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Vol. 59, No. 3

INFECTION AND IMMUNITY, Mar. 1991, p. 1100-1105 0019-9567/91/031100-06$02.00/0 Copyright C) 1991, American Society for Microbiology

Localization of Immunogenic Regions

on

the Flagellin Proteins

of Campylobacterjejuni 81116 PIET J. M. NUIJTEN,l* BERNARD A. M. VAN DER ZEIJST,1 AND DIANE G. NEWELL2

Department ofBacteriology of the Institute of Infectious Diseases and Immunology, University of Utrecht, P.O. Box 80.165, 3508 TD Utrecht, The Netherlands,' and PHLS, Centre for Applied Microbiology and Research, Porton Down, Salisbury SP4 OJG, United Kingdom2 Received 27 August 1990/Accepted 30 November 1990

The purpose of this study was to localize antigenic regions on the flagellin protein of Campylobacterjejuni 81116. This strain has two flagellin genes, flaA and flaB, which are 95 % identical; only flaA seems to be expressed in motile C. jejuni 81116 cells. Fragments of flaA and flaB were cloned in the bacterial expression vector pEX, and the expression products were incubated with flagellin-specific antibodies. Monoclonal antibodies to broadly cross-reactive epitopes recognized fragments that are located in the termini (CF16 and CF17) and in the center (CF15) of both flageilin A and B proteins. Most of the serotype-specific monoclonal antibodies (CF1, CF2, CD3, CF4, and CF13) reacted with only the center of flagellin A in an area where flagellin A and B differ in 6 amino acid residues. The epitopes in this area were further characterized by competitive binding experiments. The charge and molecular weight microheterogeneity of flagellin, as determined by two-dimensional gel electrophoresis, were unrelated to the expression of both flagellin genes or parts thereof.

Campylobacter jejuni is a microaerobic, gram-negative, and highly motile bacterium causing severe diarrhea in humans (27). The flagella of this organism are considered important in the pathogenesis of the infection because they are essential for colonization of the intestinal mucosa (1, 13, 19). The flagellin protein is immunodominant during infections (1, 11, 15, 28); moreover, antiflagellin antibodies contribute to the protective immune responses in neonatal mice (17) and possibly humans (2). Flagellar antigens are therefore potential candidates for vaccines as well as suitable antigens for diagnostic purposes (9, 12, 15). However, flagellin expresses both serotype-specific and cross-reacting epitopes that are detectable by polyclonal and monoclonal antibodies (16, 28). Additionally, the flagellum is subject to phase variation (3, 21) and antigenic variation (6), probably as an adaptation to the environment and the immune response of the host. Recently, the two flagellin genes of C. jejuni 81116 were identified, cloned, and sequenced. Both genes are 1,731 bp in length, coding for flagellins with a very similar amino acid sequence that differs at only 32 of 576 sites. Interestingly, only one gene seems to produce mRNA at a detectable level (22). A number of monoclonal antibodies directed against the flagellin of C. jejuni 81116 have been prepared. Some of these antibodies are serotype specific, whereas others are broadly cross-reacting (16). All of these antibodies reacted to denatured flagellin and were therefore directed against conformation-independent epitopes (17). To broadly map the immunogenic regions of this protein, fragments of the flagellins of C. jejuni 81116 were expressed as fusion proteins in Escherichia coli, and the reactivity of these fusion products with 11 of the monoclonal antibodies was investigated. The topographic distribution of the epitopes of one area, identified as highly immunogenic, was further studied by competitive radioimmunoassays of the reacting monoclonal antibodies. *

