Immunoblot analysis of humoral immune responses following infection with Bordetella pertussis or immunization with diphtheria tetanus pertussis vaccine

(1)

0095-1137/88/071373-05$02.00/0

Immunoblot Analysis

of Humoral

Immune

Responses following

Infection with

Bordetella

pertussis

or

Immunization with

Diphtheria-Tetanus-Pertussis

Vaccine

STEPHEN C. REDD,l* HELLA S.

RUMSCHLAG,l

ROBIN J. BIELLIK,2 GARY N. SANDEN,' CHARLES B. REIMER,' AND MITCHELL L. COHEN'

Centerfor Infectious Diseases' and Center for PreventionServices,2 CentersforDiseaseControl,Atlanta, Georgia 30333 Received 18 November1987/Accepted 30March 1988

To help develop better diagnostic tests for pertussis, we examined the serologic response to whole-cell

proteinsof Bordetella pertussis afternaturalinfectionorvaccination with diphtheria-tetanus-pertussis vaccine.

Serum specimens collected duringapertussis outbreak investigation and from uninfectedpersons wereusedin

Western blot(immunoblot) analysestodetermine thepresenceofimmunoglobulin G (IgG) and IgA antibodies tospecific B. pertussis proteins. IgG antibodiestoproteins ofmolecularmasses220 and 210kilodaltons(kDa) weredetected in 14 of 18serumsamples obtainedfrompatients with culture-confirmed pertussis .4Qdaysafter theonsetof coughing. IgAantibodiesweredetectedin 15 of the 18 samples.Of19serumsamplesobtainedfrom

patients who hadnotbeenill with pertussis,6contained IgGantibodiestothese proteins and1containedIgA

antibodies. The two proteins bound antiserum specific for ifiamentous hemagglutinin and comigrated with purifiedfilamentoushemagglutinin. IgG antibodiesto twoadditionalprotein bands of molecularmasses84 and

75 kDa wereassociated with previousvaccination. Antibodyto the84-kDa protein was detected in 15 of 17

vaccinated, never-infected persons, and antibodytothe75-kDa protein wasdetected in 16of the17. None of

11 nonvaccinated, never-infected persons tested hadantibodies toeither protein. AUlseven fully vaccinated

personswithculture-documented infectionhadantibodies tobothproteins. Antibodiestothe84-kDa protein

weredetected in 6 of 22 nonvaccinatedandinfectedpersons,andantibodiestothe 75-kDaproteinweredetected

in 8 of the 22. Use ofWestern blot analysis in this study allowedustodistinguish antibodyresponsestoinfection and immunization.

Current laboratory methods for diagnosing pertussis are

unsatisfactory. The pathophysiology of this disease is

com-plex, involvingthreestagesof illness with differing clinical, biochemical, and hematologic manifestations (15). Orga-nismsarepresentin the nasopharynxearly in illness butmay

be absent whenclinical signs of pertussis are most distinc-tive (20). Because of this complex natural history, it is unlikely that a single test would be universally appropriate for diagnosing all stages ofpertussis. Detecting organisms early in illness and assessing hostresponses in laterstages

might be the best strategy. When these clinical, epidemio-logic, andpathologic variablesarecoupled with the inherent

methodologic limitations in available methods, it is not

surprising that in threerecentpertussis outbreaks, only51 to 67% ofclinically suspected cases could be laboratory

con-firmed(3, 4, 19).

These problems have stimulated the search for better diagnostictestsforpertussis (2, 7, 9, 10, 17, 18, 27-29).We used Western blot

(immunoblot)

analysis to measure anti-bodies towhole-cellproteins ofBordetella pertussis insera

of patients who had culture-documented pertussis and in

sera ofpersons who had not had pertussis. Results ofour

study, showing thatantibodiestospecificproteinscorrelate with infection and vaccination, should assist the

develop-mentofimproved diagnosticmethods forpertussis. MATERIALS AND METHODS

Serum samples. An epidemiologic investigation ofa

per-tussis outbreak provided the opportunity to test serologic

responses to infection. The outbreak occurred in a

mid-*Correspondingauthor.

