Enzyme linked immunosorbent assay for titration of Haemophilus influenzae capsular and O antigen antibodies

JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 1980,p.185-192

0095-1137/80/08-0185/08$02.00/0 Vol. 12, No. 2

Enzyme-Linked

Immunosorbent

Assay for Titration of

Haemophilus influenza Capsular and O Antigen Antibodies

T. DAHLBERG*AND P. BRANEFORS

Departmentof Bacteriology, Institute ofMedical Microbiology, University of Goteborg, Gôteborg, Sweden The enzyme-linked immunosorbent assay (ELISA) was elaborated for the

detection of immunoglobulinM (IgM) and IgGantibodiesagainstcapsular and O

antigens ofHaemophilus influenza. Purified capsular polysaccharide and lipo-polysaccharidewereused as antigens, with optimal coatingconcentrationsbeing

about50and100

jg/ml,

respectively.The antibody content wasexpressedas the

highest serum dilution

(-log1o)

showing an absorbance of 0.2 above the

back-ground level. The titers of hyperimmunesera (referencesera) ranged between 5

and 7

-log1o.

The sensitivity of the methodwasabout 80 ng/ml with regard to

anticapsular antibodies and3 to 5ng/mlwithregard toanti-lipopolysaccharide antibodies. For detection of antibodies against capsular polysaccharide in sera

obtained after primaryimmunization,ELISA was about 100-fold more sensitive

thanthe indirecthemagglutinationassay, whereas inhyperimmunesera, ELISA wasabout 10-foldmoresensitive thantheindirecthemagglutination assay. The

sensitivity of ELISA for detecting anticapsular antibodies after primary and booster immunizations was 50-fold higher than that of the bactericidal assay using capsulated bacteria, whereas the sensitivity ofthe two methods was the samewhen hyperimmune sera were tested. ELISAperformedwith lipopolysac-charide as the antigen was about 50- and 150-fold more sensitive than the

complement

fixationandbactericidalassaystestedwithnoncapsulated variants

afterprimary injection and hyperimmunization, respectively.

Theantibody response to the capsular poly-saccharide (CPS) of Haemophilus influenza

typeb has beenestimated

by

indirect hemagglu-tinationassay (IHA) (1, 4) and, morerecently, by

radioimmunoassay

(1, 11). Both methods

have certain disadvantages. IHA is

compara-tively insensitive,

whereas radioimmunoassay, beingsensitive,

requires

certain

precautions

in

handling

radioactivereagentsaswellas

special-izedequipment formeasurement.Inadditionto

the aformentioned

methods,

the

complement-dependent bactericidalassay

(BC)

has beenused

as a test mainly for

measuring

the amount of antibodies against the CPS

antigen (1,

5,

6a),

although

thebactericidal effect

might,

toa

cer-tain extent, bedue to antibodiesagainst other antigens as well (1, 6a). Forthe determination of antibodies

against

the

cell

wall

antigens

of noncapsulated H.

influenza,

the BC assay (5, 6a) and a

complement

fixation

(CF)

test have

been available

<4).

In contrasttotheabove-mentioned

methods,

which are

dependent

on

secondary

manifesta-tions for visualization of the

antigen-antibody

reaction, the primary bindingbetween

antigen

andantibodymayberevealed

by

the

enzyme-linked immunosorbent assay

(ELISA) (7, 15).

Bytheuseof immunoglobulin class-specific

con-jugates, this technique also offers a simple method for measuring and

differentiating

be-tween immunoglobulin M (IgM), IgG, IgA, or secretoryIgA class antibodies without laborious

separation

of serum orsecretions. The micro-ELISA variant (12) is useful when small

amountsofserumand secretionareavailable. The aim ofthe present investigation was to

elaborate suitable

experimental

conditions for

the application ofELISAto the

determination

of antibodies against H.

influenza

CPS and

lipopolysaccharide

(LPS) antigens.The sensitiv-ity of ELISA for the

detection

of H.

influenza

antibodies after primary and booster immuni-zations and

hyperimmunization

was

compared

with that of other methods such as

IHA,

CF,

and BC.

