To help develop better diagnostic tests for pertussis, we examined the serologic response to whole-cell proteins of Bordetella pertussis after natural infection or ...
JOURNAL OF CLINICAL MICROBIOLOGY,
JUlY 1988, P. 1373-1377
Vol. 26, No. 7
0095-1137/88/071373-05$02.00/0 Copyright ©D 1988, American Society for Microbiology
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' 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-cell proteins 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 in Western blot (immunoblot) analyses to determine the presence of immunoglobulin G (IgG) and IgA antibodies to 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 after the onset of coughing. IgA antibodies were detected in 15 of the 18 samples. Of 19 serum samples obtained from patients who had not been ill with pertussis, 6 contained IgG antibodies to these proteins and 1 contained IgA antibodies. The two proteins bound antiserum specific for ifiamentous hemagglutinin and comigrated with purified filamentous hemagglutinin. IgG antibodies to two additional protein bands of molecular masses 84 and 75 kDa were associated with previous vaccination. Antibody to the 84-kDa protein was detected in 15 of 17 vaccinated, 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 vaccinated persons with culture-documented infection had antibodies to both proteins. Antibodies to the 84-kDa protein were detected in 6 of 22 nonvaccinated and infected persons, and antibodies to the 75-kDa protein were detected in 8 of the 22. Use of Western blot analysis in this study allowed us to distinguish antibody responses to infection and immunization. Current laboratory methods for diagnosing pertussis are unsatisfactory. The pathophysiology of this disease is complex, involving three stages of illness with differing clinical, biochemical, and hematologic manifestations (15). Organisms are present in the nasopharynx early in illness but may be absent when clinical signs of pertussis are most distinctive (20). Because of this complex natural history, it is unlikely that a single test would be universally appropriate for diagnosing all stages of pertussis. Detecting organisms early in illness and assessing host responses in later stages might be the best strategy. When these clinical, epidemiologic, and pathologic variables are coupled with the inherent methodologic limitations in available methods, it is not surprising that in three recent pertussis outbreaks, only 51 to 67% of clinically suspected cases could be laboratory confirmed (3, 4, 19). These problems have stimulated the search for better diagnostic tests for pertussis (2, 7, 9, 10, 17, 18, 27-29). We used Western blot (immunoblot) analysis to measure antibodies to whole-cell proteins of Bordetella pertussis in sera of patients who had culture-documented pertussis and in sera of persons who had not had pertussis. Results of our study, showing that antibodies to specific proteins correlate with infection and vaccination, should assist the development of improved diagnostic methods for pertussis. MATERIALS AND METHODS Serum samples. An epidemiologic investigation of a pertussis outbreak provided the opportunity to test serologic responses to infection. The outbreak occurred in a mid*
Corresponding author.
Atlantic state in a geographically isolated population that had a low rate of immunization against pertussis. Few ill persons sought medical care or received 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-tetanuspertussis (DTP) vaccination status, and date of cough onset were known for each person from whom serum was obtained. A single serum specimen was collected from each of an additional 14 persons with culture-confirmed pertussis. Since samples were available from only two outbreak patients without exposure to pertussis, serum samples from two additional uninfected groups unrelated to the outbreak were evaluated. Nine pairs of serum samples were obtained from children before and after a complete four-dose immunization series, and samples were also obtained from eight adult 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 with outbreak-associated pertussis were used to prepare wholecell 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 phosphatebuffered saline to an optical density of 0.4 at 540 nm. One milliliter of this suspension was pelleted by centrifugation, suspended in 50 ,ul of sample buffer (0.4% sodium dodecyl sulfate, 1% mercaptoethanol, 2% glycerol, 50 mM Tris, pH 6.8) and heated to 1000C for 10 min. A 250-,ul volume of this whole-cell lysate was separated by electrophoresis in a 12.5% sodium dodecyl sulfate-polyacrylamide gel (11 by 17 cm by 0.75 mm) at 30 mA of constant current for 3 h. 1373
