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Enzyme linked immunosorbent assay to evaluate the immunogenicity of a polyvalent Klebsiella capsular polysaccharide vaccine in humans

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0095-1137/88/112257-05$02.00/0

Enzyme-Linked Immunosorbent

Assay To

Evaluate the

Immunogenicity of a

Polyvalent

Klebsiella

Capsular

Polysaccharide

Vaccine in Humans

M. GRANSTROMl.2* B. WRETLIND,3 B.

MARKMAN,'

AND S.

CRYZ4

DepartmentofVaccineProduction, National Bacteriological Laboratory, S-10521 Stockholm,l* Department of Clinical Microbiology, Karolinska Institute, Karolinska Hospital, S-104 01 Stockholm,2 and Department of

Bacteriology,

DanderydHospital, S-182 88Danderyd, Sweden, and Swiss Serum and Vaccine Institute, 3001 Bern, Switzerland4 Received29February 1988/Accepted1 August 1988

Ahighlysensitive enzyme-linkedimmunosorbentassay was developed forKlebsiellacapsularpolysaccharide (CPS) andusedtoevaluatetheimmunoglobulinG(IgG) antibody responseto a24-valentCPSvaccinein seven adultvolunteers. The median rise in titer toallvaccineantigens in samples from the volunteers was significant (twofold or greater). Significant IgG responses to 11 immunologically related serotypes not included in the vaccine were alsonoted. The mean cross-reacting IgG titer of 127.2 was only slightly lower than the mean titer of 175.7 to the serotypes in thevaccine (P< 0.05). The mean 29.9-fold increase in titer to the serotypesinthe vaccine wassignificantlyhigher than the mean 13.5-fold increase in titer to the additional antigens (P<0.001). Thedifferencewaspartly because of thesignificantlylower (P<0.01)natural antibody titers in the preimmune serato theserotypes in the vaccine, compared with those to serotypes not included in the vaccine. Theselection ofvaccineserotypes wasbased on the frequency of serotype isolationfromcases ofKlebsiela bacteremia.The above findings, which show low levels of natural antibody to these serotypes, support the hypothesis that anti-CPSantibody isprotective against bacteremic disease.

Klebsiella spp. are among the leading causes of life-threatening nosocomial infections (12, 13, 19). Mortality rates can approach50% forKlebsiella bacteremic and pul-monary infections (12, 20). Recently, multiple-antibiotic resistanceamong Klebsiella clinical isolates has been shown to complicate therapy regimens (18, 21). As an alternative, the feasibility of using an immunological approach to the prevention or treatment of Klebsiella infections has been investigated (2).

Protection against fatalexperimental Klebsiellainfections centersaround thepresence of a serospecific anti-capsular-polysaccharide (anti-CPS)antibody (4, 7, 10), which acts to promote the uptake and killing of invading bacteria by phagocytic cells (3). Several experimental Klebsiella CPS-based vaccines have been evaluated in humans and have beenshown tobesafeandimmunogenic (5,8).Basedon the seroepidemiology of Klebsiella bacteremic isolates (9), a 24-valent Klebsiella CPS vaccine has been formulated and evaluated inphase1humantrials.Previously,weemployed an enzyme-linked immunosorbent assay (ELISA) as a con-venient method by which toquantitatethe riseof anti-CPS antibodylevels(5, 8). However,theresponsestoonlyseven CPSserotypes were studied

by

ELISA.

During

the

analysis

ofthehuman immune response tothe 24-valent

vaccine,

it becameapparent that aconsiderable

proportion

ofrelevant CPS serotypes gave

artificially

high

preimmunization

anti-body levels. We describe an ELISA system

capable

of accurately measuring

antibody

levels to all serotypes of Klebsiella CPS antigens and the use ofthis assay in evalu-ating the

immunogenicity

of a 24-valent Klebsiella CPS vaccine.

*

Corresponding

author.

MATERIALS AND METHODS

Purification of CPS.KlebsiellaCPS antigens were purified from culture by N-cetyl-N,N,N-trimethylammonium bro-mide-ethanol

precipitation,

extraction with chloroform-bu-tanol, and ultracentrifugation, as previously described (6). CPS

preparations

contained <3% proteinand <2% nucleic acid (wt/wt). Preparations used in the vaccine and as anti-gens in ELISA were madeinthe same manner but were not necessarilyfrom the same lot. CPS antigens werelyophilized andstored.