MATERIALS AND METHODS Bacterial strains and plasmids. C. jejuni 81116 was grown under microaerobic conditions at 42°C on saponine agar medium (21). E. coli pop2136 (constructed by 0. Raibaud, Institute Pasteur, Paris, France) was used as the host for the pEX1, pEX2, or pEX3 (25) or pEX11, pEX12, or pEX13 (7) expression vector. The pop2136 strain was grown in LB medium (10) at 37°C. However, pEX-containing E. coli pop2136 cells were grown at 30 or 42°C (see below) in the presence of 200 pg of ampicillin per ml. Subcloning flagellin gene fragments in pEX. Overlapping fragments of both flagellin genes of C. jejuni 81116 (22) were subcloned in pEX vectors. Restriction enzyme fragments were isolated from an agarose gel with a GeneClean kit (Bio 101, La Jolla, Calif.) and ligated in compatible sites of the expression vectors, ensuring a correct fusion of reading frames. Ligation samples were transformed into E. coli pop2136 by using the CaCl2 procedure (10). The constructs contained by the resulting colonies were checked by (i) gel electrophoresis of the expression product (23), (ii) restriction analysis of plasmid DNA, and/or (iii) nucleotide sequence analysis (24). Expression of flagellin fusion proteins in E. coli. The flagellin sequences are inserted at the 3' end of the cro-lacZ gene of pEX, the expression of which is under the control of the lambda PR promoter. Expression was induced by inactivation of the temperature-sensitive cI repressor by shifting the incubation temperature from 30 to 42°C (25). Recombinants were grown for 15 to 20 h in LB-ampicillin (200 ,ug/ml) at 30°C, diluted 1:50, and incubated again for 1.5 h at 30°C. The temperature was then increased to 42°C for 2 h. Cells (1.5 ml of the culture) were harvested by centrifugation and lysed in 0.1 ml of 5% sodium dodecyl sulfate (SDS)-50 mM Tris-Cl (pH 8.0) for 10 min at 95°C. Samples were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) (7.5% polyacrylamide) as previously described (23). Protein bands were stained with Coomassie brilliant blue. Western blotting. Expression products were screened with

Corresponding author. 1100

various flagellin-specific antibodies by using Western immunoblot analysis (21). Briefly, after SDS-PAGE, proteins were transferred to a nitrocellulose membrane (BA85; Schleicher & Schuell, Dassel, Federal Republic of Germany) and incubated with monoclonal antibody (1:100 dilution of ascites fluid) or rabbit polyclonal antiserum (1:1,000 dilution). Bound antibodies were detected with alkaline phosphatase conjugated to rabbit anti-mouse immunoglobulin G or goat anti-rabbit immunoglobulin G (Promega Biotec, Madison, Wis.; 1:3,000 dilution). Production and characterization of monoclonal antibodies. Mouse monoclonal antiflagellin antibodies CF1 to CF14 have been described previously (16, 17) and were derived from mice immunized with native flagella. Monoclonal antibodies CF15, CF16, and CF17 were derived from mice immunized with dissociated flagellin subunits as constituents of acidextractable material eluted from the surface of bacteria as previously described (20). To enhance the immune response to cross-reactive epitopes the mice were primed subcutaneously with acid extract (20 ,ug of protein in Freund complete adjuvant) from C. jejuni serotype 1, boosted subcutaneously with acid extracts from C. jejuni serotype 2 and then serotype 6 (20 ,ug of protein in Freund incomplete adjuvant), and finally boosted intravenously with acid extract from C. jejuni serotype 4 (20 p.g of protein aqueous). All serotype strains were kindly supplied by J. Penner, Canada. The specificities of CF1 to CF14 have been previously described (16). CF15, CF16, and CF17 were all of the immunoglobulin Gl isotype and reacted with purified flagella and acid extract from C. jejuni 81116 in an enzyme-linked immunosorbent assay (ELISA) and Western blots but reacted poorly, if at all, with antigens from the aflagellate variant of this strain. These antibodies also cross-reacted

TABLE 1. Properties of monoclonal antibodies against

Campylobacter flagellin reactingab

Reaction to flaA and/orflaB regionb

1 1 2 2 13 9 13 1 18 18 18

A IV A IV A IV A IV A IV A IV, B II-VI Several A IV A IV, B II-VI A VII, B VII A III, B III

No. of LIO serotypes

Antibody

CF1 CF2 CF3 CF4 CF5 CF6 CF7 CF13 CF15 CF16 CF17

See reference 16. Data from this study; each gene was divided into seven regions as shown in Fig. 1. a

b

with all flagellated strains of 18 serotypes of C. jejuni as well as Campylobacter laridis, C. coli, C. uppsalensis, and Helicobacter pylori but did not react with Campylobacter sputorum subsp. bubulus. The properties of all monoclonal antibodies are summarized in Table 1. Radioiodination of monoclonal antibodies. Mouse immunoglobulin was purified from ascitic fluid either by protein A affinity chromatography or by fast protein liquid chromatography on a Mono Q or Mono S column (18). Purified monoclonal antibody (100 ,ug in phosphate-buffered saline) was incubated with 200 ,uCi of carrier-free Na1251 (Amersham Corp., Amersham, United Kingdom) in the presence