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Proteins on the polyacrylamide gel were then electrophoretically 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. For comparison, whole-cell proteins of strains Tohama 1 and NIH 165 were electrophoretically separated and blotted to nitrocellulose paper under the same conditions. Silver staining was used to identify bands in the polyacrylamide 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-thimerosal buffer (14, 25) at 22°C; the nitrocellulose paper was then cut into 0.5-cm strips. The strips were incubated with human serum 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 with protein A-horseradish peroxidase conjugate (Bio-Rad Laboratories, Richmond, Calif.) or assayed for IgA antibodies with a pool of anti-human IgA mouse monoclonal antibodies followed by goat anti-mouse antibodies conjugated to horseradish peroxidase. Mouse anti-human IgM monoclonal antibodies were used to detect IgM antibodies. Specificity of anti-immunoglobulin monoclonal antibodies was confirmed by the methods of Reimer et al. (13, 23). Strips were developed with tetramethylbenzidine peroxidase developer (25 jxg of tetramethylbenzidine in 5 ml of methanol; 1.25 ml of 1 M HEPES [N-2-hydroxyethylpiperazineN'-2-ethanesulfonic acid], pH 7.5; 20 ml of 5.0% dioctylsodium sulfosuccinate sodium salt; 25 jjl of 30% H202 in 100 ml). The presence or absence of antibody binding to specific antigens was determined by visual inspection. Neither the case status nor the vaccination status of the specimen donor was known until after the presence or absence of antibodies had been 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 microagglutination test. To produce anti-PT, purified PT in phosphate-buffered saline was emulsified with an equal volume of Freund complete adjuvant. Animals received 10 ,ug of PT, administered as five 0.2-ml intradermal injections. This dose was repeated 2 and 4 weeks after the initial dose. Serum was collected 3 weeks after dose 3. Procedures for producing anti-FHA were identical to those for producing anti-PT except that each dose contained 25 ,ug of FHA. RESULTS A silver stain of a sodium dodecyl sulfate-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 intensely than those with higher molecular weights, probably because of decreased binding to the 0.45-,um-pore-size membrane. Antiserum from patients recently infected with B. pertussis bound multiple protein bands in the Western blots of
B
A 200 92.5
g t;.
45->~~~~~~~~~~~~~~~~
31->
:
_, 14 -'
_
1 2 3
1 2 3
FIG. 1. Electrophoresis of proteins from three strains of B. pertussis. (A) Silver-stained proteins in a sodium dodecyl sulfatepolyacrylamide gel. (B) India ink-stained proteins after transfer to nitrocellulose paper. Numbers to the left indicate molecular mass in kilodaltons, and numbers at the bottom indicate the strain of B. pertussis used. Strain CDC A022A was in lane 1, Tohama 1 was in lane 2, and NIH 165 was in lane 3. Slanted arrows indicate bands of molecular masses 27.5 and 17.5 kDa that stained less intensely in the CDC A022A than in the Tohama 1 or NIH 165 lane. Bands with molecular masses of less than 20 kDa stained weakly in the India ink preparation, indicating less avid binding of lower-molecular-weight proteins to the 0.45-,um-pore-size nitrocellulose paper or smaller amounts of lower-molecular-weight proteins present in the wholecell antigen preparation.
whole-cell protein preparations (Fig. 2). Both IgG and 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 lacked IgA and IgG reactivity. 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 patients (Fig. 2). These proteins are probably the same highmolecular-weight FHA bands identified by Irons et al. (12). Antibodies to proteins of 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). Studies were conducted to determine 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
VOL. 26, 1988
ANTIBODIES TO PERTUSSIS INFECTION OR DTP VACCINE TABLE 1. Antibody to FHA in persons never infected with B. pertussis
1 2 3 4 5 6 7 8 91011121314
200--116-9Q-5 -,
Serum source
Outbreak Children before immunization Children after immunization Vaccinated adult volunteers
--fk.
M&
_
_
âmo_
~~~~~~.
«I
_
66-..-. _
1375
..
No. of patients with IgG antibody
No. of patients with IgA antibody
O
0
2
0
0
9
4
0
2
1
Total no. tested
8
a Serum collected during the outbreak investigation from children who had neither had pertussis nor been exposed to other children with pertussis. b Each of these children contributed two serum samples: one before and one after a four-dose immunization series.
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 are depicted. For each serum specimen, the odd-numbered lane shows staining for IgG antibodies and the even-numbered lane shows staining for IgA antibodies. Lanes: 1 and 2, immunoblot of a serum specimen obtained more than 40 days after symptom onset from a vaccinated patient with culture-confirmed pertussis; 3 and 4, results from a specimen obtained more than 40 days after symptom onset from an unvaccinated patient 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- and 75-kDa proteins.); 5 to 8, results from two serum specimens obtained 1 month apart from a single patient with culture-confirmed pertussis who had received three doses of DTP vaccine. IgG and IgA antibodies to the 220- and 210-kDa bands are present in the later specimen (lanes 7 and 8), as are antibodies to the 84- and 75-kDa proteins. Lanes 9 to 12 show results from two serum specimens obtained from a fully vaccinated patient with culture-confirmed pertussis. IgA antibody to FHA is seen in the convalescent-phase serum specimen, and IgG antibodies to the 84- and 75-kDa proteins are seen in both serum specimens. Lanes 13 and 14 show results in a 20-month old child after completion of a 4-dose immunization series with DTP vaccine. IgG antibodies to both the 84- and 75-kDa bands are present.