Vaccine. Based on the seroepidemiology of Klebsiella bacteremia isolates (9), a 24-valent vaccine was prepared whichcontainedthefollowingcapsular serotypes: 2, 3, 5, 9, 10,15,16,17, 18, 21, 22, 25, 28,30,35, 43,52,53, 55, 60,61, 62, 63,and64. An

appropriate

amount ofeach

antigen

was treated in 0.1 N NaOH in ethanol (95%) to detoxify trace amounts of

contaminating

lipopolysaccharide

(15).

Lipo-polysaccharide

content wasmeasured

by

both

pyrogenicity

in rabbits and

quantiation

of2-keto-3-deoxyoctonate. The

individual polysaccharides

were pooled, filter

sterilized,

divided into samples under aseptic conditions, and

lyophi-lized.

Biological and chemicalcontrols.The

sterility

and

safety

of the vaccine were evaluated

by

methods described under articles V.2.1.1. and V.2.1.5. of the European Pharmaco-poeia.

Pyrogenicity

was tested

by

administering

graded

doses of vaccine

intravenously

to New Zealand White rabbits (three rabbits per group).

Body temperature

was

monitored for4h

postinjection.

A

quantity

of>24

,ug/kg

of body

weight

resulted ina

temperature

riseof<1.40C in three rabbits.

Quantitation

of

proteins,

nucleic

acids,

carbohy-drates, uronic

acids,

andresidualmoisturewas

performed

as

previously

described

(6).

Thevaccine contained 89.7%

car-bohydrate, 1%

protein,

0.84% nucleic

acid,

and 5.8% resid-ual moisture. The

partition

coefficient

(Kd)

was

<0.05,

as

determined

by

chromatography

on the

Sepharose

CL-4B

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2258 GRANSTROM ET AL.

TABLE 1. Optimal coatingdosesofKlebsiella CPSfor determinationof immune responseby ELISA

Coatingdose Serotypes

(,ug/m1)

0.2 10, 13, 16, 21,22, 30, 46

0.5 14, 18,37, 52,61, 69

1.0 2, 26, 31,55, 62

2.0 3, 11

5.0 5, 9, 15,25, 28, 35, 43, 53, 64, 68

10.0 17, 60

20.0 7,12, 63

(Pharmacia, Uppsala, Sweden).The molecularweightofthe vaccine was also determined by chromatography on the

Sepharose CL-4B (6).

Clinical studies. Informed consent was obtained from healthy Swiss adults between the ages of 16 and 54. The

vaccine was reconstituted in sterile distilled water immedi-ately priorto use, and 0.5 ml was administered

subcutane-ously into the deltoid region. This volume of vaccine

con-tained 1.2mgof total CPS (50 p.geach of the 24serotypes).

Reactions to vaccination were recorded for 5 days after

immunization. Serum samples wereobtained atthe time of vaccination and at 7 and 28 days postvaccination. The prevaccination seraandthe28-daypostvaccinationsamples

from theseven volunteerswere alsoincluded in this study.

Standard antiserum. A reference antiserum wasprepared

from samples from individuals immunized with an

experi-mental24-valent vaccine. Fourindividualswere selectedon

the basis ofhavinganacceptable(.10-fold)rise in immuno-globulin G (IgG) antibody to seven vaccine antigens in a

preliminary screening. Equal volumes ofpostimmunization

serum from each individual were mixed and lyophilized in 200-pi samples.

ELISA. The ELISA was performed essentially as

previ-ously described (15). The lyophilized antigens were

dis-solved in 36 mM triethylamine (C6H15N) to concentrations of3to6mg/ml. Dilutionstoworkingcoatingdoses(Table 1)

weremade in 0.05 M carbonate buffer (pH 9.6), and cobalt-irradiated 96-well polystyrene microplates(Dynatech, Ploch-ingen, Federal Republic of Germany) werecoated with 100

,ulperwellovernightatroomtemperature(22°C). The 100-pl volumeswereusedthroughout the study. All washingswere

donethree times in physiological saline supplemented with 0.05% Tween 20.

