Vol. 30, No. 7
JOURNAL OF CLINICAL MICROBIOLOGY, JUIY 1992, p. 1743-1751
0095-1137/92/071743-09$02.00/0 Copyright ©D 1992, American Society for Microbiology
Immunoglobulin G Antibodies to Helicobacterpylori in Patients with Dyspeptic Symptoms Investigated by the Western Immunoblot Technique LEIF PERCIVAL ANDERSEN*
AND
FRANK ESPERSEN
Department of Clinical Microbiology, Rigshospitalet, Copenhagen, Denmark Received 18 September 1991/Accepted 10 April 1992
Helicobacter pylori is a gram-negative, curved, rod-shaped bacterium known to cause gastritis and to be an important factor in the pathogenesis of peptic ulcers. Serological testing has recently been proposed as an aid in diagnosis of H. pylori infections. In this study, we used the Western immunoblot technique to evaluate the possibility of using one or more of the antigens from H. pylori for this purpose. Thirteen major bands and about 30 minor bands could be identified by Western blotting when sera from 53 consecutive dyspeptic patients, 27 healthy children, and 25 blood donors were evaluated. Antibodies against most of the major bands were found significantly more frequently in patients with H. pylori infections than in patients without such infections. However, antibodies against a single polypeptide were not produced by all patients with H. pylori infection. Polypeptides with molecular masses of 120, 50, and between 19 and 36 kDa seem to be the most specific polypeptides for the diagnosis of H. pylori infections. This study showed only minor differences in antigenic composition between different clinical isolates of H. pylori, and serological cross-reactions with other bacteria were limited. Major serological cross-reactions were found only with Campylobacterjejuni and with bacterial lipopolysaccharide. However, one band at 60 kDa reacted with antiserum to the LegioneUla micdadei common antigen, which may indicate a cross-reaction with common antigen from several other bacteria. This study demonstrates that a number of bands may be useful as antigens in serological tests after isolation and purification.
Helicobacter (Campylobacter) pylon has been established as the most common cause of gastritis (28, 33) and has frequently been found in the stomach of patients with peptic ulcer disease (45). Diagnosis of Helicobacter infection may be difficult to establish. Most of the patients have uncharacteristic dyspepsia, but no unique set of symptoms seems to be specific for this infection (1, 36). H. pylon is most frequently found in the gastric antrum, but the bacteria are not evenly distributed on the mucosal surface (5). Thus, results of culture, histological tests, and urease tests based on biopsies may be highly dependent on the amount of tissue and the number of biopsy specimens investigated. H. pylon seems, under certain conditions, to assume coccoidal forms (19) which may not be recognized as bacteria in histological sections. In addition, about 3 weeks of cultivation is required for growth of the organisms, and they are therefore not found by the usual cultivation techniques. Growth of the organism may also be impeded by transport and culture conditions, as H. pylon does not tolerate the oxygen concentration of atmospheric,air for prolonged periods (15). H. pylon produces a large amount of the enzyme urease (26). Detection of urease in biopsy specimens for the diagnosis of H. pylon infection may give false-positive or false-negative results, depending on the time at which the test is evaluated (23). The urea breath test (21, 44) can be performed without any invasive procedures and seems to be more sensitive than the urease test. Several immunological studies using different techniques and different antigen preparations have been performed to detect antibodies against H. pylon (6, 26, 32, 41). Most commonly, indirect enzyme-linked immunosorbent assays *
(ELISAs) with sonicated or acid-glycine-extracted H. pylon as an antigen have been used. Results from ELISA studies have generally shown that levels of immunoglobulin G (IgG) antibody against H. pylon increase with age and that patients with culture-proven H. pylon or Helicobacter-like organisms (HLO) in histological sections have significantly higher IgG antibody levels than patients without H. pylon or HLO. However, most serological tests developed for diagnostic use have a sensitivity and a specificity of between 70 and 90%. Thus, there is still a need for improved serological assays. Detection of antibodies by ELISA seems to have a better correlation with the urease breath test than with any other test used to diagnose H. pylon infections (39). The serological cross-reactions between H. pylon and other bacteria are, however, incompletely investigated. The aim of this study was to investigate the presence of IgG antibodies to different H. pylon antigens by the Western immunoblot technique in sera from consecutive patients with dyspeptic symptoms with and without growth of H. pylon and to compare these results with results obtained with serum from normal children and blood donors. The main purpose was to pinpoint antigens useful for the improvement of serological tests. MATERIALS AND METHODS Patients and serum samples. Serum was obtained from 53 consecutive patients (median age, 52 years; range, 18 to 85 years) with dyspeptic symptoms admitted to the Central Hospital, Hiller0d, Denmark, for gastroscopy; 27 healthy children (median age, 3 years; range, 0.5 to 12 years) whose sera were collected for other studies; and 25 adult blood donors (median age, 44 years; range, 19 to 63 years).
