JOURNAL OF CLINICAL MICROBIOLOGY, JUlY 1988, P. 1373-13770095-1137/88/071373-05$02.00/0Copyright ©D 1988, American Society for Microbiology

Immunoblot Analysis of Humoral Immune Responses followingInfection with Bordetella pertussis or Immunization with

Diphtheria-Tetanus-Pertussis VaccineSTEPHEN C. REDD,l* HELLA S. RUMSCHLAG,l ROBIN J. BIELLIK,2 GARY N. SANDEN,'

CHARLES B. REIMER,' AND MITCHELL L. COHEN'Center for Infectious Diseases' and Center for Prevention Services,2 Centers for Disease Control, Atlanta, Georgia 30333

Received 18 November 1987/Accepted 30 March 1988

To help develop better diagnostic tests for pertussis, we examined the serologic response to whole-cellproteins of Bordetella pertussis after natural infection or vaccination with diphtheria-tetanus-pertussis vaccine.Serum specimens collected during a pertussis outbreak investigation and from uninfected persons were used inWestern blot (immunoblot) analyses to determine the presence of immunoglobulin G (IgG) and IgA antibodiesto specific B. pertussis proteins. IgG antibodies to proteins of molecular masses 220 and 210 kilodaltons (kDa)were detected in 14 of 18 serum samples obtained from patients with culture-confirmed pertussis .4Q days afterthe onset of coughing. IgA antibodies were detected in 15 of the 18 samples. Of 19 serum samples obtained frompatients who had not been ill with pertussis, 6 contained IgG antibodies to these proteins and 1 contained IgAantibodies. The two proteins bound antiserum specific for ifiamentous hemagglutinin and comigrated withpurified filamentous hemagglutinin. IgG antibodies to two additional protein bands of molecular masses 84 and75 kDa were associated with previous vaccination. Antibody to the 84-kDa protein was detected in 15 of 17vaccinated, never-infected persons, and antibody to the 75-kDa protein was detected in 16 of the 17. None of

11 nonvaccinated, never-infected persons tested had antibodies to either protein. AUl seven fully vaccinatedpersons with culture-documented infection had antibodies to both proteins. Antibodies to the 84-kDa proteinwere detected in 6 of 22 nonvaccinated and infected persons, and antibodies to the 75-kDa protein were detectedin 8 of the 22. Use of Western blot analysis in this study allowed us to distinguish antibody responses to infectionand immunization.

Current laboratory methods for diagnosing pertussis areunsatisfactory. The pathophysiology of this disease is com-plex, involving three stages of illness with differing clinical,biochemical, and hematologic manifestations (15). Orga-nisms are present in the nasopharynx early in illness but maybe absent when clinical signs of pertussis are most distinc-tive (20). Because of this complex natural history, it isunlikely that a single test would be universally appropriatefor diagnosing all stages of pertussis. Detecting organismsearly in illness and assessing host responses in later stagesmight be the best strategy. When these clinical, epidemio-logic, and pathologic variables are coupled with the inherentmethodologic limitations in available methods, it is notsurprising that in three recent pertussis outbreaks, only 51 to67% of clinically suspected cases could be laboratory con-firmed (3, 4, 19).These problems have stimulated the search for better

diagnostic tests for pertussis (2, 7, 9, 10, 17, 18, 27-29). Weused Western blot (immunoblot) analysis to measure anti-bodies to whole-cell proteins of Bordetella pertussis in seraof patients who had culture-documented pertussis and insera of persons who had not had pertussis. Results of ourstudy, showing that antibodies to specific proteins correlatewith infection and vaccination, should assist the develop-ment of improved diagnostic methods for pertussis.

MATERIALS AND METHODS

Serum samples. An epidemiologic investigation of a per-tussis outbreak provided the opportunity to test serologicresponses to infection. The outbreak occurred in a mid-

* Corresponding author.