two bands. Purified PT did not comigrate with any of the bands visible after electrophoresis 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 the bands produced by electrophoresis of purified PT, indicating either nonspecific binding or complexing 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 illness from patients with culture-confirmed pertussis. From this same group of samples, 15 of 18 had IgA antibodies to FHA. In contrast, of 19 persons with no history of exposure to pertussis except through vaccination, 6 had IgG antibodies and 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 of Western blot analysis of available serum specimens by the number of days between cough onset and serum collection (Fig. 3). The likelihood of having IgG or IgA antibodies to these two bands gradually increased with the number of days between 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 were found in serum obtained from culture-positive patients at least 40 days after the onset of coughing. Despite small numbers of cases evaluated, children less than 4 years old with 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 1012141618 20
30
40
50
60
68
NUMBER 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 serum specimens 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 ONE SERUM WITH BOTH IgG AND IgA ANTIBODY TO FHA
l
0 1 2 3 4 5 6 7 8 9 10 1112131415161718218 YEARS OF AGE
FIG. 4. Antibody to FHA in serum specimens collected at least 40 days after cough onset depicted as a function of the age of the patient. Each square represents a single serum specimen; no patient contributed more than one specimen.
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 never had pertussis, 15 (88%) had antibodies to the 84-kDa protein, and 16 (94%) had antibodies to the 75-kDa protein. None of the 11 nonvaccinated, never-infected persons tested had antibodies to either of these proteins. Of seven fully vaccinated persons who had culture-documented infection, all had antibodies to both proteins. Of 22 nonvaccinated and infected persons, 6 (27%) had antibodies to the 84-kDa protein and 8 (36%) had antibodies to the 75-kDa protein. DISCUSSION This study reports the use of Western blot analysis to evaluate the activity of human serum against a whole-cell antigen preparation of B. pertussis. This technique produced numerous bands indicative of IgG and IgA antibodies to Bordetella proteins, both from patients who had been infected and from persons who had been vaccinated. From this group of heterogeneous antibody specificities, antibodies to two high-molecular-weight bands were more common in serum samples from patients who had recently had cultureconfirmed infection with B. pertussis than in serum samples from uninfected patients. These bands probably represent antibodies to the FHA present in the whole-cell preparations. The electrophoretic profile of purified FHA has been TABLE 2. IgG antibody to the 75- and 84-kDa proteins of B. pertussis by vaccination status Infection and vaccination status of patients
No. of patients with antibodies
to 84-kDa protein
No. of patients with antibodies to 75-kDa protein
Total
Not infected
Vaccinateda Unvaccinated
15
16 0
17
O
Infected Vaccinateda Unvaccinated
7 6
7 8
7 22
a
Received at least three doses of DTP vaccine.
no.
of patients
il
reported to have a complex pattern, with molecular mass bands ranging from 220 to 58 kDa. The FHA bands in our immunoblot system correspond to the higher-molecularweight 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 210kDa proteins, but by 40 days after cough onset the specimens contained both 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 with B. pertussis. This observation is consistent with previously reported studies with purified FHA as an antigen in enzyme-linked immunosorbent assays (2, 7, 9, 10). IgA antibody to FHA appears to be a more specific indicator of recent 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, only one developed IgA and IgG antibodies against FHA. This is an important group, because over 60% of recently reported pertussis cases were in children less than 5 years old (5). More young children should be studied to better define the
antibody response to infection in this age group. In our comparison of vaccinated and unvaccinated patients, we identified antibodies to two specific proteins that were associated with vaccination. These proteins had molecular masses of 84 and 75 kDa, and antibodies were found in most vaccinated persons, regardless of whether 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, appear to develop IgG antibodies to these proteins in response to infection. Evaluation of additional serum samples from infected and unvaccinated persons is needed to confirm this observation. Age did not appear to be an important determinant of this response to vaccination, because children as young as 2 years old formed antibodies to the 84- and 75-kDa proteins. These antibodies seem to persist long after vaccination, since the likelihood of negative tests did not increase with increasing age of the person tested. Recently, immunoblot analysis has been used to show that vaccination of human infants rarely results in production of antibodies to FHA (21). As in the present study, IgG antibodies to several outer membrane proteins with molecular masses of 50 to 100 kDa were detected (21). The data we report confirm these observations and also demonstrate that IgA antibodies to FHA are not produced as a result of vaccination. Western blot analysis has some advantages over existing methods for serodiagnosis. By using this method, it was possible to separate multiple antigens and analyze serologic responses to them without having to use additional isolation and purification procedures (1). The immunoblot system distinguishes different humoral immune responses that vaccination with DTP vaccine and natural infection with B. pertussis generate. LITERATURE CITED 1. Beisiegel, U. 1986. Protein blotting. Electrophoresis 7:1-18. 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 and lymphocytosis-promoting toxin of Bordetella pertussis. Infect. Immun. 41:1150-1156. 3. Centers for Disease Control. 1983. Pertussis-Maryland, 1982.
