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0095-1137/81/061080-08$02.00/0 Vol.13,No.6

Quantitation

of

Antibodies

to

Haemophilus

influenza

Type

b

in

Humans by Enzyme-Linked

Immunosorbent

Assay

TERESA DAHLBERG

Institute of MedicalMicrobiology, UniversityofGôteborg,S-41346Gôteborg, Sweden

Received11November1980/Accepted10March1981

Theenzyme-linkedimmunosorbentassaywasadaptedtodetect serum

immu-noglobulinG, immunoglobulinM,immunoglobulinA,andsecretory

immunoglob-ulinA antibodiesto

Haemophilus

influenza

typeb

capsular

polysaccharide in

humans. I studied serum samples from 92 healthy children ofvarious ages, 50

healthy adults,24 patients with various H. influenza typebinfections, and 16

patientswithclinicalsignsofepiglottitis andcellulitissuspectedtobecausedby

H.

influenza

typeb.Themeanantibodytitersoftheserafromhealthy children

increased withageandreachedadultlevelsinchildrenmorethan 6yearsold.A significantantibodyresponsetocapsularpolysaccharidewas observedinserum

samplesfrom the majority ofpatientswithinfectionsdue to H. influenzatype

b and in 4 of 16patients with clinicalsignsofepiglottitisandcellulitis.Inaddition

to the enzyme-linkedimmunosorbent assay, the antibodyresponses ofpatients

weretestedbyabactericidalassay.Whenthetwo methodswerecompared, there was no evident correlation (r, about 0.22). The enzyme-linked

immunosorbent

assaywasfurtheradaptedto testsecretoryimmunoglobulinAantibodiesspecific

tocapsularpolysaccharide innasopharynx secretions and inmilk samplesfrom

lactatingwomen.Antibodiesweredetectedin 12 of 24secretions and 9 of11milk

samples.

Almost

ail

Haemophilus

influenzae infections

in children,

including

meningitis,

epiglottitis,

septic

arthritis,

pneumonia,

and

others,

are caused by capsulatedH.

influenza

type

b

(16,

20). However, except for

epiglottitis,

the

occur-renceofthesediseasesis

relatively infrequent

in

adults (9, 20).

Using different

methods,

variousworkershave

demonstrated

antibodies against the capsular

polysaccharide

of H.

influenza

type

b,

which

maybeprotective (2, 3, 10, 11, 14, 18); of these

methods, a

radioimmunoassay

modified from

the method ofFarr is the most

sensitive,

per-mittingprecisemeasurements (2, 11, 14).

In a previous study, the enzyme-linked

im-munosorbent assay (ELISA) (7) was used to

demonstrate

antibodies

against

H.

influenza

capsular

polysaccharides

(5).Thelowestlevel of

detectionwasestimatedtobe 80 ng ofantibody

per ml. Thismethodpermits separate

determi-nationsofspecific

immunoglobulin

G(IgG),IgM,

and IgA antibodies, and thus additional

infor-mationconcerning immune responses to H.

in-fluenzae

antigens may be

obtained.

The aim ofthis study was to adapt theELISA

method forquantification ofIgG,IgM, and IgA

antibodies

against

H.

infiuenzae type b

polysac-charideinserumsamplesobtainedfromhealthy

children and

adults,

asweil asfrom

patients

with

various

types

of H.

influenza

infections.

For

comparison,

most ofthe

patient

serawere also tested

by

using

a bactericidal assay

(3).

The

ELISA was also used to measure

anticapsular

secretory

IgA

antibodies in

nasopharynx

secre-tionsfromchildren of

various

ages andin

milk

samples

from

healthy

women.

MATERIALS AND METHODS

Antigens. Capsular polysaccharide

was

prepared

from H.

influenza

type b strain RAB which was

grown

overnight

in

liquid

antigen-free medium AFH modifiedasdescribedpreviously (5).Theconcentrated culturesupernatantwasfilteredthrough Sephadex

G-150,andthepolysaccharide-containingfractionswere

purifiedfurther

by

using

diethylaminoethyl-Sepharose

chromatography

andanNaCIconcentration

gradient

(designated

preparationCPSMbJ).

Capsular

polysac-charidewasalsoprepared

by

precipitationofaculture supernatantwith Cetavlonandsubsequentgel

filtra-tion

through Sephadex

2-B(preparation CPS MbII).