Atlantic state in a geographically isolated population thathad a low rate of immunization against pertussis. Few illpersons sought medical care or received antimicrobial drugsfor the illness. Paired serum samples were obtained at1-month intervals from 13 persons with clinical signs andculture confirmation of pertussis. Age, diphtheria-tetanus-pertussis (DTP) vaccination status, and date of cough onsetwere known for each person from whom serum was ob-tained. A single serum specimen was collected from each ofan additional 14 persons with culture-confirmed pertussis.Since samples were available from only two outbreak pa-tients without exposure to pertussis, serum samples fromtwo additional uninfected groups unrelated to the outbreakwere evaluated. Nine pairs of serum samples were obtainedfrom children before and after a complete four-dose immu-nization series, and samples were also obtained from eightadult volunteers who had been fully immunized and had no

known pertussislike illness.Western blot analysis. Cells of B. pertussis CDC A022A,

isolated from nasopharyngeal mucus of one patient withoutbreak-associated pertussis were used to prepare whole-cell protein antigens. The cells were stored in horse blood at-700C and grown in 2.5% C02 on Regan-Lowe medium (22)at 350C for 72 h. Whole cells were suspended in phosphate-buffered saline to an optical density of 0.4 at 540 nm. Onemilliliter of this suspension was pelleted by centrifugation,suspended in 50 ,ul of sample buffer (0.4% sodium dodecylsulfate, 1% mercaptoethanol, 2% glycerol, 50 mM Tris, pH6.8) and heated to 1000C for 10 min. A 250-,ul volume of thiswhole-cell lysate was separated by electrophoresis in a12.5% sodium dodecyl sulfate-polyacrylamide gel (11 by 17cm by 0.75 mm) at 30 mA of constant current for 3 h.

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Proteins on the polyacrylamide gel were 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)containing 192 mM glycine and 20% (vol/vol) methanol. Forcomparison, whole-cell proteins of strains Tohama 1 andNIH 165 were electrophoretically separated and blotted tonitrocellulose paper under the same conditions. Silver stain-ing was used to identify bands in the polyacrylamide gel (16),and India ink staining was used to confirm the transfer ofproteins to the nitrocellulose paper (11).Unbound sites on the nitrocellulose paper were blocked

overnight with casein-thimerosal buffer (14, 25) at 22°C; thenitrocellulose paper was then cut into 0.5-cm strips. Thestrips were incubated with human serum at a single dilutionof 1:50 in casein-thimerosal buffer for 2 h at 37°C. Stripswere washed and assayed for immunoglobulin G (IgG)antibodies with protein A-horseradish peroxidase conjugate(Bio-Rad Laboratories, Richmond, Calif.) or assayed for IgAantibodies with a pool of anti-human IgA mouse monoclonalantibodies followed by goat anti-mouse antibodies conju-gated to horseradish peroxidase. Mouse anti-human IgMmonoclonal antibodies were used to detect IgM antibodies.Specificity of anti-immunoglobulin monoclonal antibodieswas confirmed by the methods of Reimer et al. (13, 23).Strips were developed with tetramethylbenzidine peroxidasedeveloper (25 jxg of tetramethylbenzidine in 5 ml of metha-nol; 1.25 ml of 1 M HEPES [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid], pH 7.5; 20 ml of 5.0% dioctylso-dium sulfosuccinate sodium salt; 25 jjl of 30% H202 in 100ml).The presence or absence of antibody binding to specific

antigens was determined by visual inspection. Neither thecase status nor the vaccination status of the specimen donorwas known until after the presence or absence of antibodieshad been determined. Proportions were compared by usingFisher's exact test (8).

Purified filamentous hemagglutinin (FHA) was obtainedfrom the Michigan Department of Public Health, Lansing,and purified pertussis toxin (PT) was from the MerieuxInstitute, Lyons, France. Antiserum to these antigens wasprepared in adult New Zealand rabbits that were free ofanti-Bordetella antibodies as determined by slide microag-glutination test. To produce anti-PT, purified PT in phos-phate-buffered saline was emulsified with an equal volume ofFreund complete adjuvant. Animals received 10 ,ug of PT,administered as five 0.2-ml intradermal injections. This dosewas repeated 2 and 4 weeks after the initial dose. Serum wascollected 3 weeks after dose 3. Procedures for producinganti-FHA were identical to those for producing anti-PTexcept that each dose contained 25 ,ug of FHA.