Location of flagellin fragments

Name of

Cloning site and expression vector used

flagellin fragneint

478

A5 340

A4

Al

1naI EcoRf->Spel

pEX2 pEX3 PEX 11

Baffr-II->Pstl

pEX 1

Smnal

pDE3

Smnal

pEX3

Psti

pEX2 pEX11

95 D

B

Il

D

El

II

IV

E5

V

l 111 1

____n----COOH _

1

1111111 1111

flagellin r

Ht2N

S R bZ ! 4

Vil

V

I-------------

flagellin A

--S--

r--%

E5

D Bl

PstI

576

478

55 1

B2 B3 B4 B5

576

410

A3 A2

1101

IMMUNOGENIC REGIONS ON C. JEJUNI FLAGELLIN PROTEINS

VOL. 59, 1991

COOH p

X

95

1

478

79

410

145 340

478

576 576

EcoRI->Spel Smnal PstI

pEX3 pEX2

FIG. 1. Construction and production of C. jejuni 81116 flagellin gene fusion proteins in pEX: localization of the flagellin fragments and subcloning strategy for insertion into pEX vectors. Restriction enzyme fragments encoding polypeptides Al to AS and Bi to BS were isolated from the available flagellin clones (22) and ligated into pEX vectors as indicated. Each flagellin protein was thereby divided into seven regions (I to VII). Fusion products A2 and B2 are different because of the use of different restriction enzymes. The location of amino acids at junctions between regions is indicated with each restriction site. B, BglII; D, DraI; El, EcoRI; ES, EcoRV; P, PstI; X, XbaI. Differences in amino acid sequences between flaA and flaB proteins are shown as lines connecting both flagellins.

1102

NUIJTEN ET AL. A

INFECT. IMMUN.

B

A 1 2 3 4 5 1 2 3 4 5

D

1

IEF

A B 2 3 4 5 12 3 4 5

->

BASIC

p

SDS PAGE

anti-flagellin A

B

1

2 3 4 5

1

CP1. 2. 3.

B 2 3 4 5

E

1

C

1

4. 5. and 13

A B 2 3 4 5 1 2 3

CF17 A

CF6

F

4

5

and 15

A

B

2 3 4 5 1 2 3 4 5

B

I

60-0

':

FIG. 3. Western blot of two-dimensional SDS-PAGE of flagellin from C. jejuni 81116 incubated with monoclonal antibody CF3. IEF, Isoelectrofocusing.

1 2 3 4 5 1 2 3 4 5

a

id4 CF 16

ACDIC

CF7

FIG. 2. Western blots of C. jejuni flaA and flaB gene fusion proteins. Identical blots were incubated with polyclonal antiflagellin serum (A) or with ascites fluid of 11 different monoclonal antibodies as indicated below each panel (B to F). One representative blot of each of five groups of antibodies is shown. The A2 lanes show multiple banding in most panels; all tested A2 constructs containing the correct restriction fragment showed this phenomenon.

of lodobeads (Pierce, Chester, United Kingdom) for 10 min at 20°C. The iodination mixture was applied to a column of Sephadex G-25 (1 by 10 cm) and eluted with phosphatebuffered saline, and 0.5-ml fractions were collected. Radioiodinated immunoglobulin eluted from the column after 2 to 4 ml. The specific activity of the radiolabeled antibodies was 0.1 to 0.8 mCi/mg. The 1251I-labeled antibodies were stored at 4°C in phosphate-buffered saline containing 0.05% (wt/vol) sodium azide until required. Radioimmunoassay and competitive binding experiments. Micro-ELISA plates were coated with 5 ,ug of C. jejuni 81116 acid extract antigen (20) per jil in 100 ,ul of 0.1 M carbonate buffer (pH 9.6) overnight at 20°C. The plates were washed with ELISA wash (0.85% [wt/vol] NaCl containing 0.05% [vol/vol] Tween 20). Serial fivefold dilutions of 1251I-labeled antibody in ELISA diluent (ELISA wash containing 1% [wt/vol] bovine serum albumin and 0.6% [wt/vol] Tris hydrochloride [pH 7.6]) were added to the wells and incubated at 4°C overnight. After wells were washed with ELISA wash the bound radioactivity was solubilized by incubation with 1% SDS in 0.1% NaOH and counted. For each 125I-antibody the minimum concentration required for saturation was determined. Serial fivefold dilutions of competing antibody, as ascitic fluid, were made in ELISA diluent and mixed with an equal volume of radiolabeled antibody at the minimal saturating concentration. This antibody mixture was then incubated in antigen-coated wells overnight at 4°C. After