Theseprocedureshave beendescribed previously(5).

Theoptimal coatingconcentrationforCPSMbIlwas

lowerandthecoatingtimewasshorter thanforCPS Mb I.CPSMb Iwasusedonlyinthe studies

concern-ing

the prevalenceofantibodiesinhealthy

children,

whereas CPSMbIlwasused inthe other

experiments.

Study

groups.Serumsamplesfrom the

following

groupsof

healthy

individualsandpatientswere

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VOL. 13, 1981

lyzed.

Group I included 92 healthy infants and children ranginginagefrom 1day to 14yearsand50healthy adults (blood donors).Theserafrominfantsand chil-drenwere obtained from the Department of Pediat-rics, EasternHospital,Goteborg, Sweden, whereas the blooddonorserawereobtainedfrom theBloodBank, Sahlgren's Hospital, Goteborg.

Group Il included 20 adult patients with clinical and serological signsofvariousviralinfections.Paired acuteandconvalescentserumsampleswereobtained

fromtheVirologicalLaboratory,Sahlgren's Hospital. GroupIHIincluded 24patients,each ofwhom had

oneof thefollowingdiseasesduetoH.influenzatype

b: meningitis, epiglottitis, pericarditis, arthritis, or

pneumonia.Theagesof thesepatients rangedfrom 6

monthsto61years.Morethan70%of thesepatients

were morethan 4yearsold, andtheywerehospitalized atSahlgren'sHospital, EasternHospital,orMolndal

Hospital.H. mfluenzaetypebwasisolated from the

spinalfluidand fromthe bloodofallof thesepatients. Group IV included 16patientswithclinicalsignsof epiglottitis or cellulitis. The ages of these patients rangedfrom 5 to 75years,andtheywerehospitalized

atSahlgren'sHospital.H.influenza typebcouldnot be isolated fromthebloodornasopharynxofanyof thesepatients.

Exocrine secretions from the following groups of individualswereanalyzed.

GroupVincludednasopharynxsecretionsobtained

from 24children(age range, 11 months to 13years) withprevious episodesofotitismedia.Thesechildren

were admitted to Lundby Hospital, Goteborg, for treatmentofseriousotitis media. H.influenza typeb

orother bacteriawhichcouldcauseotitismediawere

notisolatedfrom thenasopharynxesof thesechildren atthe timeofsamplingof the secretions.

Group VI included breast milk from 11 lactating

women after child delivery at Eastern Hospital, Goteborg.

Generally,theacute-phaseserumsamplesfromthe

patientsingroupsIII and IVwereobtained within3

daysafteradmission,andconvalescentserumsamples

wereobtained4to31 dayslater. Thesera werekept

frozenat-25°Cuntil the testswereperformed.Serum samplesobtainedfrom thesamepatientwerealways testedonthesameday.

Nasopharynxsecretionswereobtainedbysuction, and eachwasmixed withanequal partofphysiological salinebeforeanalysis.

Breast milksampleswereobtainedbyusingabreast pump 2 to 4 days after nursing had started. The

specimenswerecentrifugedat10,000xgfor10min at

4°Ctoremovecellsandlipids.

The antibody specificityofeach milksample was

verified byabsorption experiments performedinthe

followingway. Onepartof the milksamplewas pre-cipitated with 45% ammonium sulfate and washed. Thisprocedurewasrepeatedtwice. Eachprecipitate

was dissolved in the original volume of phosphate-buffered saline, and eachsample was absorbed with 260,ugof H.influenzatypebcapsularpolysaccharide

permlof milk.Allnasopharynx secretions andmilk

sampleswerekeptat-25°Cuntiltheywereanalyzed.

1081

Bacterialcultivation technique. Samples for

cul-tivation of H.

influenza

and other bacteria were obtained at the first examination. The cultivation and identification of the bacteria were performed as pre-viously described (4).