MATERIALS AND METHODS

Strains. H.

influenza type a,

strainSmith

(Ma),

H.

influenza

type

b, strain RAB

(Mb),

and their respective noncapsulatedvariants (Sa and Sb) were

thesameasthoseused inpreviousstudies(3-5, 6a). Antisera. Antisera against Ma, Mb, Sa, and Sb bacteria, obtainedasdescribedpreviously(5,6a),were

usedin thestudy.Theimmunization scheduleandthe bleedingsareshowninFig.1and2.

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186 DAHLBERG AND BRANEFORS

TITRE

- 10

D.-8

o.-IgM

,.

V

;t4

X

g~~~~~

M0-0

B-IHA

O 4 8 12 16 weeks

£ A , , à injections

y

' bleedings

FIG. 1. Antibody response of one rabbit during immunizationcoursewith Mbbacteriaastestedwith ELISA, IHA, and BC. Theimmunizationprocedure was asfollows. The rabbit wasgiven oneprimary immunization and,4weekslater,aboosterdosewith about 1 x 108 bacteria. After another 3 weeks, a hyperimmunization course was started, and eight injectionsweregiven. The bacterialamountateach injectionwasincreased intwofoldstepsfromIx 10' to 6x109bacteria.

TITRE

- log10

ELISA IgG--gMo--O BCF

-C F

O 4 8 12 16weeks

à à à à à à à à à à injections

yy y y y 'v y*y,y*bleedingsy ,y

FIG. 2. Antibody response ofone rabbit during

immunizationcoursewithSb bacteriaastestedwith

ELISA, CF, and BC. Immunization procedureisas

describedinthelegendtoFig. 1.

Additionally,aseries ofhyperimmuneseraagainst

the following H. influenzastrainswas employedto

study the specificity of ELISA:H. influenzaetypesc

(NCTC 8469), d(Garf),e(NCTC8472), andf(NCTC 8473).HyperimmuneseraagainstStreptococcus

pneu-moniae(types 5, 6, 21, and 27),Klebsiella pneumoniae (types O?:K1 andO?:K9), Escherichia coli(types 016: KI, 04:K12, and 075:K100), Proteusvulgaris (types 03H1, 03H2, 01OH3, and 013H4), and Vibrio

chol-erae were also utilized. The antisera against E. coli 075:K100 and thepneumococcalstrains wereobtained from StatensSeruminstitut, Copenhagen, Denmark;

antiseraagainsttheremainingstrains wereobtained fromthe Institute for MedicalMicrobiology, Univer-sityofGoteborg, Goteborg,Sweden.

Antigenic preparations for ELISA. Purified CPS waspreparedfromtheMaandMbsupernatants of shaken 10-h-old fluid cultures in antigen-free me-dium (2). These preparations were designated CPS Ma and CPSMb, respectively. Aslightmodification of the medium was utilized; the medium materials, caseinhydrolysateandyeastautolysate,weredialyzed against distilled water at 4°C for 1 week (dialysis tubes; pore size, 240 nm; Kebo-Grave, Spànga, Swe-den), insteadofbeing filteredthrough SephadexG-25, forobtaininglowmolecular medium.Saltsandgrowth

factors were added before sterilization as previously described (2). During the earlier part of the study, CPS (designated CPS I) was prepared from 10-fold-concentrated culturesupernatantswilAchwere filtered through Sephadex G-150 (3). Fractions tllat eluted with the voidvolume, containingthecapsularantigen togetherwithsomecontaminants (LPSandprobably adhering mediumproteins), werepooled andfurther purifiedby diethylaminoethyl-Sepharose chromatog-raphy using an NaCl gradient (O to 2 M). The O antigen eluted before the main part of the capsular antigen astested by immunodiffusion analyses (16). Capsularantigen-containing fractions, withno absor-bance oronly absorbance below 0.03 at260 and280 nm, werepooled andlyophilized,constituting theCPS I antigen. Its protein content was about 1% as esti-mated bythe method ofLowryetal. (10).