Pre- andpostimmunization serum samples were tested in

parallel as single-point determinations at dilutions of 10-1, 10-2, 10-3, and if necessary, 10-4 and 10-5. The serum

dilutions, made in phosphate-buffered saline with 0.05% Tween 20, were tested in triplicate and incubated with the

antigen for 1 h at 22°C. Alkaline phosphatase-conjugated

goatanti-human IgG (SigmaChemical Co., St. Louis, Mo.)

wasused ina10-3dilution in phosphate-buffered salinewith 0.05% Tween 20 and was incubated with the conjugate

overnight at 22°C. The substrate, p-nitrophenyl phosphate dissolvedin 1 Mdiethanolamine,wasadded, and the

absorb-ance was read in a Titertek Multiscan (Flow Laboratories,

Irvine, Scotland) when the standard antiserum reached

predeterminedvalues. Theassaythuscalibrated againstthe

standard, the subtrate incubation times for the serotypes

variedfrom20to 90min.

The choice of absorbance values for the standard

antise-rum was determined inrelation toa negative control serum

included in each assay in a 10-1 dilution. The negative control was identified by the screening of serum samples

from blood donors. The serum with the lowest natural antibody levels to most serotypes was chosen. The 10-1 dilution of thisnegative sample had toreach anabsorbance value of 0.1 to 0.2. With the assay thus

optimized

for sensitivity in the low-antibody ranges, the positive standard

was tested in serial twofold dilutions from a 10-2 dilution. The dilutiongivinganabsorbance value between 0.8 and 1.0 wasthen chosenasthepredetermined valuetobe reachedin each assay.

Theserumdilution in the linear part of the titrationcurve wasused for calculation of ELISA titers. The ELISA titers represented themean

A405

value of thetriplicate determina-tions multiplied by the serum dilution factor, e.g., a mean

A405

of0.4 at a10-2 dilution representedan ELISA titer of 40. Asignificant rise in titer was defined as at least a twofold increase. Thedefinition ofasignificant risewasbasedonthe intra-assay variation, which is less than 10% in single-point determinations. Although any difference >20% would be significant, a100%difference, i.e., a twofold rise, was used as a safety margin for a possible nonspecific polyclonal stimulation. The interassay variationwas 20to 25%.

Statistical analysis. The number of observations in seven volunteers was too small to allow for statistical analysis of IgG responsestoindividual serotypes. Data in the tablesare therefore presented asmedian values and ranges. When the differences in IgG responses to vaccine and nonvaccine serotypes are compared, the number of observations was large, i.e., 7 subjects x 24 antigens = 168 observations, and 7 subjects x 11 antigens = 77 observations, respectively. These data allowed for the calculations ofgeometric mean titers(GMT) and geometric mean fold rises and for compar-isonbytheStudent ttest.

RESULTS

The 24-valent vaccine was composedprimarily of carbo-hydrate with trace amounts (21% [wt/vol]) of protein and nucleic acids. The average molecularweight was >2 x 106 The vaccine was nonpyrogenic at a dose of 24 kg/kg per vaccine serotype. No systemic reactions were noted after vaccination. Two vaccinees reported mild transient pain at theinjection site.

Optimal conditions for CPS ELISA. All CPSantigens were dissolvedtoconcentrations of 3to6mg/ml in triethylamine, and thereafter, twofold serial dilutions were made from a stock (100 ptg/ml) in buffers of different pHs. Results of testing fixed dilutions of thepositive standard and anegative serum sample against different concentrationsof K25 poly-saccharide antigen are shown in Fig. 1 as an example. The difference between negative and positive samples increased as the pH of the coating buffer increased. The carbonate buffer (pH 9.6) giving the greatest difference between the positive standard and thenegative sample was chosen for the determination ofworkingdilutions, i.e., coating doses of the variousCPS antigens. The coating dose for each antigen was chosento correspond to thebeginning of the plateau of the positive standard serum at a concentration giving the maxi-maldifference between the standard and the negative sam-ples. The optimal coating doses thus determined showed a considerable variation, ranging from 0.2 to 20 F.g/ml (Table 1).

Human IgG antibody responses to vaccination. The post-immunizationIgG antibody responses to the 24 CPS antigens contained in the vaccine are shown in Table 2. There was considerable variation in the magnitudes of the immune responseselicited by theindividual CPS serotypes, both in J. CLIN. MICROBIOL.