Serological methods. The ELISA (7) was used to measure

serumn

IgG,IgM, and IgA antibodiesagainst

H. influenzatype b capsularpolysaccharideantigen

asdescribed previously (5). In addition, this method was used to measure secretory IgA antibodies. The assay was carried out in tubes (Heger Plastic AB, Stallarholmen, Sweden) (macro-ELISA) or on micro-plates (Dynatech, Novakemi AB, Enskede, Sweden) (micro-ELISA) coated with solutions containing 100 ugof CPS Mb I per ml or 30 ,g of CPS Mb II per ml. Coating was performed overnight at 37°C and for about 2 weeks at 4°C, as described previously (5). The micro-ELISA was used in all tests except those mea-suring theprevalence of antibodies in healthy group I children. All testswere performed with 10-fold

dilu-tions of serum. Purified immunoglobulin fractions of rabbit antisera monospecific for the heavy chains of human IgG, IgM, and IgA, as well as the secretory

component of IgA (Dakopatts, Copenhagen, Den-mark), were used for conjugation with alkaline phos-phatase (type VII; Sigma Chemical Co., St. Louis, Mo.) by the glutaraldehyde method. Generally, the

conjugateswereused in dilutions of 1:400, when the enzyme-substrate reaction time was about 60 min.

Alkaline phosphatase-coupled swine antibodies against human IgG, IgM, and IgA (Orion, Helsinki, Finland) were also used in this study; theseconjugates

wereused atdilutions of 1:100 and with reaction times of30 to 40 min to obtain similar sensitivity in the assay.Theseconjugateswereused in the majority of experiments. None of the conjugatescontained anti-bodiestoH. influenzatype bcapsular

polysaccha-ride.

A convalescent

serumn

obtained from one patient after H. influenzaetype b epiglottitis (see Table 2, patient 7) wasused asareferenceserumfor specific IgG, IgM, and IgA antibodies. The antibody content in serum was expressed as the -logio value of the highest

serumn

dilutionshowinganextinctionvalue of 0.2above thebackground.Titer value

(21-logio)

was

designatedaspositive.

Thestandarddeviation wascalculated from 10 ti-trations of thereferenceserumperformedonseparate days with two lots of conjugates and one antigen

preparationandwas0.2-logioforallimmunoglobulin

classes.

Thelargestdifferencebetweenacuteand convales-cent serafrom patients in control groupIl was 0.3 -logo, whichcorresponds to atwofold dilution step. An increaseofthreefold (0.5

-logwo)

or more in the

tier of the IgG classor the IgM class or bothwas

consideredasignificant antibodyresponse.

Determinations of secretory IgA antibodies were

performedby the micro-ELISA with twofold dilutions ofmilksamplesornasopharynxsecretions.The anti-bodycontent in asecretionsormilksamplewas

ex-pressed as the highest twofold dilution showing an absorbance of0.2or moreabove thebackground ab-sorbance level. Titers of 1:2 in milk and 21:4 in

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secretionsweredesignatedaspositive.

The ELISA titerassignedto eachsamplewasthe

meanoftwoorthree determinations.

Bactericidal assay. The bactericidal assay was

performed by mixing twofold serial dilutions of the

serum sampleswithappropriatelydilutedguinea pig

serumandsuspensionsof H.influenza typebstrain RAB on microtiter trays. The procedure has been

describedin detail elsewhere(3).

A significant antibody response was defined as a

titer increase of fourfoldor more when acute-phase andconvalescentsera werecompared,or,when

acute-phasesera weremissing,titer valuesexceeding1:4 and 1:8inserafrom children andadults, respectively.The

titerassignedto eachsamplewasthemeanof twoor

three determinations.

Mean values, standard deviations of titer values, and coefficients of correlationbetween the ELISA and bactericidalassaytiterswerecalculated.

RESULTS

Prevalence of serum antibodies against

type b capsular antigen in healthy adults and children. Table 1 shows the ranges and

themeanvalues of the IgGandIgMantibodies

against type b capsular polysaccharide in the

serafrom50healthyadults (groupI).Ail serum

sampleshad detectable amounts ofspecificIgG, IgM, and IgA antibodies. The mean IgG and

IgM titer valueswere 2.2 and2.6, respectively. ThemeanIgAtiterwas 1.9(range, 1.1 to2.9).

The 92 healthy children in group I were

di-vided into seven subgroups according to age

(Table 1).All20 newborn infants hadIgG

anti-bodies against type b capsular polysaccharide,

and themeantiterwas ofthesame magnitude

asthetiters of adult individuals.Of11 children whowere 1to11monthsold,9 hadpositiveIgG titers, and themean titer was about six times

lower than themeantiter ofnewborninfants. In thenext twoagesubgroups (children between 1

and4yearsold),themeanIgG titersincreased,

and forserafrom children 6yearsold orolder,

the IgG titers reached the same levels as in

adults

(Table

1).

