,andCVdd are given as percentages with 95% confidence limits. For CV values, only the upper

limit isgiven.

bSpearman rank correlation test; asterisks indicate P<0.05.

colitica infection,asevaluated by the scores, decreased with time but exceededthe scores obtained by most blood donor serafor thewhole 12-month observation period (Fig. 1).

ELISA. The backgrounds (median and range) in the ELISA systems, as evaluated by the OD values of wells containing buffer only, were 0.087 and0.038 to 0.160 (LPS ELISA),0.164 and 0.082 to 0.287 (pYV+ ELISA), and 0.090 and0.079to0.105(pYV- ELISA).

(i)LPS ELISA. LPS extracted frombacteriagrown with andwithout thevirulenceplasmid and at different tempera-tures appeared to be identical in SDS-PAGE after silver staining andgave veryclose comparable results when used in the ELISAto assay a numberof sera from blood donors and patients with recent Y. enterocolitica serotype 0:3 infections (datanotshown). Therefore,LPS extracted from plasmid-cured cells grown at20°C was used for the rest of the study, assuchcells give the highest yield of

serogroup-specific

sugars(36).

Figure 2A shows the results obtained with the different groups ofserainthe LPS ELISA. The PVneg and

PVpos

of the methodswerehigh, and the variations weresatisfactory (Table 1). Persistence ofIgA, IgG, and IgM antibodies in longitudinally observed patients isalsoillustratedin Fig. 2A. Almost no sera were positive with respect to IgA or IgM antibodies formore than 3 months postinfection. IgG

anti-bodies,

ontheotherhand,persisted for longer periods in all the patients, still giving positive results by 12 months in severalcases.

(ii) pYV+ ELISA. Y.enterocoliticaserotype0:3 cells used as antigen for the pYV+ ELISA

expressed

several ofthe

pYV-encoded antigens (Fig.

3).

Figure

2B

gives

theresults obtained with the pYV+ ELISA. PVneg and

PVpOS

for detectionofIgA and

IgG

antibodies weresomewhat lower thanin the LPS ELISA.However, thiswas notthecasefor IgM

antibodies

(Table 1).

Variationwaslow

(Table 1). IgA

andIgMantibodylevelsdeclinedfaster than

IgG

levels

(Fig.

2B),as wasthe casefor theLPSELISA. Ontheother

hand,

the pYV+ ELISA revealed several

patients

with sera that were stillpositivein all three

immunoglobulin

classes after 12months.

(iii) Detection ofpYV-specific antibodies. After

adsorption

withformalinized pYV-cells, theseracontained

only

small amounts ofLPS antibodiesand antibodies

against

chromo-somally encoded surface

antigens

(both

IgG class),

as

eval-uated by LPS ELISA (rangesforgroups I, II, andIlIaand IlIb were0.041 to0.074, 0.043 to 0.066, and 0.027 to 0.075, respectively, with no significant differences between the groups) 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, and 0.077 to 0.268, respectively, with no significant differences between the groups).

Whentestedfor IgGclass antibodies inthe pYV+ ELISA (Fig. 4),serafromgroup IIIaadsorbedwith pYV- cells gave significantly higher values of ELISA units than sera from groups I and II. As a control, the sera were also adsorbed with a mixture (1:1) ofpYV+ and pYV- cells (to remove antibodies to pYV as well as chromosome-encoded Y. en-terocolitica serotype 0:3 antigens) and were thereafter as-sayed inthepYV+ ELISA.The results thusobtained didnot differ between the fourgroups (totalrange, 0.112 to0.283). Results obtained with seraadsorbed with pYV- cells were considered to be positive for pYV Ab if the test result exceeded the upper limit of this range, i.e., 0.283.

pYV-specific

IgG antibodies reached a maximum at 5 weeks postinfection.

Thereafter,

the antibody levels gradually

de-clined,

but pYV Ab were still detectable in most patients after12 months.

Tubeagglutination. The results obtained with traditional tubeagglutination areshown in

Fig.

5.

PVpOS

PVneg,

CVid,

andCVddwere very

satisfactory (Table

1). The correlations between results obtained with XIE and the ELISAs and those obtained withtube agglutination are also includedin Table 1.

Reactive arthritis. Withingroup

Illa,

thelevels of

IgA

and IgG antibodies didnotdifferamong

patients

witharthritis

(n

= 5) and

arthralgia

(n = 7) and those without

joint

com-plaints (n = 13). Neitherwas

persistence

of suchantibodies found in group

IIlb

when

patients

with

arthralgia

(n

=

3)

were compared with those without (n = 6) (no

patients

developed

arthritisin this

group).

