Copyright © 1991, AmericanSociety forMicrobiology
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
predominantlyofthe 0:3serotype.The Yersiniavirulence
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.
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),
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
on April 12, 2020 by guest
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°Cin a water bath with shaking.
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°Cinparallel, 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-)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
A260andA280whichconfirmedthat 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
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
increasingamounts of the reference rabbit antiseruminthe intermediategel (1, 22). Amounts were0, 0.1, 0.2,
20, and 40
RI1/cm2.Foreachprecipitinin apatient's serum a
(1, 22).These titers were added togive the total score of thispatient's serum.
IgMantibodies in human sera were analyzed
bythree different indirect ELISAs. Fortheassayof antibodies toLPS
96-well polystyrene microtiter
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
immediatelywere washed three times withPBS and dried. Dried
platescould be storedat4°C foratleast 2 monthswithno
Test sera werediluted1:6,000 and 1:750inPBS
0.1% Tweenfor detection of
respectively, and 100 ,ulwas incubated in eachcoated well for 2 h. After
PBS-Tween,wells were incubatedfor1 hwith100,ul of
Denmark)diluted 1:5,000 in PBS-Tween or 100 ,ul of
Plateswere then washed
mg/ml)in 0.1 M citrate-phosphate buffer, pH 5.0,
H202.The color reactionwas
of1M sulfuric acidtoeach well.
densityat 492nm (OD492)wasmeasured witha
For assays of antibodies to whole Y. enterocolitica
(Maxisorb; Nunc)werecoated with forma-linized whole cells
YeO3O1P-:pYV- ELISA)diluted to an
OD540of 0.2 in carbonate
buffer,pH 8.6. After
overnightincubation as described above,
plateswere washed three times with PBS containing
5%lactose and 0.5% bovine serum albumin. Thereafter,theassay
procedurewascarriedout asdescribed above, except that serum
1:6,000and 1:3,000 for assays of
IgA/IgM antibodies,respec-tively. In all cases, the dilutions of serum
bringthefinal OD of the
majorityofserawithin the 0 to 2.0range of the
Optimalconcentration ofreagentswas determined
All samples were tested in
triplicate,andthe mean value was calculated. Positive and
negativecontrol sera were
samplesof control sera with low
1)OD values were
included oneveryplateforconstructionofastandardcurve. TheODofevery
adjustedtothestandard curve, and the results are
arbitraryELISA units. Back-ground was the OD of wells incubated with buffer alone. Results are
givenwithout subtraction of the
ml)of the final prepara-tion ofethanol-killed cells were added to 0.2 ml of twofold dilutionsofserumin
startingwithadilution of 1 in 10.
The results were read as the
highestfinal dilution in the
on April 12, 2020 by guest
granular agglutinationafter incubation for 20 hat
samplesfrom group I, 27 from group
II,and all serafrom groups Illaand IlIb were
pYV-Y. enterocolitica serotype 0:3 cellsto remove
incubating2mlofa1:100dilution oftheserum in
Tween)with 1 g
pelletedformalinized YeO301P- cells grownat
performedfor 4 hat
tumblingend 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
Germany)before storage at
-20°Cuntiluse. The adsorbed sera were
assayedinafinal dilution of1:500 inthe
since that dilution gave OD values within the 0 to 2.0
controls,sera were also adsorbedwithamixture
pYV-Y. enterocolitica cells to remove anti-bodiesto chromosomeaswellas
adsorptionwas carried out
accordingto the schedule described above.
sam-pleswere dissolvedin denaturation buffer
by boilingfor 10 min,
separated by SDS-PAGE,and electroblotted onto nitrocellulose paper as described
immunoblottingwas obtained aftersubcutaneous vaccination of three rabbitswithstrainYeO301P+
to the schedule used
verti-callycut nitrocellulose paper
stripswith sera and with horseradish
immunoglobulins (Dakopatts, Glostrup, Denmark)were
performedfor 2 h at room temperature with three
stripswere visualized with
tetramethylbenzidine (Merck, Darmstadt,Federal
sensitivityofthe methods were determined as described
by Wulff (40) by usingthe results obtained with sera from groups II and
diagnostic sensitivityis the
predictivevalue of a
nosographic sensitivityis the percentage of
patientswith the disease who have a
specificityis the percentageof
withoutthe disease who have a
On the basis of
repeateddeterminations of 10 serum
3),medium(n = 4),and
day-to-daycoefficients of variation for the methods
(CVidand CVdd, respectively)
weredetermined. The standard deviationwascalculated by
S/27,where d2is the sumof squared differences between double determinations of the same
sampleand n is the number ofobservations.