Atlantic state in a geographically isolated population that

had a low rate ofimmunization against pertussis. Few ill

persons sought medicalcare orreceived antimicrobial drugs

for the illness. Paired serum samples were obtained at

1-month intervals from 13 persons with clinical signs and

culture confirmation of pertussis. Age, diphtheria-tetanus-pertussis(DTP) vaccination status,and dateof coughonset

were known for each person from whom serum was

ob-tained. Asingle serumspecimen wascollected from each of

an additional 14 persons with culture-confirmed pertussis.

Since samples were available from only two outbreak

pa-tients without exposure to pertussis, serum samples from

twoadditional uninfected groups unrelatedto the outbreak

wereevaluated. Ninepairs ofserumsampleswereobtained from children before and afteracomplete four-dose

immu-nization series, and samples werealso obtained fromeight

adult volunteers who had beenfullyimmunized and had no

known pertussislike illness.

Westernblot analysis. Cellsof B. pertussis CDC A022A, isolated from nasopharyngeal mucus of one patient with outbreak-associated pertussis were used toprepare

whole-cellprotein antigens.Thecellswerestoredinhorsebloodat

-700Candgrownin2.5%C02onRegan-Lowemedium(22)

at350C for 72 h. Wholecellsweresuspended in phosphate-bufferedsaline toan optical density of 0.4 at 540nm. One

milliliter of this suspension was pelletedby centrifugation,

suspended in 50 ,ul ofsamplebuffer (0.4% sodiumdodecyl sulfate, 1% mercaptoethanol, 2%glycerol,50 mMTris,pH

6.8) andheatedto 1000Cfor 10 min. A

250-,ul

volume ofthis

whole-cell lysate was separated by electrophoresis in a

12.5% sodium dodecylsulfate-polyacrylamide gel (11 by17

cm by 0.75 mm) at 30 mA of constant current for 3 h.

1373

on April 11, 2020 by guest

http://jcm.asm.org/

(2)

1374 REDD ET AL.

Proteinson thepolyacrylamidegelwere then electrophore-tically blotted to 0.45-,um (pore size) nitrocellulose paper (24, 26) at 7 V/cm for 3 h in 25 mM Tris buffer (pH 8.6)

containing192 mMglycineand20%

(vol/vol)

methanol. For

comparison, whole-cell proteins of strains Tohama 1 and NIH 165 were electrophoretically separated and blotted to nitrocellulosepaperunder thesameconditions. Silver stain-ing was used toidentify bandsin thepolyacrylamide gel(16), and India ink staining was used to confirm the transfer of proteins to the nitrocellulose paper (11).

Unbound sites on the nitrocellulose paper were blocked overnight with casein-thimerosalbuffer(14, 25)at22°C;the nitrocellulose paper was then cut into 0.5-cm strips. The stripswereincubated with humanserum at a

single

dilution of 1:50 in casein-thimerosal buffer for 2 h at 37°C. Strips were washed and assayed for immunoglobulin G (IgG) antibodies withprotein A-horseradish

peroxidase

conjugate (Bio-RadLaboratories,

Richmond, Calif.)

or

assayed

forIgA antibodieswithapoolofanti-human IgAmousemonoclonal

antibodies followed by goat anti-mouse antibodies

conju-gated to horseradish peroxidase. Mouse anti-human IgM monoclonalantibodieswere usedto detectIgM antibodies.

Specificity of anti-immunoglobulin monoclonal antibodies was confirmed by the methods of Reimer et al. (13, 23). Stripsweredeveloped with

tetramethylbenzidine

peroxidase developer(25

jxg

of

tetramethylbenzidine

in 5 mlof metha-nol; 1.25 ml of1 M HEPES

[N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid], pH 7.5; 20 mlof5.0%

dioctylso-dium sulfosuccinate sodioctylso-dium salt;25 jjl of30%

H202

in 100 ml).

The presence or absence of

antibody

binding

to specific antigens was determined by visual

inspection.

Neither the casestatus northevaccinationstatus ofthe

specimen

donor wasknown untilafterthe presenceorabsenceofantibodies hadbeen determined. Proportions were compared

by using

Fisher's exact test(8).

Purified filamentous hemagglutinin (FHA) was obtained from the Michigan Department of Public Health,

Lansing,

and purified pertussis toxin (PT) was from the Merieux

Institute, Lyons, France. Antiserum to these antigens was prepared in adult New Zealand rabbits that were free of

anti-Bordetella antibodies as determined by slide microag-glutination test.