Corresponding author. 1743
1744
ANDERSEN AND ESPERSEN
Endoscopy, histology, and growth of H. pylori. Esophago-
gastroduodenoscopy was performed on the 53 patients with dyspeptic symptoms, and four biopsy specimens were obtained from the antral part of the stomach and duodenum of each patient, about 3 cm on each side of the pyloric sphincter
(3).
Histological sections of formalin-fixed biopsy specimens
were stained with hematoxylin-eosin to evaluate the morphology and the presence of HLO. In addition, immunohistochemical staining (2) for H. pylori was performed. Biopsy specimens from all patients, corresponding to the biopsy specimens obtained for histological tests, were cultured under microaerophilic conditions (5% 02) on chocolate agar plates (Statens Seruminstitut) at 37°C for 3 to 6 days. H. pylori was identified by conventional laboratory methods
(38). Bacterial strains. Clinical isolates of H. pylon from human gastric mucosal biopsy specimens (CH-20429, CH-48250, CH-00859, and CH-01497 [from four adults with peptic ulcers], CH-43509 [from an adult with esophagitis], CH44032 [from an adult with gastroduodenitis], CH-04756 [from an adult with a normal gastric mucosa], and Ge-41001, Ge-41002, and Ge-41003 [from three children with abdominal pain and normal gastric mucosae] were subcultured microaerophilically at 37°C on chocolate agar plates for 18 to 40 h. One clinical strain (CH-20429) was selected as the test strain. This strain gave a high positive rate (14 strong reactions and 1 weaker reaction out of 15 reactions, compared with the homologous reaction) when heat-stable antigens from 15 clinical strains of H. pylori were used as antigens in ELISAs and the 15 corresponding serum samples were tested (unpublished data). The usefulness of this strain was evaluated further in this study. Clinical isolates of Campylobacter jejuni and Campylobacter coli (SSI-8759, SSI-10776, and SSI-12131 [isolated from feces of adults with acute diarrhea]) were subcultured microaerophilically on 5% blood agar plates (Statens Seruminstitut) for 18 h. Clinical isolates of Eschenichia coli (laboratory strain), Enterobacter cloacae (RH-3221 C), Klebsiella oxytoca (RH-3725), Proteus vulgaris (RH-3511 B), Serratia marcescens (laboratory strain), Yersinia enterocolitica (RH-0301 P+), Salmonella typhi (BL-4), Salmonella typhimurium (B-Y), Haemophilus influenzae type b (RH-19648), Neisseria meningitidis (RH-BB II), Enterococcusfaecalis (RH-3691), Staphylococcus aureus (E 1369; protein A deficient [10]), and Staphylococcus epidermidis (S 8) were subcultured aerobically on 5% blood agar plates for 18 h. Whole-cell preparations. The bacterial cultures were harvested and washed two times in sterile distilled water. The preparations were centrifuged at 7,000 x g for 10 min, and the bacterial pellet was stored at -20'C. The pellets were resuspended in sterile water to a concentration of 1 g (wet weight) per ml of sterile water when used. Sonicated cell preparations. Whole-cell preparations (1 g [wet weight] per ml of sterile water) were broken by sonication at 20,000 Hz for 45 s; this process was performed a total of five times with a Rapidis 300 19-mm probe with a 9.5-mm tip. The preparation was cooled during sonication by immersion in ice water. The sonicated suspensions were stored at -200C. H. pylori-positive serum pool. Sera from seven patients with dyspeptic symptoms were selected. These sera contained antibodies against all visible bands in Western blots (fig. 1). Equal amounts of serum from each patient were pooled and stored at -20'C. Serum samples corresponding to H. pylori strains. Serum