Duringthelatterpartof thestudy, capsularantigen (CPSMbII)wasobtainedby precipitationofdialyzed culture supernatant with 0.5% cetyltrimethylammo-niumbromide(finalconcentration)at40C for2weeks. The capsular material was dissolved in 0.4 M NaCl and furtherpurifiedby threerepeatedprecipitations in3volumes of ethanol with solutionindistilledwater. Centrifugationanddiscarding of water-insoluble ma-terialwereperformed between eachprecipitation.The materialwasfinally purified by filtrationon a Sepha-rose-2Bcolumn, and fractionscontainingthecapsular antigen, asdetermined by immunodiffusion analysis, werecollectedasshown inFig.3.The pooled capsular materialwasdialyzedandlyophilized.Itsprotein con-tent wasestimatedat0.2% bythemethod of Lowry et al. The preparation did not contain detectable amounts ofO antigen, asestimatedby immunodiffu-sion analysis with 1 mgof CPS Mb II per ml. The immunodiffusionmethodindicatedabout15

gg

ofLPS per ml.

LPS was prepared fromtwononcapsulatedvariants, Sa and Sb, ofthe capsulated strains (5, 6a) by hot phenol-waterextraction (17). The crude LPS extracts were treated with ribonuclease (type 1-A; Sigma Chemical Co., St.Louis, Mo.) at aconcentrationof 0.1 mg of enzyme permg of LPS (pH 7.8) for 7 h and then centrifugedat105,000x g for 4 h. Thesepreparations were designated LPS Sa and LPS Sb, respectively. One part of LPSwaslyophilizedtoestimatethe con-centrationofantigen. The part of LPS used for ELISA

6

5.

4-

3-2

o

6

5

4

3

2

o

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VOL.12,1980

o

OD

(nm>

0I3

-0.2

-ELISA FOR H. INFLUENZAE ANTIBODIES 187

8

f

FIG. 3. Sepharose-2B filtration of30

methylammonium bromide-precipitatec material (column size, 2.6 by100cm;ph

M]-buffered saline [0.5

MI,

pH 7.1,, elution;flowrate, 20ml/h). Fractions taining all detectableO antigen ands

materialwerediscarded. Fractions38

bydotted line underfigure) werecollect asCPS MbIIantigen. It containedal

purifiedCPSMbIIperml. A 30-mlar

Mb II materialcorresponded to abou

culturefluid.

wasnotlyophilizedbecause of incomplel H. influenza LPS after lyophilization

contentwas2%astested by the metho

al.

Serological methods. (i) ELISA.El formed essentiallyasdescribed by Eng mann(8).

(a)Antigen-coatedtubes. Disposab tubes (HegerPlastic AB, Stallarholmen, coatedwith0.5or1mlof antigen solutiî

trations ranging from 1 to 1,000

jig/ml.

antigen concentrations weredetermine

of IgM as well as IgG antibodies. Th

coated overnight at37°Cand thenkept sealed tubesfor from afew days up tc

evaluate theefficiency and stability oft

(b) Enzyme-conjugated anti-imm Sheeporswineantisera specifictorabbi

immunoglobulin (Nordic Immunologi ries,Tilburg,TheNetherlands)were co

alkalinephosphatase(calf intestinalmu( SigmaChemical Co.) with the aid ofg asdescribed byHolmgren andSvenner

suitable concentration of the conjugate zyme-substrate reaction time were det

hyperimmunereferencesera.Theconjul

usedgenerally indilutions of about1:30

and 1:400 for anti-IgG, with the enz: reactiontime beingabout 50 min.

(c)Standardizationof ELISA. Ra

munesera wereusedasreferencesina]

forthedifferentserologicalsystems.Th.

strate reactions were allowed to conti tubes containing the reference serum

reachedappropriateabsorbancevalues, imately thesameendpointtiters (seeTi

separate experiment. An antigen-coat batedwith theenzymesubstrate(blanh

tube incubated with conjugates and phosphate-260nm

buffered

saline

instead

ofserum (background absor-280 nm - bance) were included as controls in each set of

exper-iments.