DISCUSSION

In this study we have

compared

results obtained with differentmethodsto

study

antibody

response after infection with Y. enterocolitica serotype

0:3,

andwehave

compared

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(5)

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* ~~~~~~~~~0 * 0

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Gr.I Gr.II Gr. Illa 3 5 7 12 26 52 (weeks aftarstartofsymptoms)

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Gr.I Gr.ll Gr. lIla 3 5 7 12 26 52 (weeks after start ofsymptoms)

Gr. lllb

FIG. 2. Levelsof IgA,IgG, and IgM antibodies against Y. enterocolitica serotype 0:3 as measured in LPS ELISA and pYV+ ELISA. For

description of patientgroups, 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 antibody response against the individual antigens in acomplex mix-ture without the need for prior purification and therefore

may be used to pinpoint antigens useful for serology. Fur thermore,XIE enables asemiquantitation of the antibody-response detected(1, 22).

The antibody response in patients with Y. enterocolitica

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B

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3 5 7 12 26 52 (weeksafterstart of symptoms)

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FIG. 3. Immunoblottingof rabbit antiserumagainstwhole bacte-rial cells. Antisera raisedagainst Y. enterocolitica YeO3O1P+

sero-type 0:3 (not adsorbed, lanes A and B; preadsorbed with cells of strainYeO3O1P-,lanesC andD)weretestedagainstcells ofstrain YeO3O1P+(+)orstrain YeO3O1P-(-).Arrowheadsmark themost

prominent pYV-encoded antigens,and their sizesaregivenin kilo-daltons. The double arrowheads mark thepYV-encodedouter

mem-braneprotein, YadA, and its subunitasvisualizedbya monoclonal YadAantibody(31)testedagainstcells ofstrain YeO3O1P+(laneE).

serotype 0:3 infections detected by means of XIE was

directed against a large number ofantigens, as previously

demonstrated inimmunoblotting(15, 33, 34). Inaccordance with our previous observations that many antigens of Y.

U,

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Gr. I Gr.II Gr. Ilia 3 5 7 12 26 52

(weeks afterstartofsymptoms)

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FIG. 4. LevelsofpYV-specific IgGantibodiesagainstY.

entero-colitica serotype 0:3 as measured in pYV+ ELISA. Sera were

preadsorbed withformalinized cells of strainYeO301P- (see text) toremoveantibodiesagainst chromosomallyencodedantigens. For descriptionofpatientgroups,seethelegendtoFig. 1. The dashed

line indicates theupperlimit of the range obtainedwith sera that

were preadsorbed with a mixture of strains YeO301P+ and YeO301P-toremoveantibodiesagainst plasmid-encodedaswellas

chromosomally encoded antigens.

FIG. 5. Levels of antibodies against Y. enterocolitica serotype

0:3 as evaluated by tube agglutination by using ethanol-killed

bacterial cellsasantigen. Fordescriptionof patientgroups, see the

legendtoFig.1.The dashed line indicates the diagnostic cutoff limit.

enterocolitica 0:3 serologically cross-reacted with other bacterial species, in particular members of the family En-terobacteriaceae (27), patients with Y. enterocolitica 0:3 infections differed from patients with other acute infectious diseasesmainlyintotal titerscoresand not in the numbers of precipitins. Consequently, simple counting of precipitins in a given serum sample was not a suitable diagnostic method. Fordiagnostic purposes we had to use the intermediate gel technique, whichenables calculation of individual titers and the total score. This technique is time-consuming and re-quiresanexperienced laboratory. Therefore, we would not recommend XIEfor routine diagnostic purposes.

Identification of single precipitins specific for infection with Y. enterocolitica serotype 0:3 was not successful. Precipitates that in ourpreviouscross-reaction studies were

specific

for Y. enterocolitica 0:3, Y. enterocolitica sero-groups, or Yersinia species (27) were not suitable for diag-nosticpurposesin this study, either because of an inexpedi-ent position in the central part ofthe reference pattern or because ofunexpected precipitins in some serafrom group II.