J. CLIN. MICROBIOL.
samples were obtained from the patients from whom H. pylon CH-48250, CH-00859, CH-01497, CH-43509, CH44032, CH-04756, Ge-41001, Ge-41002, and Ge-41003 were obtained. Absorption of sera. Twofold dilutions of the whole-cell preparation of H. pylori CH-20429, as well as the ultrasonicated preparation of the same strain, were prepared with concentrations ranging from 1.0 to 0.004 g (wet weight) per ml in phosphate-buffered saline (PBS), pH 7.4. Equal volumes of each of these antigen suspensions and the H. pylon-positive serum pool diluted 1:25 in PBS (pH 7.4) were mixed in a Vortex mixer for 30 s. The suspensions were incubated overnight at 4°C, centrifuged at 12,000 x g for 10 min and stored at 4'C. The pooled sera absorbed with the different antigen preparations were evaluated by Western blotting to define the optimal concentration of whole cells and sonicated bacteria for the absorption. All bands could be absorbed from the serum pool by 0.25 g (wet weight) of whole-cell preparations of H. pylori per ml in equal volumes or by 0.5 g (wet weight) of sonicated preparations of H. pylon per ml in equal volumes. On the basis of these results, three parts of the positive serum pool diluted 1:25 in PBS (pH 7.4) were absorbed with both one part of the whole-cell preparation and two parts of the ultrasonicated preparations. The absorption of the H. pylori-positive serum pool was repeated with all bacterial strains. Sera from the patients from whom the H. pylon strains were obtained were absorbed in the same manner with the homologous strain of H. pylon as well as with the reference strain (CH-20429). These absorbed sera were used in blotting studies with the reference strain as well as the individual strains from the patients. Polyclonal and monoclonal antibodies to detect H. pylori proteins. Monoclonal antibodies against the 28-kDa urease subunit (CP14 [17-3F5]) and the 54-kDa flagellar polypeptide (CP14 [32-3F5]) were generous gifts from Diane Newell, Porton Down, Salisbury, United Kingdom (17). Polyclonal antibodies against the 60-kDa common antigen from Legionella micdadei (ATCC 33218) were kindly donated by Jette Bangsborg, University of Copenhagen (4). Human antilipopolysaccharide (anti-LPS) serum, pooled polyspecific rabbit antibodies against LPS from three different strains of Pseudomonas aeruginosa (12), and monospecific polyclonal rabbit antibodies against Y. enterocolitica 0:3 LPS, Shigella dysenteniae LPS, and Chlamydia trachomatis LPS were kindly donated by Geoffrey Shand, Dako, Copenhagen, Denmark. SDS-PAGE. The whole-cell preparation, the ultrasonicated cell preparation, and the boiled cell preparation were diluted 10-fold from 1:10 to 1:10,000 in PBS (pH 7.4) and mixed with equal volumes of sample buffer containing 0.4% sodium dodecyl sulfate (SDS) and 4.8% (wt/vol) DL-dithiothreitol. The suspensions were boiled for 5 min in a water bath. SDS-polyacrylamide gel electrophoresis (PAGE) was carried out as described by Laemmli (22) with a 15% polyacrylamide separation gel and a 5% polyacrylamide stacking gel. Relative molecular weight (Mr) was determined with reference proteins (low-molecular-weight kit; Pharmacia Fine Chemicals). Coomassie brilliant blue staining was performed as described by Weber and Osborne (43). Silver staining was performed with silver nitrate after fixation with 10% glutaraldehyde, and the autoradiograms were developed with 75 ,ul of 24.5% formalin in 100 ml of 3% sodium carbonate (29). Western blot analysis. Western blot analysis was carried out as described previously (10). Electrophoretic transfer of protein from unstained SDS-polyacrylamide gels was per-
WESTERN BLOT FOR H. PYLORI IgG ANTIBODIES
VOL. 30, 1992
TABLE 1. Characteristics of 53 patients with dyspepsia' Endoscopic diagnosis (no. of patients)
Normal (14) Gastroduodenitis (11) Ulcer (15) Miscellaneous (13)d Total (53)
Mr
No. (%) positive for H. pylon or HLO by: No. (%) with active gastritis* HE' ImmunoCulture staining staining
8 8 13 8
(57) (73) (87) (62)
37 (70)
6 6 12 6
(43) 4 (29) 4 (56) 5 (45) 5 (80) 10 (67) 11 (46) 7 (54) 7
1745
(29) (45) (73) (54)
30 (57) 26 (46) 27 (51)
a Thirty-three patients were positive for H. pylon or HLO. b As determined by histological tests. c HE, hematoxylin-eosin. d Includes 10 patients with esophagitis, 1 patient with Menetriere's disease, 1 patient with nontropical sprue, and one patient with gastric cancer.