To

produce anti-PT, purified PT in phos-phate-bufferedsalinewasemulsifiedwithanequalvolume of

Freund complete adjuvant. Animals received 10 ,ug of PT,

administeredasfive 0.2-mlintradermal injections.This dose wasrepeated 2and 4 weeksafter the initial dose. Serum was

collected 3 weeks after dose 3. Procedures for producing anti-FHA were identical to those for producing anti-PT exceptthat each dosecontained 25 ,ug of FHA.

RESULTS

Asilver stain ofasodium dodecylsulfate-polyacrylamide

gel containing electrophoresed whole-cell proteins showed no qualitative

differences

among B. pertussis strains CDC

A022A, Tohama 1, and NIH 165 (Fig. 1). However, an apparent quantitative difference was noted; two bands, of molecular masses 27.5 and 17.5 kilodaltons (kDa), stained less intensely in strain CDC A022A than in strain Tohama 1 or NIH 165. An India ink stain of transferred proteins

showed that lower-molecular-weight proteins stained less

intenselythan those with higher molecular weights, probably

becauseofdecreasedbinding to the0.45-,um-pore-size

mem-brane.

Antiserum from patients recently infected with B. per-tussis bound multiple protein bands in the Western blots of

A

200 g

92.5 t;.

45->~~~~~~~~~~~~~~~~

31->

:

B

_,

14

-' _

1 2 3

FIG. 1. Electrophoresis of proteins from three strains of B. pertussis. (A) Silver-stained proteins in asodiumdodecyl sulfate-polyacrylamide gel. (B) India ink-stained proteins after transferto nitrocellulosepaper. Numberstothe leftindicatemolecularmass in kilodaltons, and numbers at the bottom indicate the strain of B.

pertussisused. Strain CDC A022Awasin lane 1, Tohama1wasin

lane2,andNIH 165wasin lane 3. Slantedarrowsindicate bandsof

molecularmasses27.5 and17.5 kDa that stained lessintensely inthe

CDC A022A than in the Tohama 1 or NIH 165 lane. Bands with

molecularmasses ofless than 20 kDa stainedweakly in the Indiaink preparation, indicating less avid binding of lower-molecular-weight proteins to the 0.45-,um-pore-size nitrocellulose paper or smaller

amounts oflower-molecular-weight proteins presentin the

whole-cell antigen preparation.

whole-cell protein preparations (Fig. 2). Both IgGand IgA antibodies were detected, but no consistent activity was detected with an anti-IgM conjugate at the 1:50 dilution of serum. In contrast, patients who had neither a history of pertussis nor vaccination lackedIgA andIgGreactivity. The IgA and IgG reactivity to specific proteins varied among persons with different histories of disease and vaccination. Antibodies to proteins of molecular masses 220 and 210 kDa were more frequently identified in serum samples from pertussis patients than in samples from never-infected pa-tients (Fig. 2). These proteins are probably the same

high-molecular-weight FHAbands identified by Irons et al. (12). Antibodies toproteinsof B. pertussis with molecular masses of 84 and 75 kDa were found more frequently in serum samples from vaccinated patients than in samples from unvaccinated patients (Fig. 2).

Studieswereconducted todetermine whether any of these bands were associated with previously described cellular constituents of B. pertussis. Purified FHA comigrated with the 220- and 210-kDa bands of the whole-cell preparation, and rabbit polyclonal antibodies to FHA also bound these

J. CLIN. MICROBIOL.

on April 11, 2020 by guest

http://jcm.asm.org/

(3)

1 2 3 4 5 6 7 8 91011121314

200---

116--9

_ _ ~~~~~~. «I

âmo_ _

_ ..