washing, the bound radioactivity was solubilized and counted as described above. The results are expressed as percent inhibition of binding of '25I-labeled antibody by unlabeled antibody. Western blotting of two-dimensional gels. Flagella of C. jejuni 81116 were prepared (20) and separated by isoelectrofocusing with a Protean II vertical electrophoresis cell with tube adapters. The isoelectrofocusing gels contained 5.5% acrylamide, 12 M urea, and 7.5% ampholytes (Bio-Rad Laboratories, Richmond, Calif.). Isolated flagella (20) were solubilized in 16% 2-mercaptoethanol and 33% Triton X-100 at 10 jig of protein per tube (1.5 mm by 10 cm). Gels were run at 400 V for 16 h and then at 800 V for 2 h. The isoelectrofocusing tubes were placed on the stacking gel of an SDSPAGE gradient gel (10 to 25% polyacrylamide) and run under standard conditions (20). Proteins were blotted onto a nitrocellulose membrane (18). Bound mouse immunoglobulin was detected with 125I-labeled sheep anti-mouse immunoglobulin G (Amersham). RESULTS Construction and production of flagellin fusion proteins. Five partly overlapping fragments, derived from each of the two flagellin genes (flaA and flaB) of C. jejuni 81116, were inserted into pEX. The locations of these fragments and the restriction enzymes used are shown in Fig. 1. The fusion proteins were characterized by SDS-PAGE; each fusion protein comprised a single polypeptide. The putative difference in codon usage between E. coli and Campylobacter spp. (5) does not seem to be a limiting factor for expression. This technique allowed the investigation of seven different polypeptide regions of the flagellin proteins (I to VII; Fig. 1). Localization of monoclonal antibody-specific regions on flagellins A and B. The reactivities of the monoclonal antibodies directed against fusion products offlaA andflaB were determined by Western blotting (Fig. 2). The unfused crolacZ protein did not react with any of the antibodies. The monoclonal antibodies recognized at least three immunogenic areas on the flaA flagellin: region III, between amino acids 95 and 145 (CF17); region VII, between amino acids 478 and 576 (CF16); and region IV, between amino acids 145

VOL. 59, 1991

IMMUNOGENIC REGIONS ON C. JEJUNI FLAGELLIN PROTEINS

1103

(c) CF2

C

:5

1

2

120

3

(d)

4

6

7

2

3

(e)

CF6

4

5

6

7 1

2

3

5

6

7

(CF15

CF13

100,

80 60

40' 20

1

2

3

4

5

6

7 1

2

3

4

5

6

7 1

2

3

4

5

6

7

log S (dilution) FIG. 4. Competitive radioimmunoassay. "25I-labeled antibody preparations (a through f) at minimal saturating concentrations were incubated with serial fivefold dilutions of ascitic fluid in antigen-coated wells. Competing antibodies: 0, CF1; A, CF2; *, CF3; A, CF4; O, CF6; *, CF13; , CF15. For clarity, only antibodies showing competition are shown.

and 340 (CF1, CF2, CF3, CF4, CF5, CF6, CF13, and CF15). Only CF7 (Fig. 2F) reacted with nonoverlapping fragments. Since these fragments lack any similar amino acid sequences, there is no explanation for this reaction. Polyclonal antiserum raised against denatured flagellin (21) recognized epitopes within regions III and IV (Fig. 2A). The monoclonal antibodies with broad specificity (CF15, CF16, and CF17) and also CF6 recognized flaB products. Reactivity of monoclonal antibodies with subspecies of the flagellin of C. jejuni 81116. Isolated flagella from C. jejuni 81116 were separated by two-dimensional gel electrophoresis and then Western blotting. Incubation with flagellinspecific monoclonal antibodies exhibited both charge and molecular weight heterogeneity. In agreement with earlier findings (4, 14, 17), two distinct bands (60 and 62 kDa) were observed, both of which consisted of multiple acidic spots: five or six for the 62-kDa protein and two or three for the 60-kDa protein. There were no differences observed in the reactivity patterns of the antibodies with these spots; all acidic spots reacted with the monoclonal antibodies tested (CF1, CF2, CF3, CF4, CF6, and CF13; Fig. 3). Earlier data (17) suggested that CF3, CF4, and CF13 only reacted with the 62-kDa flagellin band in immunoblots. The enhanced resolution of the two-dimensional gel system or the improved antibody detection method indicates a reinterpreta-