formed by a modification of the technique described by Towbin et al. (40). A nitrocellulose gel was assembled, and the protein was transferred to the nitrocellulose paper (HAWP 2930; pore size, 0.45 ,um; Millipore) at 20°C for 18 h at 24 V in Tris-hydrochloride (25 mM)-glycine (0.192 M; pH 8.4) containing methanol (4.9 M). After transfer, the remaining binding sites on the paper were blocked by incubation with Tween 20 (2%, wt/vol) for 30 min. The nitrocellulose sheets were then incubated for 1 h at 20°C with human sera diluted 1:100 in Tris-HCI buffer (pH 7.4) with 2% (wt/vol) Tween 20. The nitrocellulose sheet was then washed three times in Tris-HCI buffer (pH 7:4) with 2% (wt/vol) Tween 20 and 10% NaCI and then incubated for 1 h at 20°C with horseradish peroxidase-conjugated rabbit anti-human IgG antibodies (Tago, Inc., Burlingame, Calif.) diluted 1:2,000 in Tris-HCl buffer (pH 7.4) containing 2% (wt/vol) Tween 20. The washing was repeated as described above, and the sheets were incubated in a citrate-phosphate buffer pH 5.0 with H202 (5 mM)-tetramethylbenzidine (Merck cat. no. 8622) and dimethyl sulfoxide (Merck cat. no. 2931) for 10 min at 20°C. The enzyme reaction was stopped by washing the sheets in distilled water. Statistics. Fisher's exact probability test was used to evaluate the results.
RESULTS Detection of H. pylori in biopsy specimens from patients with dyspeptic symptoms. H. pylon was cultured or HLO were seen in sections from 33 (62%) of 53 consecutive patients who underwent gastroscopy (Table 1). H. pylon was cultured from 30 (91%) of these 33 positive patients. HLO were found in 26 (79%) by microscopy of hematoxylin-eosinstained sections and in 27 (82%) by microscopy of sections immunostained for H. pylon. Three patients were culture negative but HLO positive. Two of these were identified as positive only with sections immunostained for H. pylon. Antibodies to H. pylori proteins in serum samples. In the 105 serum samples, 13 major bands (A to M) could be identified, as shown in Fig. 1. In addition, about 30 minor bands were found. More than 10 bands were found in serum samples from 28 of 33 (85%) dyspeptic patients with cultureproven H. pylon or HLO in histological sections, in 3 of 20 (15%) dyspeptic patients without H. pylon or HLO, in 5 of 25 (20%) blood donors, and in 2 of 27 (7%) children (Table 2). The total number of bands was significantly higher in samples from dyspeptic patients with verified H. pylon infection
A
-B _C
97,400-
-D -E _F -G
66,20042,899
20
25 1 1
Total
27
"
Number of IgG antibodies
=
Blood donors (age, 19-63 yr)
dyspepsia Without H. pylon
With
and HLO
H. pylon or HLO
20 5 0
17 2 1
5 15 13
25
20
33
number of bands.