45-*

31

FIG. 2. Selected immunoblots. Numbers to the left indicate molecular mass in kilodaltons; heavy arrows to the left indicate bands with molecular masses of 220 and 210 kDa, and open arrows to the right indicate bands of molecular masses 84 and 75 kDa. Seven serum specimens that were obtained from five different persons aredepicted. For each serum specimen, the odd-numbered lane shows staining for IgG antibodies and the even-numbered lane showsstaining for IgA antibodies. Lanes:1and 2, immunoblot ofa

serumspecimen obtained morethan40daysafter symptom onset

fromavaccinated patient withculture-confirmed pertussis; 3and4, results from aspecimenobtained more than 40 days after symptom

onsetfromanunvaccinatedpatient with culture-confirmed pertussis

(Lanes 1 to 4 show IgG and IgA antibodies to FHA, but the unvaccinated patient did not have antibodies to the 84- and75-kDa proteins.); 5 to 8, results from two serum specimens obtained 1 month apart fromasingle patient with culture-confirmed pertussis who had received three doses of DTP vaccine. IgG and IgA antibodiesto the 220- and 210-kDa bands arepresent in the later specimen (lanes 7and8), as areantibodies tothe 84- and 75-kDa proteins. Lanes 9to 12 show results from two serum specimens obtained from a fully vaccinated patient with culture-confirmed pertussis. IgA antibody to FHAis seen in the convalescent-phase

serumspecimen, and IgG antibodiestothe 84- and 75-kDaproteins

are seeninbothserumspecimens.Lanes 13and14show results in

a 20-month old child after completion of a4-dose immunization series withDTPvaccine.IgGantibodiestoboth the84-and 75-kDa bands arepresent.

TABLE 1. Antibody to FHA inpersonsnever infected with B.pertussis

No. of No. of

Serumsource patients with patients with Totalno. IgG antibody IgA antibody tested

Outbreak O 0 2

Children before 0 0 9

immunization

Children after 4 0

immunization

Vaccinatedadult 2 1 8

volunteers

aSerum collected during the outbreakinvestigation from children who had neither had pertussisnor beenexposedto other children with pertussis.

b Each of these children contributedtwo serum samples: one beforeand oneafter afour-dose immunization series.

two bands. Purified PT did not comigrate with any of the bands visible afterelectrophoresis of the whole-cell antigen,

suggesting that too little PT was present in the whole-cell preparation for detection. It is possible that the static culture method we used inhibited production of PT (6). The rabbit polyclonal antibodies to PT bound bands of higher molecular weight than that of thebands produced by electrophoresis of purified PT, indicating either nonspecific binding or

com-plexing ofPTI with higher-molecular-weightproteins.

IgG antibody to FHA was detected in 14 of 18 serum

samples obtained atleast 40 days afterthe onset of illness from patients with culture-confirmed pertussis. From this samegroupofsamples,15of 18 hadIgAantibodies to FHA. In contrast, of 19 persons with no history ofexposure to

pertussis except through vaccination, 6 had IgGantibodies and 1 had IgAantibodiesto FHA(Table 1).

Todetermine the effect oftime since symptom onset on

production of antibodies toFHA, we plotted the results of Western blotanalysis of available serum specimens bythe numberofdays betweencough onsetand serum collection (Fig. 3). The likelihoodofhaving IgG orIgA antibodies to these twobandsgraduallyincreased with the number ofdays betweencough onsetandserumcollection.

Todeterminethe effect ofpatientage on developmentof antibodies to FHA,we stratified theresults by patientage.

Figure 4 shows how frequently antibodies to FHA were found in serum obtained from culture-positive patients at

least 40 days after the onset of

coughing. Despite

small numbers ofcases evaluated, children less than 4 years old with positive cultures were

significantly

less

likely

to have

E ONE SERUM WITHOUT19G OR IgA ANTIBODY TOFHA ONESERUM WITH IgG ANTIBODYTO FHA

ONESERUM WITH 1gA ANTIBODY TO FHA

S ONE SERUMWITH BOTHIgG ANDIgAANTIBODY TO FHA

-2 0 2 4 6 8 101214161820 30 40 50 60 68

NUMBER OF DAYSAFTER THE ONSET OF COUGH

FIG. 3. AntibodytoFHAdepictedas afunction of

days

aftercoughonset.Eachsquarerepresentsasingleserumspecimen. Two serum

specimenswereobtained fromeachof14patients, andone serum specimenwasobtained from each of 13 patients.

--fk.