In 6 of the 20serafrom newborn infants, IgM

titers ofabout 1.2wererecorded.Aboutone-half

of the children in the next age subgroup had

demonstrable IgM antibodies, and in the chil-dren 1 to4 yearsold IgM antibodies were

de-tectable inthesera fromnearlyallindividuals.

Intheserafrom childrenmorethan 6yearsold,

themeanIgM titerswerealmostashighasthose

in the adult sera (Table 1). However, a wide

variation in IgM and IgG titers was observed

within eachagegroup.

One child in thesubgroup containing children

1 to11months old andonechild in the subgroup

containingchildren 2to4yearsold had neither

IgGnorIgMantibodies againsttypebcapsular polysaccharide.

Antibodyresponse toH.

influenza

type

b inpatients with infections duetoH.

influ-enzae type b. Ail epiglottitis patients (group III) except onehad detectable amountsof IgG and IgM antibodies specific totype b capsular polysaccharide in their acute-phaseserum

sam-ples (Table 2). For themost part, the titers of thefirstserumsamples takenafter hospital

ad-missionwere comparabletothe titers found in

healthy individuals (Table 1). When

convales-cent sera were analyzed, asignificant antibody response wasregistered in9of12patients (Table

2). The three remaining patients (Table 2,

pa-tients6, 8, and 10) had high antibodylevels in thesamples obtained 2 and 3 days after

admis-sion.

The highest increases in IgG and IgM anti-body titers observed (about 80- and 50-fold,

re-spectively) occurred in two children who were

morethan 6yearsold (Table 2,patients 3 and

4). An IgAresponse wasobserved in four of nine

epiglottitispatientstested, and the highest titer

increase recordedwasabout 90-fold,whichwas

observed in a child who was 4 years and 7

monthsold.

TABLE 1. Serumantibody titers(-loglq)totypeb capsular polysaccharide in 92 infants and children and 50adults (group I) tested by the ELISA

IgG IgM

Age

sub-group No.

positive/no.

Meantitera Titerrange No.positive/no.

titerd

Mean Titerrange

1-7days 20/20 2.3 1.2-3.5 6/20 1.2 <1.0-1.3

1-11months 9/11 1.5 <1.0-2.6 6/11 1.4 <1.0-2.0

1-2years 10/12 1.7 <1.0-2.7 11/12 1.7 <1.0-2.5

2-4years 15/18 1.7 <1.0-2.8 16/18 1.6 <1.0-2.4

4-6years 12/14 1.8 <1.0-2.7 12/14 1.6 <1.0-3.3

6-10years 9/9 2.2 1.0-3.0 8/9 2.1 <1.0-2.5

10-14years 8 8 2.6 1.7-3.3 8/8 2.3 1.3-2.9

Adults 50/50 2.2 1.3-3.25 50/50 2.6 1.6-3.9

aMeantiter calculated with positivesera.

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TABLE 2. Serum antibody responses to type b capsular polysaccharide in patients with epiglottitis due to H.

influenza

type

b (groupIII)

Patient

uno.

Age Daysafteradmis- ELISA titers

(-loglo)

Bactericidal titer

Years Months 8i0n IgG IgM IgA

1 4 7 0 1.8 2.25 NDa ND

1 6

5 2

6 4

6 10

9

O

9

O

5

O

19 25

O

8

1.8 2.7

3.0 3.65

<1 2.1

1.65 3.6

2.3 <1

3.1 <1

1.5 1.75

1.5 3.5

ND ND

1.8 2.0 ND

3.0 2.4 ND

3.7 2.6 ND

2.2 1.7 ND

3.2 3.0 ND

ND 1:128 1:2 1:64 1:2 1:64

ND ND ND

ND ND

6 il 10

7 25

8 25

9 28

10 29

2 il

31

3 7 4 7 3

7

il 56 O

8

12 61 O

3 6

2.85 2.9 2.25 3.05 2.6

3.5 2.7

3.4 2.65

1:2 1:2

1.8 1.7 1:32

2.9 3.1 1:64

2.6 2.2 1:64

2.7 2.8 1.4 ND

2.5 1.9 1.9 ND

2.0 2.5 1.9

2.7 2.9 <1

1:16 1:64

2.7 4.0 1.9 1:256

2.2 3.2 2.7 1:8

2.0 2.6 1.6 ND

2.7 3.2 1.8 ND

1.9 2.3

2.0 2.25

2.9 2.6

1.3 1:2

1.3 1:2

1.7 1:8

aND,Notdone.

bThereferenceserumusedintheELISA.