1746
ANDERSEN AND ESPERSEN
J. CL-IN. MICROBIOL.
TABLE 3. Number of patients with IgG antibodies against major H. pylon antigens No. (%) of patients with antibodies
Western blot band
to indicated band Mr3(10:8) M,(10 1)1 H. pylori or HLO H. pylon and HLO
positive (n = 33)
A B C D E F G H I J K L M
150 120 75 60 56 50 47 36 30 27 25 19 15
28 32 28 31 28 27 27 25 27 22 15 21 28
negative (n = 20)
(85) (97) (85) (94) (85) (82) (82) (76) (82) (67) (45) (64) (85)
14 7 8 12 10 6 9 1 3 1 1 1 12
(70) (35) (40) (60) (50) (30) (45) (5) (15) (5) (5) (5) (60)
NS
JOURNAL OF CLINICAL MICROBIOLOGY, JUIY 1992, p. 1743-1751
0095-1137/92/071743-09$02.00/0 Copyright ©D 1992, American Society for Microbiology
Immunoglobulin G Antibodies to Helicobacterpylori in Patients with Dyspeptic Symptoms Investigated by the Western Immunoblot Technique LEIF PERCIVAL ANDERSEN*
AND
FRANK ESPERSEN
Department of Clinical Microbiology, Rigshospitalet, Copenhagen, Denmark Received 18 September 1991/Accepted 10 April 1992
Helicobacter pylori is a gram-negative, curved, rod-shaped bacterium known to cause gastritis and to be an important factor in the pathogenesis of peptic ulcers. Serological testing has recently been proposed as an aid in diagnosis of H. pylori infections. In this study, we used the Western immunoblot technique to evaluate the possibility of using one or more of the antigens from H. pylori for this purpose. Thirteen major bands and about 30 minor bands could be identified by Western blotting when sera from 53 consecutive dyspeptic patients, 27 healthy children, and 25 blood donors were evaluated. Antibodies against most of the major bands were found significantly more frequently in patients with H. pylori infections than in patients without such infections. However, antibodies against a single polypeptide were not produced by all patients with H. pylori infection. Polypeptides with molecular masses of 120, 50, and between 19 and 36 kDa seem to be the most specific polypeptides for the diagnosis of H. pylori infections. This study showed only minor differences in antigenic composition between different clinical isolates of H. pylori, and serological cross-reactions with other bacteria were limited. Major serological cross-reactions were found only with Campylobacterjejuni and with bacterial lipopolysaccharide. However, one band at 60 kDa reacted with antiserum to the LegioneUla micdadei common antigen, which may indicate a cross-reaction with common antigen from several other bacteria. This study demonstrates that a number of bands may be useful as antigens in serological tests after isolation and purification.
Helicobacter (Campylobacter) pylon has been established as the most common cause of gastritis (28, 33) and has frequently been found in the stomach of patients with peptic ulcer disease (45). Diagnosis of Helicobacter infection may be difficult to establish. Most of the patients have uncharacteristic dyspepsia, but no unique set of symptoms seems to be specific for this infection (1, 36). H. pylon is most frequently found in the gastric antrum, but the bacteria are not evenly distributed on the mucosal surface (5). Thus, results of culture, histological tests, and urease tests based on biopsies may be highly dependent on the amount of tissue and the number of biopsy specimens investigated. H. pylon seems, under certain conditions, to assume coccoidal forms (19) which may not be recognized as bacteria in histological sections. In addition, about 3 weeks of cultivation is required for growth of the organisms, and they are therefore not found by the usual cultivation techniques. Growth of the organism may also be impeded by transport and culture conditions, as H. pylon does not tolerate the oxygen concentration of atmospheric,air for prolonged periods (15). H. pylon produces a large amount of the enzyme urease (26). Detection of urease in biopsy specimens for the diagnosis of H. pylon infection may give false-positive or false-negative results, depending on the time at which the test is evaluated (23). The urea breath test (21, 44) can be performed without any invasive procedures and seems to be more sensitive than the urease test. Several immunological studies using different techniques and different antigen preparations have been performed to detect antibodies against H. pylon (6, 26, 32, 41). Most commonly, indirect enzyme-linked immunosorbent assays *
(ELISAs) with sonicated or acid-glycine-extracted H. pylon as an antigen have been used. Results from ELISA studies have generally shown that levels of immunoglobulin G (IgG) antibody against H. pylon increase with age and that patients with culture-proven H. pylon or Helicobacter-like organisms (HLO) in histological sections have significantly higher IgG antibody levels than patients without H. pylon or HLO. However, most serological tests developed for diagnostic use have a sensitivity and a specificity of between 70 and 90%. Thus, there is still a need for improved serological assays. Detection of antibodies by ELISA seems to have a better correlation with the urease breath test than with any other test used to diagnose H. pylon infections (39). The serological cross-reactions between H. pylon and other bacteria are, however, incompletely investigated. The aim of this study was to investigate the presence of IgG antibodies to different H. pylon antigens by the Western immunoblot technique in sera from consecutive patients with dyspeptic symptoms with and without growth of H. pylon and to compare these results with results obtained with serum from normal children and blood donors. The main purpose was to pinpoint antigens useful for the improvement of serological tests. MATERIALS AND METHODS Patients and serum samples. Serum was obtained from 53 consecutive patients (median age, 52 years; range, 18 to 85 years) with dyspeptic symptoms admitted to the Central Hospital, Hiller0d, Denmark, for gastroscopy; 27 healthy children (median age, 3 years; range, 0.5 to 12 years) whose sera were collected for other studies; and 25 adult blood donors (median age, 44 years; range, 19 to 63 years).