(d) Micro-ELISA. Micro-ELISAwasperformedon microplates (Dynatech, Micro-ELISA; Nova Kemi AB,Enskede, Sweden) using a total reactant volume of 0.2ml. Otherwise,experimentalconditions wereas described above. Absorbances were registered byan r-r----.- automatic spectrophotometer (Titertek Multiskan; o 90 100 Flow Laboratories,

Solna,

Sweden). The reference raction number anti-Ma, anti-Mb, anti-Sa, and anti-Sb sera were 5ml each) testedsimultaneously by micro- andmacro-ELISA for comparison andstandardization ofexperimental

con-mi

olceyMbtrII

ditions so as to obtaincomparablesensitivity.

ioCPS'

Mb05

(i)

Quantitative

precipitin

determination.

osp

ated

0.0 Quantitative precipitin determination (9) was per-was used

for

formed toestimate the amount ofspecific antibodies 25 to 37 con- against CPS Ma, CPS Mb, LPS Sa, and LPS Sbin

omre

capsular

homologous hyperimmune antisera. A0.2- or

0.25-ml

to 75(marked amount of serum was mixed with equal volumes of

acted

andused antigen dilutions with concentrations ranging from

°out

160

,gp

of 0.002 to 1mgincubated for1 hat37°C andwasthen t1.7 I

PtS

kept about1 week at 4°C. Themaximumamount of

t *.7

lters

of

precipitated

antibodieswasdetermined

by

the method ofLowry etal.

tesolubilityof (iii) Absorption of serum. The anti-Mb serum mThe protein diluted 1:1,000 was mixed with various dilutions of

d

ofhe

Lowrtent

CPSMb I or CPS Mb IIantigens ranging in concen-trations from0.2 to 200

gg.

After incubation for1hat LISA was per

37°C,

thetubeswerestoredat2°C for about1 week.

vail

and

Perl-

Thesupernatantswerethentested for remaining

anti-,vall and Perl- bodies bymicro-ELISA using the IgG-conjugate.The ole

polystyrene

antigen

concentration

giving

50%

reduction

of the Sweden) were extinction at 400 nm was used as a measure of the onsin concen-

inhibiting

capacity.

The optimal (iv) Other serological methods. BC, used for d for titration determination of bactericidal antibodies

against

cap-ie tubes were sulated and

noncapsulated

bacteria, IHA, used for

tat 4°C

hi

the

measuring

antibodies

against capsular antigen,

and 3 months to

CF,

used for

measuring

antibodies

against

cell wall ;he coating. antigens, were performed as previously described (4, unoglobulin. 5)

tIgG and IgM Statistical methods. Calculation of mean and

ical

Laborato- standard deviation of titer values wasperformed ac-njugatedwith cording to standard methods.

cosa, typeVII;

,lutaraldehyde RESULTS

holm (8).The A

series

of experiments was performed to

is

and the en- studyvarious factorshavinga

possible

influence

gates

cwuldbe

on the results obtained

by

ELISA as used for

O

foruanti-IgM

detection

of antibodies

against

H.

influenza

yme-substrate CPS and LPS.

Antigenconcentration and

coating

time.

Lbbithyperim- ForCPS Ma I and CPS Mb 1,increasing absor-Ilexperiments bance wasregisteredwhenincreasing concentra-eenzyme-sub- tions ofantigen were used for the coating of the nue until the

tubes,

with no leveling of absorbance

being

giving approx

reacheduptoan

antigen

concentration of 1 mg/

able 1) at each

ml

(Fig. 4). To obtain the highest titers of the ed tube incu- homologous reference sera, coating with CPS

o)

andanother MaI preparations should be

performed

at

4°C

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188 DAHLBERG AND BRANEFORS

E400nm

O.---....o

f..

