Article Eur. J. Clin. Microbiol. Infect. Dis., July 1992, p. 595-601 0934-9723/92/07 0595-07 $ 3.00/0
---.__
Vol. 11. No. 7
595
Serodiagnosis of Helicobacter pylori Infections with an Enzyme Immunoassay Using the Chromatographically Purified 120 Kilodalton Protein B. G e r s t e n e c k e r 1, B. Eschweiler 1, H. V/3gele I, H.K. Koch 2, U. Hellerich 3, M. Kist I*
A membrane-associated 120 kDa protein of Helicobacterpylori with known speciesspecificity was isolated and used in an enzyme immunoassay (EIA) for the detection of ttelicobacterpylori-specific IgG antibodies in patient sera. The EIA was compared with two other methods used for serodiagnosis of HeHcobacterpylori infections: an EIA using sonicated whole Helicobacterpylori cell antigen and Western immunoblot. In a prospective study 127 unselected patients (76 patients with antrum gastritis, 51 patients without gastritis) who underwent gastroscopy were studied histologically and serologically. The EIA using the purified 120 kDa protein had the highest specificity (92 %) compared with the EIA using a whole cell sonicate of a single Helicobacter pylori strain as antigen (60.7 %) and the immunoblot (90.2 %). The sensitivity was 96 %, 100 % and 92 %, respectively. Sera of three control patients reacted strongly in all three methods, indicating possible Helicobacterpylori infection with negative histological findings. The EIA using the 120 kDa protein as antigen was shown to be a specific and sensitive technique for the serodiagnosis of Helicobacter pylori infections.
Helicobacter pylori, a spiral-shaped gram-negative microaerophilic organism, can be isolated from gastrointestinal biopsies in more than 50 % of patients undergoing endoscopy (1, 2). Various Studies have demonstrated a strong association between colonization of the gastric mucosa with Helicobacterpylori and the occurrence of chronic antrum gastritis and peptic ulceration (3-6). Helicobacterpylori infections are predominantly diagnosed by histological examination of gastric biopsies (7), furthermore by detection of urease activity in biopsy material and by culture of the microorganism. The 13C-urea or 14C-urea breath test is used as a non-invasive method for diagnosis of Helicobacter pylori infections (8-10). Because they are costly or time-consuming or involve expOSUre to radiation, the diagnostic methods mentioned are less suitable for screening of patients prior to endoscopy and for follow-up studies after treatment of the infection. Therefore, inexpensive, non-invasive, sensitive and specific methods 2Institute of Medical Microbiology, and 3Institute of Pathology, University of Freiburg Hermann-Herder-Str. 211.. 7800 Fre~bmg, Germany ' rman . Pt lvate Institute for Pathology,7800 Frelburg, Ge y
such as enzyme immunoassays (E/As) are desirable. Most cases of Helicobacter pylori infection in adults seem to be chronic (11). Antigens persisting in the gastrointestinal tract obviously provoke high titers of circulating Helicobacter pylorispecific antibodies (12, 13). Various crude antigen preparations such as sonicated whole bacterial cells, acidic glycine extracts and formalin-treated cells have been used as antigen for the detection of specific antibodies in patient sera. Furthermore, mixtures of whole cell antigen from different strains of Helicobacter pylori have been tested (14-16). However, due to cross-reacting proteins such as the flagellin (17) and other possibly membrane-associated proteins (18) included in these antigen preparations, the sensitivity and specificity of such tests vary considerably. Therefore, efforts have been made to prepare more defined protein antigens. Recently, individual proteins of molecular weights between 24.7 kDa and 128.8 kDa of Helicobacter pylori were purified by SDS-PAGE and electroelution and tested as solid-phase immobilized antigens with sera from infected and non-infected patients (19). Good discrimination between positive and
596
E u r , J. Clin. M i c r o b i o l . Infect. Dis.