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Morbid. Mortal. Weekly Rep. 32:297-305. 4. Centers for Disease Control. 1985. Pertussis-Washington, 1984. Morbid. Mortal. Weekly Rep. 34:390-400. 5. Centers for Disease Control. 1987. Pertussis surveillanceUnited 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 factor-hemagglutinin from Bordetella pertussis. Semin. Infect. Dis. 4:371-379. 7. Finger, H., and C. H. Wirsing Von Koenig. 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. Enzymelinked immunosorbent assay for detection of pertussis immunoglobulin A in nasopharyngeal secretions as an indicator of recent infection. J. Clin. Microbiol. 13:286-292. 10. Granstrom, M., G. Granstrom, A. Lindfors, and P. Askelof. 1982. Serologic diagnosis of whooping cough by an enzymelinked immunosorbent assay using fimbrial hemagglutinin as antigen. J. Infect. Dis. 146:741-745. 11. Hancock, K., and V. C. W. Tsang. 1983. India ink staining of proteins on nitrocellulose 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. Microbiol. 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 for human IgG sub-classes: results of an IUIS/ WHO collaborative study. Immunol. Lett. 10:223-252. 14. Kenna, J. G., G. N. Major, and R. 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 diseases of children and adults, 5th ed., p. 175-176. C. V. Mosby Co., St. Louis. 16. Morrissey, J. H. 1981. Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Anal. Biochem. 117:307-310. 17. Nagel, J., S. de Graff, and D. Schijf-Evers. 1985. Improved
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serodiagnosis of whooping cough caused by Bordetella pertussis by determination of IgG anti-LPF antibody levels. Dev. Biol. Stand. 61:325-330. Nagel, J., and E. J. Poot-Scholtens. 1983. Serum IgA antibody to Bordetella pertussis as an indicator of infection. J. Med. Microbiol. 16:417-426. 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 in preventing transmission. Am. J. Dis. Child. 140:433-437. Pittman, M. 1979. Pertussis toxin: the cause of the harmful effects and prolonged immunity of whooping cough. A hypothesis. Rev. Infect. Dis. 1:401-412. Redhead, K. 1984. Serum antibody responses to the outer membrane proteins of Bordetella pertussis. Infect. Immun. 44: 724-729. Regan, J., and F. Lowe. 1977. Enrichment medium for the isolation of Bordetella. J. Clin. Microbiol. 6:303-309. 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 human IgG epitopes. Hybridoma 3:263-275. Renart, J., J. Reiser, and G. R. Stark. 1979. Transfer of proteins from gels to diazobenzyloxymethyl-paper and detection with antisera: a method for studying antibody specificity and antigen structure. Proc. Natl. Acad. Sci. USA 76:3116-3120. Spinola, S. M., and J. G. Cannon. 1985. Different blocking agents cause variation in the immunologic detection of proteins transferred to nitrocellulose membranes. J. Immunol. Methods 81:161-165. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nictrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76:4350-4354. Viljanen, M. K., J. Mertsola, T. Kuronen, and O. Ruuskanen. 1985. Class-specific antibody response to lymphocytosis-promoting factor (LPF) and fimbrae (F) in pertussis. Dev. Biol. Stand. 61:337-340. Winsnes, R., T. Lonnes, B. Mogster, and B. P. Berdal. Antibody responses after vaccination and disease against leukocytosis promoting factor, filamentous hemagglutinin, lipopolysaccharide and a protein binding to complement-fixing antibodies induced during whooping cough. Dev. Biol. Stand. 61:353-366. Zackrisson, G., T. Lagergard, and I. Lonnroth. 1986. An enzyme-linked immunosorbent assay method for detection of immunoglobulin to pertussis toxin. Acta Pathol. Microbiol. Immunol. Scand. Sect. C 94:227-231.