/~~~o

CPS Ma

CPS Mb Ic

CPSMaill

CPSMbIlc .. w

E400nm

1,2-1

-

0,8-

0.6-

0.4-0.2

-O -*-e

o-o

...

o-....o

10 100 1000

ANTIGEN ug/ml

/"

e~~~~~~~~~~

1 110 100 1000

ANTIGEN ug/ml

FIG. 5. Determinationofoptimalamount ofLPS Sbantigen for coating oftubes. The anti-Sbserum wasdiluted1:1,000. TheIgG conjugatewasusedin thetest.

FIG. 4. DeterminationofoptimalamountsofCPS MaI,CPS MbI,CPS MaIH,and CPS MbIIantigens for coating oftubes. Theanti-Ma and anti-Mbsera were diluted 1:1,000. Asecond anti-Mb serum was

tested with CPSMb IIandwasdiluted 1:100. The IgG conjugatewasused in thetest.

for 1to2days, whereascoatingwith CPS Mb I should be performed for about 2 to 3 weeks. When the CPSIantigenwasused forcoatingat

aconcentration of100 to200,ug/ml,theIgGand IgM titers of thereferencesera wereaboutthe same asthose observedwhenhigher concentra-tions ofantigen were used for coating. In the earlierpart ofthestudy,100 to200,tg of CPSI

permlwasfoundtobesuitable for determina-tionof bothIgGandIgM antibodies.

TheCPSII antigenpreparations adsorbedto thetubes in lower concentration than did CPS I. Optimalcoatingwasobtained byusing about

50,ug/ml

asdetermined by IgG conjugate (Fig. 4). In addition, the CPS II preparationsadsorbed moreeffectively (overnight incubation at37°C and 1to2daysat4°C) than didtheCPS I. To

savematerial,asuboptimal concentration of30

,ug/ml

wasutilized for furtherassays.

Theinhibitionstudy with CPS Mb I and CPS Mb IIantigen preparations andan anti-Mb

se-rumdilution of 1:1,000revealed that the

inhibi-tory concentrations causing 50% reduction of extinction at400 nm wereabout250,g/ml for theCPSMb I andabout30,g/mI forthe CPS MbIIantigen preparations.

Optimal coating of tubes with LPS was

ob-tained by usingasolution containing 100

,ug/ml

astested with 10-fold dilutions of LPS antigen (Fig. 5).

E400nm

1.6 1.44

1 2 3 4 5 1 2 3 4 5 6 SERUMDILUTION-bogl

FIG. 6. ELISA titration curvesforIgGandIgM class antibodies as obtained with rabbit antisera

testedagainstCPS MbII antigen. (a) After primary injection. IgG titer, <1 -loglo; IgM titer, 2.5-log1o.

(b) Afterhyperimmunization (referenceserum). IgG titer, 4.5-logio; IgMtiter, 3.8-log1o.

Principles forquantitation of antibodies. Thesteepnessof the linearportions of the titra-tion curves increased during the course of the immunization. Thisincreasewaspronounced for IgG antibodies. The IgG curves were steeper

than those obtained with anti-IgM conjugates. Figures6a and 6bgiverepresentative examples of the titration curves of sera obtained after primaryinjection and hyperimmunization using CPS MbIIantigen. For ELISAperformed with H. influenza CPSandLPSantigens, the anti-bodycontentwasexpressedasthe

-loglo

of the serum dilution titerr) which showed an

absor-1,6

1,4-

1,2-1

-

0,8-

0,6-

0.4-

0,2-1

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ELISA FOR H. INFLUENZAE ANTIBODIES 189

bance of0.2 above the background level. The

background absorbance for the CPS as well as theLPSantigens was generally low, about 0.01 to0.07.

Precision of ELISA. The rabbit reference

serumsamplewastitrated 10 times on separate

days with two lots ofconjugates and two CPS

MbIIantigenpreparations.Thestandard

devia-tioncalculated from these experimentswas 0.21

-log1o.