Of12 epiglottitis patients, 8weretested with

regardtothepresenceof bactericidal antibodies to a capsulated typeb strain. A significant

in-creaseinbactericidal antibodiesor ahighinitial

bactericidaltiterwasobservedinsevenof these

patients. One child, who was 11 years and 10

months old, showednoincrease in bactericidal titerdespite high anticapsulartiters as

demon-stratedbythe ELISA.Thehighestbactericidal

titerrecorded in thisgroupofpatientswas1:256,

which was observed in one adult who was 29 yearsold.

Generally, the amounts of specific IgG and

IgAantibodiesinacute-phaseserafrompatients

who hadmeningitis (Table 3) werecomparable

to the amounts in the corresponding healthy

group(Table 1). IgMtiterswerehigherinserum

samples from about one-half of the diseased children than for the corresponding healthy

group. Asignificant antibodyresponse was ob-served in three of seven cases, whereas two children (2 yearsand 9 months old and 3years

old) did not show significant titer increases. However, IgM levels were already high in the acute serum of the older child. High antibody

levelswerefound inthe serafrom the patients

from whomnoacute-phaseserumsampleswere

obtained (patients 2 and 7). The greatest

in-2

3

4

5

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TABLE 3. Serumantibody responsetotypebcapsularpolysaccharide in patients withmeningitis,

pericarditis, arthritis, andpneumoniadue toH.influenzatype b(groupIII) Age Dasatra- ELISA titers(-logio)

Disease Patientno. Days after ad- Bactericidal titer

Years Months mission IgG IgM IgA

Meningitis 1 6 0 1.0 1.5 <1 1:8

9 1.7 2.9 2.3 1:2

2 2 6

3 2 9

4 3

4 2.8 3.2 1.5 1:64

7 2.8 3.2 1.45 1:64

0 1.2 2.3

6 1.45 2.75

<1 1.0

1:8 1:2

0 2.4 3.3 2.3 1:64

8 2.6 3.7 2.2 1:64

5 4 8 0 1.6 3.1 <1

14 3.3 3.4 2.6

1:4 1:8

0 2.05 3.15 2.6 1:8

5 2.6 3.2 2.7 1:128

5 3.45 3.0 3.3 1:32

8 3.3 2.75 2.9 1:8

2 3.0 1.75 ND" ND

20 3.5 2.25 ND ND

12 3.5 2.9 ND

14 3.5 3.0 ND

ND ND

0 2.5 2.7 1.75 1:2

7 3.0 3.25 3.3 1:2

14 3.7 3.3 2.8 1:2

21 3.4 3.2 2.4 1:2

O 1.9 1.8 ND 1:16

7 2.3 1.7 ND 1:16

12 3.0 2.0 ND 1:32

0 2.0 2.8 ND ND

6 2.6 2.7 ND ND

aND, Not done.

crease in IgGtiters obtained inserum samples

from these patients was about 50-fold, which wasobserved ina childwhowas4 yearsand8

months old, and the greatest increase in IgM

titerswas about25-fold, whichwasobserved in

the youngestchild (6months old). Increases in IgA antibody levelswerefound inserumsamples

fromtwoof thesevenpatients (Table3).

Atiter increaseoraninitialhigh bactericidal

titerwasrecordedforfiveof thesevenmeningitis

patients tested. The highest bactericidal titer

foundwas 1:128, whichwas observed in serum

samplesfroma9-year-old child(Table 3).

A significant ELISA antibody response was

recorded fortwoof three patients with

pericar-ditis (Table 3). No significant bactericidal

re-sponsecould be demonstrated inserumsamples

fromonepatient(patient 3). High IgM and IgG

antibody levelswereobservedinserumsamples

taken 12daysafter admission ofthethird

peri-carditis patient. A significant IgG antibody

re-sponsewasalso observed intwoother patients,

onewith arthritisandonewith pneumonia.