tion of these results. The 60- and 62-kDa protein bands seem to be modifications of the same gene product. Radioimmunoassay and competition studies. Results from the interaction of the monoclonal antibodies with the flagellin fusion proteins indicated that one area (region IV) was highly immunogenic, reacting with eight of the monoclonal antibodies investigated. To study the topography of the antigenic sites within this part of the protein, these antibodies were labeled with 1251I and used in competitive radioimmunoassays (Fig. 4). CF1 and CF5 could not be successfully iodinated with this technique. By incubating heterologous antibodies with the predetermined minimum saturation dilution of 1251I-labeled antibody, inhibition of binding could be observed. The homologous antibody was used as a positive control and caused 80 to 100% inhibition of labeled antibody

binding. On the basis of strong mutual inhibition of binding and similarities in their pattern of competition with other antibodies, CF3 and CF4 are directed against the same epitope. The epitopes defined by CF1 and CF13 are sufficiently closely related to each other and to that of CF3/4 for the antibodies to exhibit strong competitive inhibition. Conversely, CF15 competed strongly with CF1 but only partly with CF13, suggesting a slightly distant epitope. The absence of strong competition with heterologous antibody by CF2

1104

NUIJTEN ET AL.

flaA

259

-

T I G K I E Y K D G D G N G S L I *

flaB

259

-

INFECT. IMMUN.

*

*

*

*

-

275

-

275

*

V I G QI N Y K D G D N N G Q L V

FIG. 5. Amino acid sequence of the possible serotype-specific flagellin IV domain of the flagellins A and B. Differences are indicated with asterisks (*).

and CF6 indicated the epitopes in this region.

presence

of two

more

unrelated

DISCUSSION

The reactivities of 11 monoclonal antibodies with five overlapping polypeptide fragments of each of the two flagellins (A and B) of C. jejuni 81116 have been determined by using a fusion protein expression system (pEX) in E. coli. The fusion proteins produced by pEX recombinants precipitate inside the cell and are therefore protected against proteolysis. This approach has been previously useful in the mapping of conformation-independent epitopes of the coronavirus infectious bronchitis virus (8). The patterns of reactivity between the antibodies and recombinant proteins indicate the presence of at least four distinct immunogenic areas. Two of these areas were located in the amino-terminal end (region III) and the carboxyterminal end (region VII) and reacted with antibodies CF17 and CF16, respectively. Interestingly, these epitopes were only detected by antibodies induced by immunization with flagellin subunits rather than intact flagella. This is in agreement with the findings of Mills et al. (11, 12) that a flagellum antigen common to campylobacters is not surface exposed but is still immunogenic during infection. Both of these epitopes must be highly conserved, since CF17 and CF16 react with all tested serotypes of C. jejuni as well as other Campylobacter species and closely related bacteria. This antigenic conservation suggests that the polypeptides located at the ends of the flagellin would be suitable antigens for a broad-spectrum serodiagnostic test, providing these areas prove to be immunogenic during infection. A third area occurred in the center of the flaA protein (region IV), recognized by CF1, CF2, CF3, CF4, CF5, and CF13. The homologous region of the flaB protein, however, did not react with these antibodies. In this region IV, the amino acid sequences of the flaA andflaB proteins differ significantly at two places (Fig. 1). The first difference is at position 157, where a valine in flaA is substituted by a structurally analogous isoleucine in flaB. It seems unlikely that such a Val-Ile substitution would cause any great change in the properties of an epitope. The other changes occur between amino acids 259 and 275 (Fig. 5). Interestingly, the monoclonal antibodies reacting with this region are serotype restricted, except for CF5 (Table 1); this suggests the presence of at least one conserved epitope in region IV. DNA hybridization data with different parts of the flagellin genes as a probe (26) are consistent with our finding that serotype specificity is located in the center of the flagellin genes. More information on the location of the epitopes in region IV was obtained by competitive radioimmunoassays. CF3 and CF4 are directed against the same epitope. The epitopes detected by CF1, CF2, CF13, and CF3/4 are all sufficiently close to allow strong competitive inhibition of binding by the heterologous antibodies. Nevertheless, differences in other properties of the antibodies, particularly immunogold localization and serotype specificity (16, 17), indicate that there are at least three serotype-restricted epitopes recognized by