Sensitivity

and specificity of ELISA. To estimate the sensitivity of ELISA, quantitation of thespecific antibodycontentinrabbit

hyper-immunesera wasperformedinparallelby means

of thequantitative precipitationtest.The lowest

amountof antibodies detectable with ELISAfor

H.

influenza

CPS and LPSwascalculated from

themaximumamountofprecipitated antibodies and the corresponding ELISA titer by using

IgGconjugate. The lowestamountof antibodies detectable with ELISA wasabout 80 ng/ml for

anti-CPS Ma and Mb and about 3 to 5 ng/ml foranti-LPS Sa and Sb (Table 1).

Tostudy the specificityofELISAconcerning

anti-capsular

b antibodies, in addition to the

homologous

antisera, a number of antisera against variousgram-negative and gram-positive bacteria, including five remaining types of H.

influenzae,

weretested for

presence of IgG

anti-bodies against CPS Mb II antigen by micro-ELISA

(Table

2). Demonstrable titerswere

ob-tained

in sera

against

H.

influenza

type

a

(about

2.4

-logwo),

S.

pneumoniae type

6

(about

2.2

-logio),

and E. coli 075:K100 (about 3.4

-logio).

Asregards antibodies against LPS Sb antigen, theaforementioned sera were employed in the study.Nodemonstrable titerswerenotedin any

of them.

TABLE 1. Comparative IgG antibodyclass determinationof anti-Ma, anti-Mb,anti-Sa,and

anti-Sbhyperimmuneseratestedagainst homologous CPS(I andII)andLPSantigens,

respectively

Specific Lws e

precipi- ELISA end- Lowest

de-Antisera tated anti- point titer

amt

of anti-bodies (IgG) bodies (ng)

(mng/mld)

Anti-MaI 5.2-5.4 60,000 86-90" Anti-MbI 3.1 30,000 103" Anti-MbII 4 50,000 80" Anti-SaI 4.4 1,000,000 4.4 Anti-SbI 3.6 1,000,000 3.6

aCPSIusedinELISA andquantitative

precipita-tion.

bCPS II used inELISA andquantitative

precipi-tation.

TABLE 2. ELISAtiters(-logîo)against CPS MbII antigenin antisera against various bacteria

Antiseraagainst: Straintype Titer (-logo)

H.influenza b 4.5

a 2.4

c <1

d <1

e <1

f <1

S. pneumoniae 5 <1

6 2.2

21 <1

27 <1

E. coli 016:K1 <1

04:K12 <1 075:K100 3.4 K.pneumoniae O?:K1 <1

O?:K9 <1

P.vulgaris 03H1 <1

03H2 <1

010H3 <1

013H4 <1

V. cholerae <1

Comparison

ofmacro-and micro-ELISA. Parallel experiments were performed with

ref-erence seraby using optimal concentrations of the corresponding antigens and the same

con-jugate concentration and enzyme-substrate

re-action time. The greatest

differences

recorded between resultsobtained bymacro- and micro-ELISA techniques were limited to 0.2

-log1o

when CPS and LPS antigens were used in the assays.Therewas notendency foranytechnique

togivegenerally higher values.

Antibody response in rabbits against

capsulated

bacteria studied

by

means of ELISA,

IHA,

and BCassays.The four rabbits immunized withcapsulated bacteria hadno de-monstrable antibodies against CPSMaandCPS Mb in their preimmune serum

samples

as

re-vealed by ELISA. The primary

immunization

with Ma orMb bacteriacausedan

IgM

antibody

response within 2 to 4 days, whereas an

IgG

antibodyresponsewas notdemonstrable until7 to 14days. TheIgM titersrapidly decreased to lowlevels

before

the boosterdose,incontrast to theIgG titers,whichincreased

continuously.

The booster dose caused an

IgM antibody

response of the samemagnitudeasthat obtained after the primary immunization, whereas the

IgG titer levels increased only

slightly. During

the

hyperimmunization

course,the

IgM

and

IgG

antibody levels increased

gradually

totiter val-uesof about4and5

-logio,

respectively.

Figure

1gives thedevelopmentofELISA

IgM

and

IgG

antibody titers in a rabbit

given

Mb

bacteria,

the titer development being

representative

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the other rabbitsaswell.