Bac-tericidalantibodiesweredemonstrated inserum

samples from the former patient.

TheELISAIgG and IgM antibody titerswere

6 9

7 61

Pericarditis 1 6

2 10

3 56

1 50

Arthritis

Pneumonia 1 2 1

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DAHLBERG

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ANTIBODY QUANTITATION BY ELISA 1085

comparedseparatelywiththebactericidaltiters

determinedin serum samples frompatients(Fig. 1). There was no obvious correlation between ELISA titers (IgG and IgM) and bactericidal titers (r = 0.23 and0.22,

respectively).

Antibody responsetoH.

influenza

type

b in patients with suspected but not

con-firmedinfections. Sera from 14 patients who

had clinical signs ofepiglottitis and 2 patients

who showed clinical signs of cellulitis but in

whom H.

influenza

type b could not be isolated

(group IV) were tested by the ELISA and the

bactericidal assay. A significant antibody

re-sponse to type b capsular polysaccharide was found for 2 ofthe 14 patients with epiglottitis

and for bothpatientswithcellulitis.However,a

significant bactericidal response to the

capsu-latedtype bstrainwasfoundonlyintheserum

samples from thetwoepiglottitis patients.

Demonstration of secretory IgA

anti-bodiesagainst

capsular

polysaccharide.

Se-cretoryIgAspecifictotype bcapsular

polysac-charide was tested in nasopharynx secretions

from 24children with previous episodes of otitis

media (group V) and in milk samples from 11

women (group VI).

Secretory

IgAwas

demon-strated in secretions from 12ofthe 24children;

10 of these 12patientswere more than4years

old. The titersrangedfrom 1:4 to1:8.

Milk secretory IgA antibodies were

demon-strated in9of11

samples,

and the titers

ranged

from1:2 to 1:64. Figure2showstitrationcurves

for a milk

sample

obtained before and after

absorption

with

capsular polysaccharide.

4

-o,

w,

2-cn -'

1-

VI'-o

e

. 0

o 0

o

o

o

g *

* O

0

o o

.

.

.

I T I I

<1 1 2 3

BC TITER(-1og10)

FIG. 1. Relationship between titers obtainedwith theELISA and the bactericidal(BC)assay inserum

samples ofpatientsfromgroupIII. Symbols:*,IgG

(r=0.23);O,IgM(r=0.22).

E(400nm)

1,2

\

0,8

-0,6

04 2eO

0,2-0---

---'' I T T T1 T T T

2 4 8 16 32 64 128 256 516

MILK DILUTION FIG. 2. Secretory IgA levels in one milk sample before (solid line) (titer, 1:32) and after (dashed line) absorption with type b capsular polysaccharide an-tigen.

DISCUSSION

This study examined the ability of the ELISA (5) todemonstratehumanserum IgG, IgM, IgA, and secretory IgA antibodies against H. influ-enzaetypeb capsular polysaccharide.

The large amounts of specific antibodies of

the IgG class observed in serum samplesfrom

newborninfantswere mostlikely due to

trans-placentaltransfer of IgG from themothers.The

lowest IgG andIgM antibody levels were found

in serum samples from children 1 month to 1 yearold. The mean IgG and IgM titers increased inchildrenmore than 1 year old,reachingadult

levels at about 6 years of age. A few of the

children in each ofthe groups containing chil-drenup to 6 yearsolddid not have anyspecific serum antibodies, as revealed by the ELISA.

Generally, these results agree with the results

obtained in otherstudiesoftheage-related

prev-alence of H.

influenzae

type

b

antibodies

as

estimated by

radioimmunoassay

(10) and by

other methods (8, 17). Antibodies specific to type b capsular polysaccharide could be

regis-teredin serumsamplesfromalladults testedby

the ELISA. When the radioimmunoassay

method was used, the presence of these anti-bodies in the majority of serum

samples

from

adultswasreported (14).

Antibody responses to type bcapsular

poly-saccharide were observed in a

majority

of the

patients with various infectious conditions

caused

by

H.

influenzae

type b,

asestimated

by

the ELISA. Asignificant

antibody

responsewas

also observed inall

epiglottitis

patients.

Gener-ally, the titer increases found in this

study

were

comparabletothoseobserved inprevious

stud-13,1981

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DAHLBERG

ies in which the

radioimmunoassay

method was used (11).