RESULTSA silver stain of a sodium dodecyl sulfate-polyacrylamide

gel containing electrophoresed whole-cell proteins showedno qualitative differences among B. pertussis strains CDCA022A, Tohama 1, and NIH 165 (Fig. 1). However, anapparent quantitative difference was noted; two bands, ofmolecular masses 27.5 and 17.5 kilodaltons (kDa), stainedless intensely in strain CDC A022A than in strain Tohama 1or NIH 165. An India ink stain of transferred proteinsshowed that lower-molecular-weight proteins stained lessintensely than those with higher molecular weights, probablybecause of decreased binding to the 0.45-,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 g92.5 t;.

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

31-> :

B

_,14-' _

1 2 3 1 2 3FIG. 1. Electrophoresis of proteins from three strains of B.

pertussis. (A) Silver-stained proteins in a sodium dodecyl sulfate-polyacrylamide gel. (B) India ink-stained proteins after transfer tonitrocellulose paper. Numbers to the left indicate molecular mass inkilodaltons, and numbers at the bottom indicate the strain of B.pertussis used. Strain CDC A022A was in lane 1, Tohama 1 was inlane 2, and NIH 165 was in lane 3. Slanted arrows indicate bands ofmolecular masses 27.5 and 17.5 kDa that stained less intensely in theCDC A022A than in the Tohama 1 or NIH 165 lane. Bands withmolecular masses of less than 20 kDa stained weakly in the India inkpreparation, indicating less avid binding of lower-molecular-weightproteins to the 0.45-,um-pore-size nitrocellulose paper or smalleramounts of lower-molecular-weight proteins present in the whole-cell antigen preparation.

whole-cell protein preparations (Fig. 2). Both IgG and IgAantibodies were detected, but no consistent activity wasdetected with an anti-IgM conjugate at the 1:50 dilution ofserum. In contrast, patients who had neither a history ofpertussis nor vaccination lacked IgA and IgG reactivity. TheIgA and IgG reactivity to specific proteins varied amongpersons with different histories of disease and vaccination.Antibodies to proteins of molecular masses 220 and 210 kDawere more frequently identified in serum samples frompertussis patients than in samples from never-infected pa-tients (Fig. 2). These proteins are probably the same high-molecular-weight FHA bands identified by Irons et al. (12).Antibodies to proteins of B. pertussis with molecular massesof 84 and 75 kDa were found more frequently in serumsamples from vaccinated patients than in samples fromunvaccinated patients (Fig. 2).

Studies were conducted to determine whether any of thesebands were associated with previously described cellularconstituents of B. pertussis. Purified FHA comigrated withthe 220- and 210-kDa bands of the whole-cell preparation,and rabbit polyclonal antibodies to FHA also bound these

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FIG. 2. Selected immunoblots. Numbers to the left indicatemolecular mass in kilodaltons; heavy arrows to the left indicatebands with molecular masses of 220 and 210 kDa, and open arrowsto the right indicate bands of molecular masses 84 and 75 kDa.Seven serum specimens that were obtained from five differentpersons are depicted. For each serum specimen, the odd-numberedlane shows staining for IgG antibodies and the even-numbered laneshows staining for IgA antibodies. Lanes: 1 and 2, immunoblot of aserum specimen obtained more than 40 days after symptom onsetfrom a vaccinated patient with culture-confirmed pertussis; 3 and 4,results from a specimen obtained more than 40 days after symptomonset from an unvaccinated patient with culture-confirmed pertussis(Lanes 1 to 4 show IgG and IgA antibodies to FHA, but theunvaccinated patient did not have antibodies to the 84- and 75-kDaproteins.); 5 to 8, results from two serum specimens obtained 1month apart from a single patient with culture-confirmed pertussiswho had received three doses of DTP vaccine. IgG and IgAantibodies to the 220- and 210-kDa bands are present in the laterspecimen (lanes 7 and 8), as are antibodies to the 84- and 75-kDaproteins. Lanes 9 to 12 show results from two serum specimensobtained from a fully vaccinated patient with culture-confirmedpertussis. IgA antibody to FHA is seen in the convalescent-phaseserum specimen, and IgG antibodies to the 84- and 75-kDa proteinsare seen in both serum specimens. Lanes 13 and 14 show results ina 20-month old child after completion of a 4-dose immunizationseries with DTP vaccine. IgG antibodies to both the 84- and 75-kDabands are present.