For eachmethod the cutoff level(to discriminate between sera thatwere
positiveand negative forrecent Y. enteroco-litica
infection)that gave the best PVneg and
PVpOSwas chosen. Discriminationbetween serathat were positive and
pYVAb in the pYV+ ELISA was, however, handled
differently.Antibodiesto pYV as well as chromo-some-encoded
antigenswereremovedfromseraby means of
adsorptionwithformalinizedpYV+andpYV- Y. enteroco-litica cells. The upperlimitobtained with these preadsorbed
serawas chosenas the cutoff.
Gr.I Gr.II Gr. Illa 3 5 7 12 26 52 (weeksafterstart ofsymptoms)
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.
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-spondingprecipitates 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
gastrointestinalorinvasiveinfectious 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
yersineaeand othermembersof thefamily Enterobac-teriaceae.
PVneg'CVdd,andCVdd for the method are
givenin Table 1. None of the individual antigens proved
specificfor Y. enterocolitica serotype 0:3infection. The
antibodylevels inserafrompatients with Y.
0 04o S
- - -.- - --&
on April 12, 2020 by guest
TABLE 1. Clinicalapplicabilityofand variation andcorrelation among resultsofthetubeagglutination, XIE,andELISAsfor detection ofrecent Y.enterocolitica serotype0:3 infectiona
Assay Nosographic Nosographic PV PV *
CVidCVdd 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*
IgA100(86-100) 97(87-100) 100(91-100) 96
(80-100)15 (40) 17(43) 0.67*
IgG100(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*
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
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
Figure 2A shows the results obtained with the different groups ofserainthe LPS ELISA. The PVneg and
PVposof 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
pYV-encoded antigens (Fig.3).
givestheresults obtained with the pYV+ ELISA. PVneg and
PVpOSfor detectionofIgA and
IgGantibodies weresomewhat lower thanin the LPS ELISA.However, thiswas notthecasefor IgM
(Table 1). IgA
2B),as wasthe casefor theLPSELISA. Ontheother
the pYV+ ELISA revealed several
patientswith sera that were stillpositivein all three
immunoglobulinclasses after 12months.
(iii) Detection ofpYV-specific antibodies. After
withformalinized pYV-cells, theseracontained
onlysmall amounts ofLPS antibodiesand antibodies
againstchromo-somally encoded surface
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
ITlbwere 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-specificIgG 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
satisfactory (Table1). The correlations between results obtained with XIE and the ELISAs and those obtained withtube agglutination are also includedin Table 1.
Reactive arthritis. Withingroup
IgAand IgG antibodies didnotdifferamong
= 5) and
arthralgia(n = 7) and those without
com-plaints (n = 13). Neitherwas
persistenceof suchantibodies found in group
3)were compared with those without (n = 6) (no
In this study we have
comparedresults obtained with differentmethodsto
antibodyresponse after infection with Y. enterocolitica serotype
on April 12, 2020 by guest
IPS ELISA IgA
I * g
I-I_ - I- -r
LPS ELISA IgG
* ~~~~~~~~~0 * 0
0 * S~~~0
1 * *
~~~00 0 * 0*
Gr.I Gr.II Gr. Illa 3 5 7 12 26 52 (weeks aftarstartofsymptoms)
8 0 0
* 0 0
>2.0I I I I
Gr.I Gr.ll Gr. lIla 3 5 7 12 26 52 (weeks after start ofsymptoms)
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
I IIl k I I I I
-1 G >2.0
i I I i i v
f . i i i
on April 12, 2020 by guest
- + - + l.4
46- 4 -- 4 4
Gr. I Gr. II Gr. Ilia
I I I I I 1
3 5 7 12 26 52 (weeksafterstart of symptoms)
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.
Gr. I Gr.II Gr. Ilia 3 5 7 12 26 52
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
specificfor 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)
(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
sampleswithin a short
period of time. ThePVnegand
PVPOSfordetection ofrecent infection were ingeneral high, and the variationwas
satis-factory. Wefound thePVnegand
PVP.Stoslightly favor the LPS ELISA
pYV+ELISA. On the other
antigenwaseasierto prepare than the LPS antigen, which
requiresarather troublesome prepara-tion that mostlaboratories would liketoavoid.
The ELISAs enabled the detection of antibodies of
on April 12, 2020 by guest
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
PVPOSof 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%  and 43% ; Belgium, 46% ; andFinland, 22% ).
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
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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