Corresponding author. 1743
1744
ANDERSEN AND ESPERSEN
Endoscopy, histology, and growth of H. pylori. Esophago-
gastroduodenoscopy was performed on the 53 patients with dyspeptic symptoms, and four biopsy specimens were obtained from the antral part of the stomach and duodenum of each patient, about 3 cm on each side of the pyloric sphincter
(3).
Histological sections of formalin-fixed biopsy specimens
were stained with hematoxylin-eosin to evaluate the morphology and the presence of HLO. In addition, immunohistochemical staining (2) for H. pylori was performed. Biopsy specimens from all patients, corresponding to the biopsy specimens obtained for histological tests, were cultured under microaerophilic conditions (5% 02) on chocolate agar plates (Statens Seruminstitut) at 37°C for 3 to 6 days. H. pylori was identified by conventional laboratory methods
(38). Bacterial strains. Clinical isolates of H. pylon from human gastric mucosal biopsy specimens (CH-20429, CH-48250, CH-00859, and CH-01497 [from four adults with peptic ulcers], CH-43509 [from an adult with esophagitis], CH44032 [from an adult with gastroduodenitis], CH-04756 [from an adult with a normal gastric mucosa], and Ge-41001, Ge-41002, and Ge-41003 [from three children with abdominal pain and normal gastric mucosae] were subcultured microaerophilically at 37°C on chocolate agar plates for 18 to 40 h. One clinical strain (CH-20429) was selected as the test strain. This strain gave a high positive rate (14 strong reactions and 1 weaker reaction out of 15 reactions, compared with the homologous reaction) when heat-stable antigens from 15 clinical strains of H. pylori were used as antigens in ELISAs and the 15 corresponding serum samples were tested (unpublished data). The usefulness of this strain was evaluated further in this study. Clinical isolates of Campylobacter jejuni and Campylobacter coli (SSI-8759, SSI-10776, and SSI-12131 [isolated from feces of adults with acute diarrhea]) were subcultured microaerophilically on 5% blood agar plates (Statens Seruminstitut) for 18 h. Clinical isolates of Eschenichia coli (laboratory strain), Enterobacter cloacae (RH-3221 C), Klebsiella oxytoca (RH-3725), Proteus vulgaris (RH-3511 B), Serratia marcescens (laboratory strain), Yersinia enterocolitica (RH-0301 P+), Salmonella typhi (BL-4), Salmonella typhimurium (B-Y), Haemophilus influenzae type b (RH-19648), Neisseria meningitidis (RH-BB II), Enterococcusfaecalis (RH-3691), Staphylococcus aureus (E 1369; protein A deficient [10]), and Staphylococcus epidermidis (S 8) were subcultured aerobically on 5% blood agar plates for 18 h. Whole-cell preparations. The bacterial cultures were harvested and washed two times in sterile distilled water. The preparations were centrifuged at 7,000 x g for 10 min, and the bacterial pellet was stored at -20'C. The pellets were resuspended in sterile water to a concentration of 1 g (wet weight) per ml of sterile water when used. Sonicated cell preparations. Whole-cell preparations (1 g [wet weight] per ml of sterile water) were broken by sonication at 20,000 Hz for 45 s; this process was performed a total of five times with a Rapidis 300 19-mm probe with a 9.5-mm tip. The preparation was cooled during sonication by immersion in ice water. The sonicated suspensions were stored at -200C. H. pylori-positive serum pool. Sera from seven patients with dyspeptic symptoms were selected. These sera contained antibodies against all visible bands in Western blots (fig. 1). Equal amounts of serum from each patient were pooled and stored at -20'C. Serum samples corresponding to H. pylori strains. Serum