ANTIBODIES TO PERTUSSIS INFECTION OR DTP VACCINE 1375

TABLE 1. Antibody to FHA in persons neverinfected with B. pertussis

No. of No. ofSerum source patients with patients with Total no.

IgG antibody IgA antibody tested

Outbreak O 0 2Children before 0 0 9immunization

Children after 4 0immunization

Vaccinated adult 2 1 8volunteers

a Serum collected during the outbreak investigation from children who hadneither had pertussis nor been exposed to other children with pertussis.

b Each of these children contributed two serum samples: one before andone after a four-dose immunization series.

two bands. Purified PT did not comigrate with any of thebands visible after electrophoresis of the whole-cell antigen,suggesting that too little PT was present in the whole-cellpreparation for detection. It is possible that the static culturemethod we used inhibited production of PT (6). The rabbitpolyclonal antibodies to PT bound bands of higher molecularweight than that of the bands produced by electrophoresis ofpurified PT, indicating either nonspecific binding or com-plexing of PTI with higher-molecular-weight proteins.IgG antibody to FHA was detected in 14 of 18 serum

samples obtained at least 40 days after the onset of illnessfrom patients with culture-confirmed pertussis. From thissame group of samples, 15 of 18 had IgA antibodies to FHA.In contrast, of 19 persons with no history of exposure topertussis except through vaccination, 6 had IgG antibodiesand 1 had IgA antibodies to FHA (Table 1).To determine the effect of time since symptom onset on

production of antibodies to FHA, we plotted the results ofWestern blot analysis of available serum specimens by thenumber of days between cough onset and serum collection(Fig. 3). The likelihood of having IgG or IgA antibodies tothese two bands gradually increased with the number of daysbetween cough onset and serum collection.To determine the effect of patient age on development of

antibodies to FHA, we stratified the results by patient age.Figure 4 shows how frequently antibodies to FHA werefound in serum obtained from culture-positive patients atleast 40 days after the onset of coughing. Despite smallnumbers of cases evaluated, children less than 4 years oldwith positive cultures were significantly less likely to have

E ONE SERUM WITHOUT 19G OR IgA ANTIBODY TO FHA

ONE SERUM WITH IgG ANTIBODY TO FHA

ONE SERUM WITH 1gA ANTIBODY TO FHA

S ONE SERUM WITH BOTH IgG AND IgA ANTIBODY TO FHA

-2 0 2 4 6 8 101214161820 30 40 50 60 68NUMBER OF DAYS AFTER THE ONSET OF COUGH

FIG. 3. Antibody to FHA depicted as a function of days after cough onset. Each square represents a single serum specimen. Two serumspecimens were obtained from each of 14 patients, and one serum specimen was obtained from each of 13 patients.

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D] ONE SERUM WITHOUT IgG OR IgA ANTIBODY TO FHA

P ONE SERUM WITH IgA ANTIBODY TO FHA

l ONE SERUM WITH BOTH IgG AND IgA ANTIBODY TO FHA

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

FIG. 4. Antibody to FHA in serum specimens collected at least40 days after cough onset depicted as a function of the age of thepatient. Each square represents a single serum specimen; no patientcontributed more than one specimen.

antibodies to FHA (P = 0.004 for IgA antibody and IgGantibody).IgG activity against the 84- and 75-kDa proteins was

independently associated with vaccination (Table 2). Of 17vaccinated persons who had never had pertussis, 15 (88%)had antibodies to the 84-kDa protein, and 16 (94%) hadantibodies to the 75-kDa protein. None of the 11 nonvacci-nated, never-infected persons tested had antibodies to eitherof these proteins. Of seven fully vaccinated persons who hadculture-documented infection, all had antibodies to bothproteins. Of 22 nonvaccinated and infected persons, 6 (27%)had antibodies to the 84-kDa protein and 8 (36%) hadantibodies to the 75-kDa protein.