n e g a t i v e s e r a was a c h i e v e d w i t h h i g h - m o l e c u l a r w e i g h t p r o t e i n s a b o v e 80 k D a . E v a n s e t al. (20) r e p o r t e d t h e d e v e l o p m e n t o f a n E I A using a p a r t i a l l y p u r i f i e d a d h e s i n - l i k e p r o t e i n (Nacetylneuraminyllactose-binding hemagglutinin ( N L B H ) ) . T h e a p p a r e n t m o l e c u l a r w e i g h t o f the N L B H r a n g e s b e t w e e n 50 k D a a n d 150 k D a ; t h e p r o t e i n s e e m s to b e o r g a n i z e d in f i l a m e n t o u s structures. P u r i f i e d u r e a s e c o a t i n g m i c r o t i t r e p l a t e s was also t e s t e d as a n t i g e n f o r d e t e c t i o n of Helicobacter pylori-specific s e r u m a n t i b o d i e s (21), b u t o n l y 25 % o f t h e p o s i t i v e s e r a t e s t e d r e a c t e d s t r o n g e r t h a n n e g a t i v e sera. Species-specificity of the membrane-associated 120 k D a p r o t e i n has b e e n d e m o n s t r a t e d (22) so t h a t this p r o t e i n m i g h t b e s u i t a b l e f o r s e r o d i a g nosis o f Helicobacter pylori i n f e c t i o n . In this p a p e r w e d e s c r i b e t h e d e v e l o p m e n t o f an E I A using t h e p u r i f i e d 120 k D a p r o t e i n i m m o b i l i z e d on microtitre plates, and the comparative evaluation o f t h e E I A in a p r o s p e c t i v e s t u d y using 127 s e r a f r o m h i s t o l o g i c a l l y d e f i n e d cases.
Material and M e t h o d s
Organisms, Helicobacter pylori strain 151 (Hp 151) was grown in liquid cultures in Brucella Broth supplemented with 3 % foetal calf serum under micro-aerophilic conditions (5 % oxygen). After incubation (48 h at 37 °C) bacteria were harvested by centrifugation and washed twice with sterile phosphate-buffered saline (PBS) o f p H 7.4. Cell sediments were stored at -20 °C. Patient Sera. The sera were collected by two of the authors (H.K. Koch and U. Hellerich). The patients underwent gastroscopy; histopathological examination of biopsies was performed by the same investigators to detect gastritis and Helicobacter pylori. Sera from 76 patients with histologically confirmed Helicobacter pylori-associatcd antrum gastritis and sera from 51 patients without positive histological findings (control patients) were examined for Helicobacter pylori specific IgG antibodies. The sera were taken after gastroscopy. The age of the patients ranged from 20 to 88 years (mean
age 49.5 years). The gastric biopsies were evaluated using the gastritis classification system of Whitehead which distinguishes in the pyloric and fundie region between active and inactive superficial gastritis and active and inactive chronic gastritis. Table 1 summarizes data on the histological classification of gastritis and the associated microscopic detection of Helicobacter pylori in the biopsies.
Solubilization. Five to six bacterial cell sediments (containing up to 500 mg protein) were resuspended and pooled in 30 ml homogenization buffer containing 1% (w/v) of the zwitterionic detergent (3-[(3-cholamidopropyl)-dimethylammonio]-l-propanesulfonate (CHAPS)) (23). The suspension was homogenized by two cycles of pulsed sonication (5 min for each cycle at 4 °C). Unbroken ceils and unsolubilized material were removed by centrifugation (17,000 x g, 40 min, 4 °C). The cleared supernatant was collected and stored in aliquots (5 mg protein/ml) at -20"C. Purification of the 120 kDa Protein. The 120 kDa protein was purified by size exclusion chromatography in the presence of low concentrations of sodium dodecylsulfate (SDS). Briefl}; 1 ml aliquots of the CHAPS-solubilisate were applied to the column (Superose 6 prep grade HR 16/50, Pharmacia/LKB, Sweden; separation range 5 x 106 Da to 5 x 103 Da) equilibrated with 100 mM sodium phosphate (pH 6.8) containing 0.1% SDS. Chromatography was performed at a flow rate of 0.2 ml/min. Eluted material was detected at a wavelength of 280 nm. The corresponding fractions were analyzed by SDS-PAGE (24) and Coomassie brilliant blue (25) or silver staining (26). Fractions 44 to 47 of up to 15 runs containing the 120 kDa polypeptide were pooled and concentrated to a volume of 4-5 ml by ultrafiltration (Centricon-10, Amicon, USA). The concentrate was subjected again to chromatography using identical conditions as described above. The identity of the purified protein was confirmed on immunoblot analysis (22) using a positive human control serum containing specific IgG antibodies against the 120 kDa protein and the serum of a hyperimmune rabbit immunized with the purified 120 kDa protein. Enzyme bumunoassays. For the 120 kDa E1A the purified 120 kDa antigen (2 tag/ml) was coated onto microtitre plates (100 pA/well, 48 h at 4 °C) in the presence of the chromatography buffer with the pH adjusted to 9.6. After washing the plates with PBS/1% Tween-20 (PBS-T), unspecific protein binding sites were blocked with 1% human serum albumin (HSA, fraction V; Sigma, USA) in PBS (1 h at 37 °C). Patient sera were diluted
Table I: Association of histologicallyconfirmed gastritis and bacteriological detection of Hellcobacterpylori in gastric biopsies of 127 patients taken from the antral area which were classified according to the gastritis classification system of Whitehead.