Q-5

-, M&

66-..-.

on April 11, 2020 by guest

http://jcm.asm.org/

(4)

1376 REDD ET AL.

D]

ONE SERUM WITHOUT IgG OR IgA ANTIBODY TO FHA

P

ONE SERUM WITH IgA ANTIBODYTO FHA

l

ONE SERUM WITH BOTH IgG AND IgA ANTIBODY TO FHA

0 1 2 3 4 5 6 7 8 9 10 1112131415161718218

YEARS OFAGE

FIG. 4. AntibodytoFHAinserum specimenscollected atleast 40days aftercoughonset depicted as afunctionof theageof the patient. Eachsquare representsasingleserumspecimen;nopatient contributedmorethan onespecimen.

antibodies to FHA (P = 0.004 for IgA antibody and

IgG

antibody).

IgG activity against the 84- and 75-kDa

proteins

was independently associated with vaccination

(Table 2).

Of 17 vaccinated persons who had neverhad

pertussis,

15

(88%)

had antibodies to the 84-kDa protein, and 16

(94%)

had

antibodies tothe75-kDaprotein. Noneofthe 11

nonvacci-nated, never-infected persons tested had antibodiestoeither of theseproteins.Ofsevenfully vaccinatedpersonswho had

culture-documented infection, all had antibodies to both proteins. Of22nonvaccinatedand infected persons, 6

(27%)

had antibodies to the 84-kDa protein and 8

(36%)

had

antibodiestothe 75-kDaprotein.

DISCUSSION

This study reports the use of Western blot analysis to evaluate the activity of human serum against a whole-cell

antigenpreparationof B. pertussis. Thistechnique produced

numerous bands indicative of IgG and IgA antibodies to Bordetella proteins, both from patients who had been in-fected and from persons who had been vaccinated. From this groupofheterogeneous antibody specificities, antibodies to two high-molecular-weight bands were more common in serum samples from patients who had recently had culture-confirmed infectionwithB. pertussis thanin serum samples from uninfected patients. These bands probably represent

antibodies to the FHA present in the whole-cell prepara-tions. The electrophoretic profile of purified FHA has been

TABLE 2. IgG antibody to the 75- and84-kDa proteins

ofB.pertussisbyvaccination status

Infectionand No. of patients No. of patients

vaccinationstatus with antibodies with antibodies Totalno.

ofpatients to84-kDa to75-kDa ofpatients

protein protein

Notinfected

Vaccinateda 15 16 17

Unvaccinated O 0 il

Infected

Vaccinateda 7 7 7

Unvaccinated 6 8 22

aReceivedatleast three doses ofDTP vaccine.

reported

to have a

complex

pattern, with molecular mass

bands

ranging

from 220 to58 kDa. The FHA bands in our immunoblot system

correspond

to the

higher-molecular-weight bands found

by

others

(6).

We found no consistent pattern of

activity

specific

to

lower-molecular-weight

bands in infected

patients.

Serum

specimens

drawn within the first week after

cough

onset

rarely

contained

IgA

antibodies to the 220- and 210-kDa

proteins,

but

by

40

days

after

cough

onset the

speci-menscontainedboth

IgG

and

IgA

antibodies. Since

only

one

of the control serum

samples

had

IgA

activity specific

to these

bands,

these data suggest that the presence of

IgA

antibody

specific

for these

FHA

bands indicates infection withB.pertussis. Thisobservation is consistent with

previ-ously

reported

studies with

purified

FHA as an

antigen

in

enzyme-linked

immunosorbent assays

(2,

7, 9, 10).

IgA

antibody

to FHAappears tobe a more

specific

indicator of recent

pertussis

thandoes

IgG

antibody.

Although

thefourinfectedchildren less than 4 years old in our

sample

may not be

representative

of

all

children,

only

one

developed IgA

and

IgG

antibodies

against

FHA. This is an

important

group, becauseover60% of

recently

reported

pertussis

cases were in children less than 5 years old

(5).

More young children shouldbe studied tobetter define the

antibody

responseto infectionin thisage group.

In our

comparison

of vaccinated and unvaccinated

pa-tients,

we identifiedantibodies to two

specific

proteins

that were associated with vaccination. These

proteins

had

mo-lecularmassesof84and 75

kDa,

andantibodies werefound

in most vaccinated persons,

regardless

ofwhether or not

they

had had

pertussis. Among

the

unvaccinated,

only

persons who had had

pertussis

had antibodies to these

proteins.