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Antigen coating concentration(tig/mi)

FIG. 1. Determinationof optimal coating conditions forKlebsiella CPS antigens illustrated by serial dilutions of serotype K25 in four buffers. Symbols: *,a1:500dilutionofpositive standard;O, a1:100 dilution of negative serum.

regard tofinal ELISA titer and fold rise in titer. Postimmu-nizationtiters ranged from 40 (serotype 22) to 730 (serotype 16), while fold rises ranged from 3.6-fold (serotype 53) to 150-fold (serotype 16). The majority of CPS antigens were found to be good immunogens, with 17 of 24 yielding postimmunization titers of

.100.

Similarly, 21 of 24 CPS antigens evoked 2>10-fold rises in titer. Samples from all

TABLE 2. IgGresponses toKlebsiella CPS elicitedby a

24-valentvaccine inseven humanvolunteers

Antigen Median ELISA titer (range) Median foldrise

(serotype) Preimmunization Postimmunization (range)

2 1.4(0.5-6.0) 230(60-870) 140(42.9-870) 3 5.0(1.0-7.0) 60(30-460) 18(5.7-115) S 8.0 (5.0-10) 310 (50-610) 47 (12.5-76.3) 9 5.0(2.0-6.0) 50(30-340) 12(8-120) 10 5.0(1.0-30) 430(100-2,500) 94(6.3-357) 15 5.0(1.5-40) 220(50-340) 25(3.8-145) 16 5.0(2.0-80) 730(320-10.250) 150(81.1-480) 17 7.5(3.0-60) 290(60-700) 29(1.2-157.5) 18 5.0(1.4-30) 300(60-940) 62.5(2.7-214.3) 21 4.5 (1.0-50) 70(60-830) 25(4.6-70) 22 3.0(1.0-30) 40(20-330) 13.3(1.3-165) 25 2.0(1.0-500) 360(50-630) 130(1.3-570) 28 40.0(3.0-140) 150(40-600) 8.6(1.0-85) 30 4.0(1.0-10) 200(60-3,500) 60(9.2-875)

35 5.0(1.5-10) 60(30-460) 17.1 (3.0-59)

43 9.0(5.0-30) 90(70-320) 12.2(2.7-53.3)

52 6.0(2.0-40) 290(140-560) 50.0(12-140)

53 8.3 (1.5-180) 50(30-210) 3.6(1.0-53.3)

55 4.2(2.0-60) 240(70-450) 43.3(5.5-120)

60 10.0(4.2-50) 280(20-800) 16.0(1.0-63.0)

61 4.0(2.0-9.0) 620(60-920) 88.6(15-460)

62 2.0(1.0-4.0) 270(70-310) 96.7(35-300)

63 7.0(2.7-100) 100(30-250) 7.9(1.0-40.0)

64 9.4(4.0-70) 220(70-1,570) 16.0(6.4-215)

seven

individuals

showed significant (twofold or greater) rises in titer ofIgG to 17of24vaccine antigens. Significant rises in titer of IgG were recorded in 158 (94%) of 168 individual responses.

The IgG antibody responses to 11 additional Klebsiella CPS antigens are shown in Table 3. These serotypes were selected according to results obtained by counterimmuno-electrophoresis with polyclonal typing sera, which showed these 11antigenstoimmunologicallycross-reactwith one or more vaccine antigens (T. Pitt, personal communication). The median fold titer rises to these cross-reactive CPS antigens ranged from 4.2-fold (serotype 14) to 42.9-fold (serotype37). SignificantrisesinIgG titerto8of11antigens were noted in all seven

vaccinees.

Significant

rises

were recorded in 73 (95%)of 77

individual

responses.

The overall responsesto the 24vaccineantigensin seven

individuals

(n = 168 observations) were compared with

responses to the 11 additional antigens (n = 77

observa-tions). The geometric mean fold rise to the 24 vaccine antigenswas29.9-fold

(+4.7-fold),

significantly higher(P<

0.001) than the 13.5-fold(+3.3-fold)risetothe 11additional antigens. Also, the GMT of IgG to the vaccine

antigens,

174.7 (±3.1), was

higher

(P < 0.05)than the GMT of 127.2

(±3.1)

to the 11antigens notincluded in the vaccine.