J. CLIN. MICROBIOL.
samples were obtained from the patients from whom H. pylon CH-48250, CH-00859, CH-01497, CH-43509, CH44032, CH-04756, Ge-41001, Ge-41002, and Ge-41003 were obtained. Absorption of sera. Twofold dilutions of the whole-cell preparation of H. pylori CH-20429, as well as the ultrasonicated preparation of the same strain, were prepared with concentrations ranging from 1.0 to 0.004 g (wet weight) per ml in phosphate-buffered saline (PBS), pH 7.4. Equal volumes of each of these antigen suspensions and the H. pylon-positive serum pool diluted 1:25 in PBS (pH 7.4) were mixed in a Vortex mixer for 30 s. The suspensions were incubated overnight at 4°C, centrifuged at 12,000 x g for 10 min and stored at 4'C. The pooled sera absorbed with the different antigen preparations were evaluated by Western blotting to define the optimal concentration of whole cells and sonicated bacteria for the absorption. All bands could be absorbed from the serum pool by 0.25 g (wet weight) of whole-cell preparations of H. pylori per ml in equal volumes or by 0.5 g (wet weight) of sonicated preparations of H. pylon per ml in equal volumes. On the basis of these results, three parts of the positive serum pool diluted 1:25 in PBS (pH 7.4) were absorbed with both one part of the whole-cell preparation and two parts of the ultrasonicated preparations. The absorption of the H. pylori-positive serum pool was repeated with all bacterial strains. Sera from the patients from whom the H. pylon strains were obtained were absorbed in the same manner with the homologous strain of H. pylon as well as with the reference strain (CH-20429). These absorbed sera were used in blotting studies with the reference strain as well as the individual strains from the patients. Polyclonal and monoclonal antibodies to detect H. pylori proteins. Monoclonal antibodies against the 28-kDa urease subunit (CP14 [17-3F5]) and the 54-kDa flagellar polypeptide (CP14 [32-3F5]) were generous gifts from Diane Newell, Porton Down, Salisbury, United Kingdom (17). Polyclonal antibodies against the 60-kDa common antigen from Legionella micdadei (ATCC 33218) were kindly donated by Jette Bangsborg, University of Copenhagen (4). Human antilipopolysaccharide (anti-LPS) serum, pooled polyspecific rabbit antibodies against LPS from three different strains of Pseudomonas aeruginosa (12), and monospecific polyclonal rabbit antibodies against Y. enterocolitica 0:3 LPS, Shigella dysenteniae LPS, and Chlamydia trachomatis LPS were kindly donated by Geoffrey Shand, Dako, Copenhagen, Denmark. SDS-PAGE. The whole-cell preparation, the ultrasonicated cell preparation, and the boiled cell preparation were diluted 10-fold from 1:10 to 1:10,000 in PBS (pH 7.4) and mixed with equal volumes of sample buffer containing 0.4% sodium dodecyl sulfate (SDS) and 4.8% (wt/vol) DL-dithiothreitol. The suspensions were boiled for 5 min in a water bath. SDS-polyacrylamide gel electrophoresis (PAGE) was carried out as described by Laemmli (22) with a 15% polyacrylamide separation gel and a 5% polyacrylamide stacking gel. Relative molecular weight (Mr) was determined with reference proteins (low-molecular-weight kit; Pharmacia Fine Chemicals). Coomassie brilliant blue staining was performed as described by Weber and Osborne (43). Silver staining was performed with silver nitrate after fixation with 10% glutaraldehyde, and the autoradiograms were developed with 75 ,ul of 24.5% formalin in 100 ml of 3% sodium carbonate (29). Western blot analysis. Western blot analysis was carried out as described previously (10). Electrophoretic transfer of protein from unstained SDS-polyacrylamide gels was per-
WESTERN BLOT FOR H. PYLORI IgG ANTIBODIES
VOL. 30, 1992
TABLE 1. Characteristics of 53 patients with dyspepsia' Endoscopic diagnosis (no. of patients)
Normal (14) Gastroduodenitis (11) Ulcer (15) Miscellaneous (13)d Total (53)
Mr
No. (%) positive for H. pylon or HLO by: No. (%) with active gastritis* HE' ImmunoCulture staining staining
8 8 13 8
(57) (73) (87) (62)
37 (70)
6 6 12 6
(43) 4 (29) 4 (56) 5 (45) 5 (80) 10 (67) 11 (46) 7 (54) 7
1745
(29) (45) (73) (54)
30 (57) 26 (46) 27 (51)
a Thirty-three patients were positive for H. pylon or HLO. b As determined by histological tests. c HE, hematoxylin-eosin. d Includes 10 patients with esophagitis, 1 patient with Menetriere's disease, 1 patient with nontropical sprue, and one patient with gastric cancer.