Some

patients,

provided

that the immunization

history

is correct, appearto

develop

IgG

antibodiestothese

proteins

in response to infection. Evaluation of additional

serum

samples

from infected and unvaccinated persons is

neededtoconfirmthisobservation.

Age

didnotappeartobe

an

important

determinant of this response to

vaccination,

because childrenas young as 2 years oldformedantibodies to the 84- and 75-kDa

proteins.

These antibodies seem to

persist long

after

vaccination,

since the likelihood of nega-tive tests did not increase with

increasing

age of the person tested.

Recently,

immunoblot

analysis

has been usedtoshow that

vaccinationof human infants rarelyresults in

production

of antibodies to FHA

(21).

As in the present

study,

IgG

antibodies to several outermembrane

proteins

with

molec-ularmassesof 50to100kDaweredetected(21).The datawe

reportconfirm theseobservationsand also demonstrate that

IgA

antibodies to FHA are not produced as a result of vaccination.

Western blot

analysis

has some advantagesoverexisting

methods for

serodiagnosis.

By using this method, it was

possible

to separate

multiple antigens

andanalyze serologic

responsestothemwithouthavingto useadditional isolation and

purification

procedures

(1). The immunoblot system

distinguishes

differenthumoral immuneresponses that vac-cination with DTP vaccine and natural infection with B.

pertussisgenerate.

LITERATURE CITED

1. Beisiegel, U. 1986. Proteinblotting. Electrophoresis7:1-18. 2. Burstyn,D.G.,L. J.

Baraif,

M.S. Peppler, R. D. Leake, J. St.

Geme, Jr.,and C. R. Manclark. 1983. Serological response to filamentous hemagglutinin andlymphocytosis-promoting toxin

ofBordetellapertussis. Infect. Immun.41:1150-1156.

3. Centers forDisease Control. 1983. Pertussis-Maryland, 1982. J. CLIN. MICROBIOL.

on April 11, 2020 by guest

http://jcm.asm.org/

(5)

Morbid. Mortal. Weekly Rep.32:297-305.

4. Centers forDisease Control. 1985. Pertussis-Washington, 1984. Morbid. Mortal. Weekly Rep.34:390-400.

5. Centers for Disease Control. 1987. Pertussis surveillance-United States, 1984 and 1985. Morbid. Mortal. Weekly Rep. 36:168-171.

6. Cowell, J. L., Y. Sato, H. Sato, B. An der Lan, and C. R. Manclark. 1982. Separation, purification, and properties of the filamentous hemagglutinin and the leukocytosis promoting fac-tor-hemagglutinin from Bordetella pertussis. Semin. Infect. Dis. 4:371-379.

7. Finger, H., and C. H. Wirsing VonKoenig. 1985. Serological diagnosis of whooping cough. Dev. Biol. Stand. 61:331-336. 8. Fleiss, J.L.1981.Statistical methods for rates and proportions,

p. 24-26. John Wiley & Sons, Inc., New York.

9. Goodman,Y.E., A. J. Wort, and F. L.Jackson. 1981. Enzyme-linked immunosorbent assay fordetection of pertussis immuno-globulin A in nasopharyngeal secretions as an indicator of recentinfection. J. Clin. Microbiol. 13:286-292.

10. Granstrom, M., G. Granstrom, A. Lindfors, and P. Askelof. 1982. Serologic diagnosis of whooping cough by an enzyme-linked immunosorbent assay using fimbrial hemagglutinin as antigen. J.Infect. Dis. 146:741-745.

11. Hancock, K., and V. C. W. Tsang. 1983. India ink stainingof proteinsonnitrocellulose paper. Anal. Biochem. 133:157-162. 12. Irons, L. I., A. E. Ashworth, and P. Wilton-Smith. 1983.

Heterogeneity of the filamentous haemagglutinin of Bordetella pertussis studied with monoclonal antibodies. J. Gen. Micro-biol. 129:2769-2778.