The antiserawerealso testedagainstLPSSb antigen. The developmentofIgM and IgG anti-bodieswas similar to that described below for the antisera against noncapsulated bacteria. However, the titers were about 10- to 100-fold

lower in the hyperimmune sera against capsu-lated bacteria than in sera against the

corre-spondingnoncapsulated variant.

In addition, theIHA and BC titersaregiven for comparison ofsensitivity. For the foursera, the IHA titers were50- to 100-fold lower after theprimaryimmunization than the correspond-ing ELISA titers. During the hyperimmuniza-tion course, the IHA titers increased to about thesamelevelsasELISAIgM titers,beingabout 70-fold lower than the ELISAIgGtiters(Fig. 1). The BC titerswere10- to 50-fold lower than the corresponding ELISA IgM titers after the pri-mary immunization. At the end of the hyper-immunization course, veryhigh BC titerswere obtained, showing even higher levels than the ELISAIgGinthecorrespondingserumsamples (Fig. 1).

Antibody response in rabbits against

noncapsulated bacteria studied by means

ofELISA, CF, and BC assays.The two rab-bits immunized with Sa bacteria had low amountsofIgM antibodies,asdemonstratedby ELISA(about 1.2 -log1o), against LPS Sa before immunization. In sera obtained after primary immunization, IgM and IgG antibody response

against Sa bacteria was obtained after 2 days (titer about 3

-logio

for both classes of anti-bodies). The preimmune sera of two rabbits immunized with Sb bacteria didnotrevealany IgM antibodies against LPS Sb. The primary immunizationcausedanIgMantibodyresponse after 2 days, whereas IgG antibodies were not revealed untilafter about 3weeks(Fig. 2). The IgM titers against LPS did not increase any further after the booster dose and during the hyperimmunization course, whereas the IgG antibodiesincreased rapidly tovalues between 6and 7

-logio

(Fig. 2). In addition, the CF and BC titersaregiven for comparison. The sensitiv-ity of the CF and BC methodswasabout50-and

150-fold lower than that of ELISA in serum

samples taken after primary immunizationand hyperimmunization, respectively.

DISCUSSION

Thepresent reportevaluates thesuitabilityof ELISA forquantitation of antibodiesagainst the CPS andLPSantigens of H. influenza inrabbit antisera.

The investigation showed that the capsular substanceof H. influenzatypesaand b (CPS

MaandCPSMb) adsorbedtothe

plastic

tubes

insufficient amounts to allowdetermination of

antibodies. Twopreparations of purified H.

in-fluenzae

capsularantigenwereutilized,one ob-tained by Sephadex G-150 filtration and di-ethylaminoethyl Sepharose chromatography (method I) and the other obtainedby cetyltri-methylammonium bromide precipitation and Sepharose-2Bfiltration (method II). Theywere

foundtodiffer inbinding capacitytotheplastic surface, with CPS IIbeing attachedmoreeasily; theoptimal concentration wasmarkedlylower, and for CPS Mb II, the necessary attachment timewasconsiderably shorter than for CPS Mb

I. Differentlots of CPS II preparations showed thesamehigh attachmentability.It wasfurther observed in inhibition studies that the immu-nological reactivity ofCPS Mb II antigen was

about 10-fold higher thanthatofCPS Mb I. In

addition, itspurity (e.g., low protein content) is

afurtheradvantage of method II for preparation of H.

influenza

capsular antigen

forELISA.

Results obtainedby ELISAmaybeexpressed

in a variety of ways (15). In this study, the antibody content was expressed as

-logio

of serum dilution representing an endpoint titer. Repeatedtitration of referenceserashowed that there was good reproducibility of results

ex-pressed in thisway.

For standardization ofthe assay, the use of referenceserum, withknown amountsofspecific antibodiesinallsetsofexperiments,was neces-sary to compensateforvariation in test param-eterssuch asantigencoatingon different

occa-sions,different lots of conjugates,etc.The stand-arddeviation of ELISA described in this study

was aslowas 0.21

-logio.