these monoclonal antibodies. In our experience, immunization with intact flagella preferentially produces antibodies against this variable region, suggesting some immunodominance. However, these epitopes are not necessarily surface exposed, as shown by the absence of antibody-immunogold labeling of the flagellum shaft (17). A final and fourth area is localized adjacent to the previous one in region IV. Antibodies CF6 and CF15 react with this area; they recognize theflaB andflaA gene products and are also significantly less serotype specific. In C. jejuni 81116, only mRNA forflaA has been detected (22), but the absence of expression of minute amounts offlaB protein cannot be excluded. By Western blotting, two flagellin bands (60 and 62 kDa) were originally observed in C. jejuni 81116 with some of the monoclonal antibodies (17). This heterogeneity of molecular weight was more readily

distinguishable by two-dimensional gel electrophoresis, which also confirmed the microheterogeneity in charge previously noted by others (1, 4, 14). These differences do not represent products from the two genes, since the flaA gene product-specific antibodies recognized both the 60- and 62-kDa bands. Therefore, the two protein bands cannot represent the two flagellins (A and B). Possibly, posttranslational phosphorylation (5) or individual amino acid substitutions due to tRNA recognition errors (4) can cause this microheterogeneity of the flagellin protein. It is notable that no monoclonal antibodies specific forflaB gene products were found, possibly reflecting the absence of flagellin B in the flagella of strain 81116. Production of antiserum against gene B-specific amino acid sequences, such as the variable part of region IV (Fig. 5), would be useful in the investigation of flagellin B. REFERENCES 1. Aguero-Rosenfeld, M. E., X.-H. Yang, and I. Nachamkin. 1990. Infection of adult Syrian hamsters with flagellar variants of Campylobacterjejuni. Infect. Immun. 58:2214-2219. 2. Black, R. E., M. M. Levine, M. L. Clements, T. P. Hughes, and M. J. Blaser. 1988. Experimental Campylobacter jejuni infection in humans. J. Infect. Dis. 157:472-479. 3. Caldwell, M. B., P. Guerry, E. C. Lee, J. P. Burans, and R. I. Walker. 1985. Reversible expression of flagella in Campylobacterjejuni. Infect. Immun. 50:941-943. 4. Dunn, B. E., M. J. Blaser, and E. L. Snyder. 1987. Twodimensional gel electrophoresis and immunoblotting of Campylobacter outer membrane proteins. Infect. Immun. 55:15641572. 5. Guerry, P., S. M. Logan, S. Thornton, and T. J. Trust. 1990. Genomic organization and expression of Campylobacter flagellin genes. J. Bacteriol. 171:1853-1860. 6. Harris, L. A., S. M. Logan, P. Guerry, and T. J. Trust. 1987. Antigenic variation of Campylobacter flagella. J. Bacteriol.

169:5066-5071.

7. Kusters, J. G., E. J. Jager, and B. A. M. Van der Zeist. 1990. Improvement of the cloning linker of the bacterial expression vector pEX. Nucleic Acids Res. 17:8007. 8. Lenstra, J. A., J. G. Kusters, G. Koch, and B. A. M. Van der Zeist. 1989. Antigenicity of the peplomer protein of infectious bronchitis virus. Mol. Immunol. 26:7-15. 9. Lior, H. 1984. Serotyping of Campylobacter jejuni and Campylobacter coli by slide agglutination based on heat-labile antigenic factors, p. 61-76. In J.-P. Butzler (ed.), Campylobacter infection in man and animals. CRC Press, Inc., Boca Raton, Fla. 10. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 11. Mills, S. D., and W. C. Bradbury. 1984. Human antibody response to outer membrane proteins of Campylobacter jejuni during infection. Infect. Immun. 43:739-743.

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