formed by a modification of the technique described by Towbin et al. (40). A nitrocellulose gel was assembled, and the protein was transferred to the nitrocellulose paper (HAWP 2930; pore size, 0.45 ,um; Millipore) at 20°C for 18 h at 24 V in Tris-hydrochloride (25 mM)-glycine (0.192 M; pH 8.4) containing methanol (4.9 M). After transfer, the remaining binding sites on the paper were blocked by incubation with Tween 20 (2%, wt/vol) for 30 min. The nitrocellulose sheets were then incubated for 1 h at 20°C with human sera diluted 1:100 in Tris-HCI buffer (pH 7.4) with 2% (wt/vol) Tween 20. The nitrocellulose sheet was then washed three times in Tris-HCI buffer (pH 7:4) with 2% (wt/vol) Tween 20 and 10% NaCI and then incubated for 1 h at 20°C with horseradish peroxidase-conjugated rabbit anti-human IgG antibodies (Tago, Inc., Burlingame, Calif.) diluted 1:2,000 in Tris-HCl buffer (pH 7.4) containing 2% (wt/vol) Tween 20. The washing was repeated as described above, and the sheets were incubated in a citrate-phosphate buffer pH 5.0 with H202 (5 mM)-tetramethylbenzidine (Merck cat. no. 8622) and dimethyl sulfoxide (Merck cat. no. 2931) for 10 min at 20°C. The enzyme reaction was stopped by washing the sheets in distilled water. Statistics. Fisher's exact probability test was used to evaluate the results.
RESULTS Detection of H. pylori in biopsy specimens from patients with dyspeptic symptoms. H. pylon was cultured or HLO were seen in sections from 33 (62%) of 53 consecutive patients who underwent gastroscopy (Table 1). H. pylon was cultured from 30 (91%) of these 33 positive patients. HLO were found in 26 (79%) by microscopy of hematoxylin-eosinstained sections and in 27 (82%) by microscopy of sections immunostained for H. pylon. Three patients were culture negative but HLO positive. Two of these were identified as positive only with sections immunostained for H. pylon. Antibodies to H. pylori proteins in serum samples. In the 105 serum samples, 13 major bands (A to M) could be identified, as shown in Fig. 1. In addition, about 30 minor bands were found. More than 10 bands were found in serum samples from 28 of 33 (85%) dyspeptic patients with cultureproven H. pylon or HLO in histological sections, in 3 of 20 (15%) dyspeptic patients without H. pylon or HLO, in 5 of 25 (20%) blood donors, and in 2 of 27 (7%) children (Table 2). The total number of bands was significantly higher in samples from dyspeptic patients with verified H. pylon infection
A
-B _C
97,400-
-D -E _F -G
66,20042,899
20
25 1 1
Total
27
"
Number of IgG antibodies
=
Blood donors (age, 19-63 yr)
dyspepsia Without H. pylon
With
and HLO
H. pylon or HLO
20 5 0
17 2 1
5 15 13
25
20
33
number of bands.
1746
ANDERSEN AND ESPERSEN
J. CL-IN. MICROBIOL.
TABLE 3. Number of patients with IgG antibodies against major H. pylon antigens No. (%) of patients with antibodies
Western blot band
to indicated band Mr3(10:8) M,(10 1)1 H. pylori or HLO H. pylon and HLO
positive (n = 33)
A B C D E F G H I J K L M
150 120 75 60 56 50 47 36 30 27 25 19 15
28 32 28 31 28 27 27 25 27 22 15 21 28
negative (n = 20)
(85) (97) (85) (94) (85) (82) (82) (76) (82) (67) (45) (64) (85)
14 7 8 12 10 6 9 1 3 1 1 1 12
(70) (35) (40) (60) (50) (30) (45) (5) (15) (5) (5) (5) (60)
NS