ELISAwasfoundtobeasensitive method for thedemonstration of antibodiestoH.

influenza

antigens. By means of this technique, it was

possible to measure about 80 ng of IgG

anti-bodies againstCPS. However, since IgM

anti-bodies present in the hyperimmune sera may

also havecontributed tothe amount of precipi-tate obtained at the quantitative precipitation analysis,thesensitivity of ELISA may have been

somewhat

underestimated. Despite this

possi-biity,

thesensitivitywasof the

same

magnitude as that of radioimmunoassays as reported by Norden andMichaels (11). However, other stud-ieshavereported higher sensitivity of

radioim-munoassay techniques (1). The sensitivity of

ELISA with regard to

determination

of anti-bodies against LPS was at least 10-fold higher than that ofantibodies against CPS. It may be notablethatthe sensitivity of the assay used in the

present

study for

demonstration

of anti-bodies against H.

influenza

LPS was about

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ELISA FOR H. INFLUENZAE ANTIBODIES 191

fourfold

higher than that registered inanassay

for determination of antibodies against

Salmo-nella O antigen(6).

ToobtainhighspecificityofELISA, aprimary requisiteis to use highly purified antigen

prep-arationsatthe coating of the tubes. The pres-enceofatraceamountofOantigen intheCPS preparationused in ELISA (less than 0.5

ig/ml)

shouldnothave influencedthe absorbance ob-tained in the testsperformedwith thisantigen

because an absorbance as low as 0.2 was ob-served with1,gofCPSperml.

ELISA performed with hyperimmune sera

against other H.

influenzae

strains and

other

species of bacteria revealed antibodies against

H.

influenzae

type a, S. pneumoniae type 6, and

E. coli K100cross-reactingwith theCPS Mb II antigen. The

capsular

antigens of these bacteria

areknowntohave somechemical similaritiesto

the

capsular antigen of H.

influenzae

type

b

(13,

14).

Using

the same

experimental

parameters in

micro-ELISAasinmacro-ELISA,nodifferences

in titer values exceeding 0.2

-logio

were

re-corded. On this

basis,

the same sensitivity can

be assumed for the micro method and macro

method.

Although the number of animals included in

the

study

wasrather

small,

some

general

conclu-sions concerning the sensitivity of ELISA as

compared with that of other serological methods mightbedrawn.

Forestimationoftheprimaryresponse (IgM class

antibodies),

ELISAwasfoundto bemore

sensitive (10-to

100-fold)

than the other meth-ods,

especially

when

compared

to IHA, which

measurescapsular antibodies,too.

After the booster dose, in addition to

IgM

class

antibodies,

ELISA

IgG

titerswererevealed whichwere

higher

than those

obtained by

other methods.

In the

hyperimmune

sera to

capsulated

bac-teria, the differencesbetweenELISA titersand

the titers of the other methodswere less

pro-nounced. Intheantisera to

noncapsulated

bac-teria,

the

higher sensitivity

ofELISAwas more

evident.The

discrepancy

in

sensitivity

between BCand ELISA may

partly

be due tothe very

high sensitivity of ELISA for the detection of

anti-LPS antibodies. Moreover, antibodies

against LPSand othercell wall

antigens

of

non-capsulated

bacteriaare

possibly

not aseffective

inthe BC assayas are

anticapsular

antibodies (6a).

ELISAwasfoundtobeasuitablemethod for

detection of

anticapsular

antibodies

against

H.

influenza

type

b

in

humans,

too

(manuscript

in

preparation). Especially,

its

high

sensitivity

in

estimation ofprimary response antibodies, as

observed forthe rabbit sera, may be valuable for

detection ofantibodies during the acute stages

of

infectious

diseases. The micro variant is easier toperformand is more economical with regard toconsumptionofantigen, conjugate, etc., and

this modification can, therefore, be

recom-mendedfor routine work.

ACKNOWLEDGMENTS

This study was supported by grants from the Swedish MedicalResearch Council (16X-04978), the Faculty of Medi-cine, University ofGoteborg,and theGôteborg Medical So-ciety.

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