DISCUSSION

This study reports the use of Western blot analysis toevaluate the activity of human serum against a whole-cellantigen preparation of B. pertussis. This technique producednumerous bands indicative of IgG and IgA antibodies toBordetella proteins, both from patients who had been in-fected and from persons who had been vaccinated. From thisgroup of heterogeneous antibody specificities, antibodies totwo high-molecular-weight bands were more common inserum samples from patients who had recently had culture-confirmed infection with B. pertussis than in serum samplesfrom uninfected patients. These bands probably representantibodies 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- and 84-kDa proteinsof B. pertussis by vaccination status

Infection and No. of patients No. of patientsvaccination status with antibodies with antibodies Total no.

of patients to 84-kDa to 75-kDa of patientsprotein protein

Not infectedVaccinateda 15 16 17Unvaccinated O 0 il

InfectedVaccinateda 7 7 7Unvaccinated 6 8 22a Received at least three doses of DTP vaccine.

reported to have a complex pattern, with molecular massbands ranging from 220 to 58 kDa. The FHA bands in ourimmunoblot system correspond to the higher-molecular-weight bands found by others (6). We found no consistentpattern of activity specific to lower-molecular-weight bandsin 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-mens contained both IgG and IgA antibodies. Since only oneof the control serum samples had IgA activity specific tothese bands, these data suggest that the presence of IgAantibody specific for these FHA bands indicates infectionwith B. pertussis. This observation is consistent with previ-ously reported studies with purified FHA as an antigen inenzyme-linked immunosorbent assays (2, 7, 9, 10). IgAantibody to FHA appears to be a more specific indicator ofrecent pertussis than does IgG antibody.Although the four infected children less than 4 years old in

our sample may not be representative of all children, onlyone developed IgA and IgG antibodies against FHA. This isan important group, because over 60% of recently reportedpertussis cases were in children less than 5 years old (5).More young children should be studied to better define theantibody response to infection in this age group.

In our comparison of vaccinated and unvaccinated pa-tients, we identified antibodies to two specific proteins thatwere associated with vaccination. These proteins had mo-lecular masses of 84 and 75 kDa, and antibodies were foundin most vaccinated persons, regardless of whether or notthey had had pertussis. Among the unvaccinated, onlypersons who had had pertussis had antibodies to theseproteins. Some patients, provided that the immunizationhistory is correct, appear to develop IgG antibodies to theseproteins in response to infection. Evaluation of additionalserum samples from infected and unvaccinated persons isneeded to confirm this observation. Age did not appear to bean important determinant of this response to vaccination,because children as young as 2 years old formed antibodiesto the 84- and 75-kDa proteins. These antibodies seem topersist long after vaccination, since the likelihood of nega-tive tests did not increase with increasing age of the persontested.

Recently, immunoblot analysis has been used to show thatvaccination of human infants rarely results in production ofantibodies to FHA (21). As in the present study, IgGantibodies to several outer membrane proteins with molec-ular masses of 50 to 100 kDa were detected (21). The data wereport confirm these observations and also demonstrate thatIgA antibodies to FHA are not produced as a result ofvaccination.Western blot analysis has some advantages over existing

methods for serodiagnosis. By using this method, it waspossible to separate multiple antigens and analyze serologicresponses to them without having to use additional isolationand purification procedures (1). The immunoblot systemdistinguishes different humoral immune responses that vac-cination with DTP vaccine and natural infection with B.pertussis generate.

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Geme, Jr., and C. R. Manclark. 1983. Serological response tofilamentous hemagglutinin and lymphocytosis-promoting toxinof Bordetella pertussis. Infect. Immun. 41:1150-1156.

3. Centers for Disease Control. 1983. Pertussis-Maryland, 1982.

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23. Reimer, C. B., D. J. Phillips, C. H. Aloisio, B. D. Moore, G. G.Galland, T. W. Wells, C. M. Black, and J. S. McDougal. 1984.Evaluation of thirty-one mouse monoclonal antibodies to humanIgG epitopes. Hybridoma 3:263-275.

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