13. Jefferis,R., C. B. Reimer, F. Skvaril, G. de Lange, N. R. Ling, J. Lowe,M. R.Walker,D.J. Phillips,C. H.Aloisio,T. W.Wells, J.P.Vaerman, C. G. Magnusson,H.Kubagawa,M.Cooper,F. Vartdal,B.Vandvik, J. J. HaarUman,O. Makela,A.Sarnesto, Z. Lando, J. Gergely,E. Rajnavolgyi, G. Laszlo, J. Radl, and

G. A. Molinaro. 1985. Evaluation of monoclonal antibodies

having specificity forhumanIgGsub-classes: results of an IUIS/ WHO collaborative study. Immunol. Lett. 10:223-252. 14. Kenna, J. G., G. N. Major, andR.S. Williams. 1985.Methods

for reducing non-specific antibody binding in enzyme-linked immunosorbent assays. J. Immunol. Methods 85:409-419. 15. Krugman,S., and R. Ward.1973. Infectious diseasesof children

andadults, 5th ed., p. 175-176. C. V.Mosby Co., St. Louis. 16. Morrissey, J. H. 1981. Silver stain for proteins in

polyacryl-amidegels: amodifiedprocedurewith enhanceduniform sensi-tivity. Anal. Biochem. 117:307-310.

17. Nagel, J., S. de Graff, and D. Schijf-Evers. 1985. Improved

serodiagnosis of whooping cough caused by Bordetella pertussis by determination of IgG anti-LPF antibody levels. Dev. Biol. Stand. 61:325-330.

18. Nagel, J., and E. J. Poot-Scholtens. 1983. Serum IgA antibody to Bordetella pertussis as an indicator of infection. J. Med. Micro-biol. 16:417-426.

19. Nkowane, B. M., S. G. F.Wassilak, P. A. McKee, D. J. O'Mara, G. Deliaportas, G. R. Istre, W. A. Orenstein, and K.J. Bart. 1986.Pertussis epidemic in Oklahoma: difficulties inpreventing transmission. Am.J. Dis. Child. 140:433-437.

20. Pittman, M. 1979. Pertussis toxin: the cause of the harmful effects andprolonged immunity of whooping cough. A hypoth-esis. Rev.Infect. Dis. 1:401-412.

21. Redhead, K. 1984. Serum antibody responses to the outer membraneproteins of Bordetella pertussis. Infect. Immun. 44: 724-729.

22. Regan, J., and F. Lowe. 1977. Enrichment medium for the isolation of Bordetella. J. Clin. Microbiol. 6:303-309.

23. Reimer,C.B.,D.J. Phillips, C.H. Aloisio,B. D.Moore, G.G. Galland,T. W. Wells,C. M.Black,andJ. S. McDougal. 1984. Evaluation ofthirty-onemousemonoclonalantibodiestohuman IgG epitopes. Hybridoma 3:263-275.

24. Renart, J., J. Reiser,and G. R. Stark.1979. Transfer ofproteins from gels to diazobenzyloxymethyl-paper and detection with antisera: amethod forstudying antibodyspecificity andantigen

structure. Proc.Natl. Acad. Sci. USA 76:3116-3120.

25. Spinola, S. M., and J. G. Cannon. 1985. Different blocking agentscausevariation in theimmunologic detection ofproteins transferredtonitrocellulose membranes. J. Immunol. Methods 81:161-165.

26. Towbin, H.,T.Staehelin,andJ. Gordon. 1979.Electrophoretic transfer ofproteins frompolyacrylamide gelstonictrocellulose sheets:procedure andsomeapplications. Proc. Natl. Acad. Sci. USA 76:4350-4354.

27. Viljanen,M. K., J. Mertsola,T. Kuronen, andO.Ruuskanen. 1985. Class-specific antibody response to lymphocytosis-pro-moting factor (LPF) and fimbrae (F) in pertussis. Dev. Biol. Stand. 61:337-340.

28. Winsnes, R.,T.Lonnes,B.Mogster,and B. P. Berdal.Antibody responses after vaccination and disease against leukocytosis promoting factor, filamentous hemagglutinin, lipopolysaccha-ride and a protein binding to complement-fixing antibodies inducedduring whooping cough. Dev. Biol. Stand. 61:353-366. 29. Zackrisson, G., T. Lagergard, and I. Lonnroth. 1986. An

en-zyme-linked immunosorbent assay method for detection of immunoglobulin to pertussis toxin. Acta Pathol. Microbiol. Immunol. Scand. Sect. C 94:227-231.