Analysisofpreimmunization titers. There was substantial individual variationin the levelsof

preexisting IgG

antibody

tothe 35 serotypes ofCPS studied

(Tables

2and

3).

This is best illustrated with serotypes 25 and 53, for which

preim-munization titers ranged from 1 to 500 and 1.5 to

180,

respectively. Also, the

median

preimmunization

titers ranged from 1.4 (serotype 2) to 40 (serotype

28)

and 50 (serotypes 14 and 31). Thedifferences in theoverall

magni-tude of the immune response to vaccine and nonvaccine antigens were due not

only

to

higher

postimmunization

levels attained

by

vaccine

antigens,

but alsotodifferencesin

preimmunization

titers

(Table 2).

Therefore,

the

overall

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2260 GRANSTROM ET AL.

TABLE 3. Responsesofcross-reacting IgGto11Klebsiella CPS antigens not included in the vaccine

Antigen (serotype) Immunologically Median ELISA titer (range) Median fold rise

notinvaccine

~related

serotype (a .

flot

in vaccine

inlaecine

Preimmunization Postimmunization

(range)

7 5, 10 7.0(5-80) 100(20-480) 11.4(3.3-55)

il 21 6.0(2-70) 30(10-490) 5.0(1.7-70)

12 3 2.6(1-20) 40(20-510) 15.4(5.3-250)

13 2 4.6(2.7-30) 120 (50-680) 29.6(7-97.1)

14 64 50.0(1.9-60) 180 (60-400) 4.2(1.2-155)

26 21 8.4(4.-250) 150(70-770) 11.0(2.1-125)

31 60 50.0(4.0-110) 270(70-1,700) 6.8 (1.6-17.5)

37 22 3.0(0.7-9.3) 150(30-310) 42.9(4.3-100)

46 5 8.0(3.6-40) 100(40-170) 11.1 (2.5-21.2)

68 3 5.0(2.0-90) 170(50-430) 17.0(4.8-84)

69 2 9.0(4.2-50) 120(60-2,500) 13.3 (9.0-125)

preimmunization GMTwassignificantlylower(P<0.01)for

vaccine antigens (5.9 ± 3.3) than that for the 11

cross-reacting CPS antigens evaluated(9.5 ± 3.5).

Interestingly, the lowest preimmunization GMT was for

serotype2, which is themostcommonantigen expressed by

Klebsiellabacteremic isolates inEurope (9).Wewere there-fore interested to determine if there was a relationship

between the frequency with which a given Klebsiella

sero-typecausesbacteremia andbase-line levels ofIgG antibody inserum. Acomparisonbetween thepreimmunizationGMT of the five most commonly observed isolates among

Euro-peanbacteremic strains (serotypes 2, 21, 55, 16, 53,and62) and the correspondingGMT for 13 serotypes (5, 11, 12, 13, 14, 17, 26, 30, 35, 43, 46, 52, and 69) with an overall

individual incidence of <1.5% showed the former to be significantly lower (P <0.01).

DISCUSSION

In this study, we have described the development of an

ELISAsystem capableofdetectingandaccurately

measur-ing IgG antibodies to a variety of Klebsieila CPSs. This ELISA was used as a means of quantitating the human immune response engendered by a 24-valent, CPS-based Klebsiella vaccine.

AilKlebsiella CPS antigens could be adsorbedto

polysty-renemicrodilution plates when the antigenswere dissolved

in triethylamine without additional modification. This is in

contrast toother bacterial polysaccharides, which must be

treated, for example, with cyanogen bromide before they

canbeusedto coat microdilution plates (1).Inaddition, we

didnot usetwocoating layers, as are often used in ELISA

systems to block residual reactive sites that remain after primary antigen adsorption. This blocking layer usually

consists of bovine serum albumin or casein. The plateau

seen on the coating curve (and chosen for the optimal

coating dose) indicates that few, ifany,reactive sitesremain

inthewell. The coatingdose thus chosen rendersablocking

layer unnecessary. On the contrary, the addition of such

extraneousproteins can yield artificially high titers because

ofthe presence of preexisting antibodies in normal human

sera(18). These antibodiesare especiallydisturbing in sera

with low specific-antibody levels such as are present in

preimmunization samples. Instead, all steps subsequent to

theinitialcoatingwereperformed in thepresenceofTween

20, which, in addition to virtually eliminating nonspecific

binding, ensured that onlyspecific anti-CPS antibodies with

high affinity would be measured.