Several ELISAs for detection of antibodies against Y.

enterocolitica have been described. LPS (3, 5, 6, 13, 14), whole cells (3, 9), SDS extracts of pYV+ and pYV-cells (11), and Yops (23, 28) were used as antigens. How-ever, thesemethods havenotbeensystematicallyevaluated and compared with respect to (i) variation, (ii) specificity againstcontrols with other diarrheal and infectiousdiseases, (iii)

sensitivity,

and

(iv)

correlation of results with those obtained with the standard agglutinationtest.

Our ELISAs were all easy to handle, permitting the

examination of a large number of

samples

within a short

period of time. ThePVnegand

PVPOS

fordetection ofrecent infection were ingeneral high, and the variationwas

satis-factory. Wefound thePVnegand

PVP.S

toslightly favor the LPS ELISA

compared

withthe

pYV+

ELISA. On the other

hand, thewhole-cell

antigen

waseasierto prepare than the LPS antigen, which

requires

arather troublesome prepara-tion that mostlaboratories would liketoavoid.

The ELISAs enabled the detection of antibodies of

spe-

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cific immunoglobulin classes. For detection ofrecent infec-tion we found that IgA and IgM antibodies did not add valuable information to that obtained by IgG antibodies alone. However, PVneg and

PVPOS

of the pYV+ ELISA for detection ofrecent Y. enterocolitica serotype 0:3 infection

were somewhat higher for IgM than for IgA and IgG

anti-bodies. Furthermore, it ispossible that measurementofIgA andIgM antibodies in the LPS ELISA may helpto discrim-inatebetweenrecent andpastinfection in patients withouta

clinical history ofrecent 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 ofwhole bacterial

cells expressing pYV-encoded antigens for the ELISA has notpreviously been studied, though Granfors etal. reported thattheuseofanSDSextractof such cells failedtoprovide information additionaltothatobtained with pYV- cells (11). We foundthat the pYV+ ELISA enabled specific detection ofpYV Ab when theseratobeexaminedwerepreadsorbed

withpYV- Y. enterocolitica serotype0:3 cells. The level of pYV Ab inourlongitudinallyobserved Y. enterocolitica 0:3

patients wasstill declining at12 months postinfection, indi-cating that such antibody response is long lasting.

Forty-eight percent of sera from groups I + II revealed positive levels ofpYV Ab. We therefore consider the possibility that these serarepresentsubjects with previous Y. enterocolitica 0:3 infection because pYV-encoded antigens have not

shown serological cross-reactions with other microbial

anti-gens (19, 24). Recent reports have revealed a very high

prevalence of pYV Ab inserafrom normal subjects(Federal

Republic of Germany, 35% [37] and 43% [28]; Belgium, 46% [32]; andFinland, 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 provide partial protection against infection in an animal model (35).

Therefore, wefeelthat the establishment ofnewmethodsto study such antibodies is important. A recent study has suggested that detection of IgA and IgG pYV Abmayalso be

useful to identify patients with chronic infection due to Y. enterocolitica (8).

The agglutinationtest, astraditionally performed in tubes

ormicrotiterplates by using 0 orOHantigens oruntreated cells, has been the reference standard for measurement of Yersiniaantibodiessince the late 1960s(e.g.,references3, 4, and 39). We found this method to give very satisfactory

results. Surprisingly, agglutinating antibodies were still

de-tectable after12months inserafrom mostofthe longitudi-nally observed patients. This method was, however, more

laborious than the ELISAs. The results obtained with the othermethods correlated well with those obtained with the tubeagglutination method.

None of the methods revealed differences in level or

durationofantibodyresponseamong Y. enterocolitica

sero-type 0:3 patients without complications or with reactive

arthritisorarthralgia. However, the numberof patientswas

small, and the existenceofastrongand prolonged IgA (and to alesserdegree also IgG) antibodyresponsein Y.

entero-colitica 0:3 patients with reactive arthritis has been well documentedin otherstudies (12, 17).

All the assays studied could be applied for detection of recent infection with Y. enterocolitica serotype 0:3. We found theLPS ELISA to be preferable because it was less

laborious than the agglutination testandXIE and carried a

somewhatgreaterdiagnostic specificity than the whole-cell pYV+ 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 of chromosomally encoded antigens, and the

pYV+

ELISA enabled detection of

specific pYV

Ab.

ACKNOWLEDGMENTS

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

This workwassupported bygrantsfrom Lwgeforeningens Forsk-ningsfond,The VilleHeiseFoundation, and Erik H0rslev og hustru Birgit H0rslevs Fond. A.P. is a research fellow supported by the

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