Themajorityof children withmeningitishad

higher IgMantibodytiters inacute-phaseserum

samples than the

corresponding healthy

group.

It is probable that the infections started a few days before admissiontothe

hospital,

thus

elic-iting the production of IgM antibodies. In a previousstudy of the

antibody

response toH.

influenza

typeb in

rabbits,

specific IgM

anti-bodieswere detectable as early as 2 to4 days afterprimary immunization (5).

Thus,

thehigh

antibodylevels recorded in the

early

serum

sam-plesobtained from the children withmeningitis

may indicate the presence ofa rapid antibody

response.

The ELISA measures antibodies

specific

to

capsular

polysaccharide,

whereas the

bacteri-cidal assaymeasuresnotonly

anticapsular

anti-bodies but also antianti-bodies

against

othercellwall antigens (5, 6).

Moreover,

the ELISA is more

sensitive than the bactericidal assay, especially

inthe detection ofprimary response antibodies (5). This may explain the lack of correlation

between theresultsobtained

by

thetwo

meth-ods. However,Norden and Michaels

(11)

found

acorrelation coefficientofabout 0.66 whenthey

compared the bactericidal assay anda

radioim-munoassay modified from the method ofFarr.

Itshouldbe noted thatthe

methodological

prin-ciples of theFarrtechnique and theELISAare

different. The former method is based on a

precipitation reaction betweenantigenand

anti-bodies, whereas in the ELISA technique the

antigenisboundto asolidphaseand isallowed

toreactwith antibodies.

TheELISAused in thisstudywaswellsuited

for detection and quantitation of the antibody

responsetocapsularpolysaccharideof H.

influ-enzaetype b inhumans.

Serological diagnosis

is

oftenusedtoconfirma suspected clinical

diag-nosis or

bacteriological

diagnosis or both ofan

infectious disease and may be suitable when

bacteriological diagnosishasfailed for some

rea-son.

However,

serodiagnosis

of

H.

influenza

type binfectionsmightbelimitedtoolder chil-dren and adults, since it has been found that

mostyoung children do not respond with

anti-body formation either to

natural

H.

influenza

infectionsortovaccination with purified

capsu-larpolysaccharide (12-14). The children with H.

influenza

infections

included

in

this

study

(even the 6-month-old child) showed specific

antibody responses. Furthermore, it is known

that several types ofbacteria possess antigens which cross-react with type bpolysaccharide (5, 18) (e.g., E. coli K-100 [15], pneumococci [19], and other bacteria [1]). Therefore, it is

conceiv-able that suchorganismscouldgiveriseto

anti-bodies that cross-reactwith H.

influenza

type

b

polysaccharide,

whichmight explaintheinitial

titers,

inadditionto

previous

H.

influenzae

type

binfections.

Basedon significant antibody responses, two cases of epiglottitis and two cases ofcellulitis couldprobably be classifiedas beingdue toH.

influenza

type b or

cross-reacting bacteria

or

both.

TheELISAwasalsoadaptedsuccessfully for detection and quantitation of anti-capsular

se-cretory IgA, as demonstrated by using

naso-pharynx secretions and milk. The clinical

use-fulness of this method should be evaluated with

a larger group of healthy individuals and

pa-tients withH.

influenza

typebinfections.

ACKNOWLEDGMENTS

I amgratefultoCarlvonSydowandOlle Nylén forsome patientmaterialandtoLars-Âke Nilsson, PaulaBranefors, andAnn-MariSvennerholm foracriticalreadingofthe

man-uscript.

Thisstudywassupported bygrantsfromtheFacultyof Medicine, UniversityofGoteborg,Goteborg, Sweden.

LITERATURE CITED

1. Agram, M. 1973. Serologic relationship between Hemoph-ilus influenza, type b, capsular polysaccharide and polyribitol teichoic acids of gram-positive bacteria,p. 49-56. In S. Sell and D. Karzon (ed.), Hemophilus influenza. Vanderbilt University Press, Nashville, Tenn.

2. Andersson, P., R.B.Johnston, Jr., and D. H. Smith. 1973.Methodologyindetectionofhumanserum anti-bodiestoHemophilusinfluenza,typeb,p.87-98. In S. Sell and D. Karzon (ed.), Hemophilusinfluenza. Vanderbilt University Press, Nashville, Tenn. 3. Branefors-Helander, P., andT.Dahlberg.1980.Serum

bactericidal effect on capsulated and non-capsulated Haemophilus influenza. Acta Pathol. Microbiol. Scand. Sect.C88:47-56.