Excellent results were obtained forall 35 Klebsiella CPS

antigens evaluated when they were dissolved in

triethyl-amineandsubsequentlydiluted inanoptimal coatingbuffer. The 0.05 M sodium carbonate buffer(pH 9.6) gavethe best resultsin the present system but maynotdosoin others. We therefore routinely check the pH optimum of the coating buffer in each new system (11, 14, 15). Dissolution of Klebsiella CPSs in triethylamine was evaluated because of prior favorable results obtained inourlaboratory with vari-ous lipopolysaccharide antigens (S. B. Svenson, personal communication). Although the precise means by which triethylamine influences the adsorption of an antigen to polystyrene isunknown, it may act to disaggregate Klebsi-ella CPS(6), thereby increasing coating efficiency.

Theextremely widerangeinoptimal coatingdoses(0.2to 20

p.g/ml)

for the 35antigens was

unexpected

and couldnot be predicted from chemical composition (16). However, all Klebsiella CPSsareof

high

molecular

weight

and possessa net negative charge. Differences incharge amongthe CPSs could account fortheir various abilities to bind to

polysty-rene. It is important to note that factors

influencing

the binding of

antigens

to

solid-phase

support systems for ELISA are largely unknown.

Therefore,

the conditions needed for optimal coating must be determined for each individualantigen,even thoughtheantigensmay be

closely

related(asarethe above-mentioned CPSs).

The previously described Klebsiella CPS vaccine was found to be well tolerated and

highly immunogenic

in hu-mans. The vaccine induced good IgG antibody titers, not only to the 24 vaccine antigens, but also to 11 additional antigensshowntobeimmunologicallyrelatedtoone or more vaccine serotypes. However, wide individual variations werenotedwhenbothpre-andpostimmunization titerswere studied. At present, theKlebsiellavaccinewouldmostlikely be used to immunize healthy donors to prepare a hyperim-muneglobulinforintravenoususe(IVIG) (3). These individ-ual fluctuations in titerwould, therefore, be ofonly minor

practical

concern

considering

theexcellentoverall response andthe fact thatasingle lot of IVIG is usually produced from pools ofnot less than 1,000donors. The sensitivity of the ELISA system would also readily allow the detection of antibody passively transferred to patients in the form of an IVIG.Thisassumptionisbased on a mean postimmunization GMT of 175, a 25- to 50-fold-concentration step in the production of IVIG, and the transfer of 100 ml to each

patient.

Thecurrentvaccine engenders an antibody response to 35 of the known 77 Klebsiella CPS serotypes and would cover 72% of all

Kiebsiella

bloodisolates and 82% of all typeable bacteremic strains. Since the ELISA system described above isfarmore sensitivethanthe

counterimmunoelectro-J. CLIN. MICROBIOL.

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phoresis technique used to initially select the 11

cross-reactive serotypes studied, we are continuing to examine additionalserotypes toaccuratelyassesstheextentof

cross-reactive antibodies induced bythecurrent24-valent vaccine. An additional interesting finding was the significantly

lower preimmunization titerstovaccineserotypescompared with titerstotheadditional 11 antigens studied. The decision

as to which serotypes of CPSs to include in the vaccine formulation was based on seroepidemiology of Klebsiella

bactereniic isolates (9). Itwas,therefore, interesting to note

that the levels ofpreexisting IgG antibodies in only seven

individuals showed sucha stronginverse correlation to the fivemostcommonly encountered isolates among Klebsiella

bacteremic strains. The results, representing the first effort

toquantitate natural IgG antibody in humanserum againsta

variety ofKlëbsiella CPS serotypes, indirectly support

ex-perimental data which demonstrate the protective capacity ofanti-CPSantibody (4, 7, 10). These data also indicate that thehighly sensitive ELISA described herecanbe appliedto

seroepidemiological studies on the occurrence of natural

antibodiesto Klebsiella CPSs.

ACKNOWLEDGMENTS

We thank Eivor Norstrom and Carina Bengtsson for excellent

technicalassistance.

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