4. Branefors-Helander, P.,O.Nylén, andP.-H. Jepp-son.1972.Acute otitis media. Abacteriologicalstudy. Pract.Oto-Rhino-Laryngol. 34:281-295.

5. Dahlberg, T., andP.Branefors. 1980. Enzyme-linked immunosorbentassayfortitrationofHaemophilus in-fluenzae capsular and O antigen antibodies. J.Clin. Microbiol. 12:185-192.

6. Dahlberg, T., andP.Branefors.1980.Characterization of thebactericidal antibody response against Haemo-philus influenza.ActaPathol.Microbiol. Scand.Sect. C88:115-120.

7. Engvall, E., and P. Perlmann. 1973. Enzyme-linked immunosorbentassay. III.Quantitationofspecific anti-bodiesbyenzyme-labeled antiimmunoglobulin in anti-gen-coatedtubes. J.Immunol.109:129-135.

8. Norden, C.W.1974.Prevalenceofbactericidalantibodies toHemophilusinfluenza, typeb.J.Infect. Dis. 130: 489-494.

9. Norden, C.W. 1978.Hemophilus influenza in adults. Med.Clin.North Am. 62:1037-1046.

10.Norden, C. W.,and H. A.Feldman. 1975.Haemophilus influenzatype bantibodyfrequencies determinedwith bactericidalandradioimmnunoassay tests. J. Clin. Mi-crobiol.2:136-138.

11. Norden, C. W.,and R.H.Michaels. 1973.Immunologic

J. CLIN. MICROIBIOL.

on February 7, 2020 by guest

http://jcm.asm.org/

(8)

responsesinpatients with epiglottitis caused by He-mophilus influenza typeb. J.Infect. Dis.128:777-780. 12. Norden,C.W.,R.H.Michaelb,and M.Melish. 1975.

Effect ofprevious infectiononantibody response of childrentovaccination with capsular-polysaccharide of Haemophilu influenza.J.Infect.Dia 132:69-74. 13.Norden, C.W.,R.H. Michaels,and M.Melish. 1976.

geologicallresponsesof children withmeningitisdue to Hemophilusinfluenzaetypeb. J. Infect. Dis. 134:495-499.

14. Robbina,J.B.,J.C.Parke, Jr.,R.Schneerson,and J.K. Whisant. 1973.Quantitativemeasurement of "natural"andimmunization-inducedHaemophilus

in-fluenzaetypebcapeularpolysaccharide antibodies. Pe-diatr.Res.7:103-110.

15. Schneerson,R.,M.Bradshaw,J. K.Whisnant,R1..

Myerowitz, J. C. Park, Jr., and J. B. Robbins. 1972.AnEscherichia coli antigencross-reactive with the capsularpolysaccharideofHaemophilus influenza typeb: occurrenceamong knownserotypes, and im-munochemicalandbiologicpropertiesof E.colantisera toward H. influenzaetypeb.J.Immunol.

108:1551-1562.

16. Se1, S. IL,D. J.Turner, andC. F. Federspiel.1973. Naturalinfections withHemophilus influenzain

chi-dren. I. Typesidentified, p. 3-12. InS. Selland D. Karzon (ed.),Hemophilusinfluenza.Vanderbilt Uni-versity Press,Nashville,Tenn.

17. Smith,D.H., S.Hamn,V. M.Howie, J. H. Ploussard, A.L. Harding,and P.Andersson.1973.Studies on theprevalence of antibodies toHemophilluinfluenza, type b, p. 175-185. In S. Sell and D. Karzon (ed.), Hemophilusinfluenza. Vanderbilt University Press, Nashville, Tenn.

18. Solotorovsky, M., and M. Lynn. 1978. Haemophilus influenza: immunology and immunoprotection. Crit. Rev.Microbiol. 61:1-32.

19. Tunevall,G. 1952. Studies on Haemophilusinfluenzae typecharacteristics. Acta Pathol.Microbiol.Scand. 30: 203-212.

20.Turk,D. C., and I. R. May. 1967.Haemophilus influ-enzae:itsclinicalimportance.English University Press, London.

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