H. pylori
Normal
status Positive
1
Active chronic gastritis
Inactive chronic gastritis
Active chronic gastritis + gastric ulcer
Active chronic gastritis + duodenal ulcer
44
18
7
6
Uncertain
-
2
2
2
-
Negative
32
5
8
-
-
Vo1.11,1992
597
Briefly, total cell protein of Helicobacter pylori strain Hp 151 was solubilized, separated by SDS-PAGE in an 8 % get and transferred electrophoreticalty to nitrocellulose membranes (BA 85, 0.45 ~tm; Schleicher & Schiill, Germany) (28). After blockage of unspecific binding sites with 10 % skimmed milk the diluted sera were incubated with the immobilized antigens. Bound antibodies were detected by the PAP-technique (22). Visualization of the immune complexes was achieved using diaminobenzidine as chromogen in the presence of H202. After the substrate reaction membrane strips were washed and airdried.
1/100 in the HSA-containing blocking solution and incubated for 1 b at 37 °C with the immobilized antigen. Antigen-bound IgG antibodies were detected with lkaline phosphatase-conjugated secondary antibodies goat anti-human IgG (H+L); Dianova, Germany). After a final washing substrate buffer (0.1% p-nitrophenylphosphate in diethanolamine buffer of pH 9.6) was added and the mixture incubated at 37 *C. Absorption was read at 405 rim. The cut-off value was defined arbitrarily as the duplicate value of absorption after reaction of the enzyme-conjugated secondary antibodies with the antigen and was set at OD (405 n m ) = 0.32 after a substrate reaction of 1 h at 37 °C. An EIA using sonicated whole Helicobacter pylori cells as immobilized antigen was used in parallel. Briefly, ttelicobacter pylori cells (strain 151) were suspended in coating buffer (50 mM sodium carbonate/bicarbonate pH 9.6) and disrupted by sonication (10 min, 4 °C). The sonicare was diluted to a protein concentration of 10 lag/ml and immobilized directly on microtitre plates (100 ~1/ well, 24 h, at 4 °C). The EIA was performed as described above. The cut-off value was identical to the value used in the 120 kDa EIA.
~
Results
Purification of the 120 kDa Protein. P r e l i m i n a r y experiments
had
shown
that
treatment
of
Helicobacter pylori cells with t h e z w i t t e r i o n i c detergent CHAPS w a s m o r e e f f e c t i v e in s o l u b i l i z i n g p a r t i c u l a r l y t h e 120 k D a p r o t e i n t h a n t h e n o n - i o n i c d e t e r g e n t s T r i t o n X-100, N o n i d e t P-40 o r o t h e r z w i t t e r g e n t s such as S u l f o b e t a i n
Western bnmunoblot Analysis. Sera were tested for antibodies to the dominant species-specific 120 kDa, 88 kDa and 86 kDa antigens by the immunoblot technique (27).
--0.5
120kD~ ~ 88kD~. i 86kD ~
~
,,~ • ~ ~
E
--0.4
1= O t",l
0
lip 46 48 50 52 54 56 58 fractions
--0.3
a ,Q
--0.2
%'__
--0.1
I
I
I
I
I
I
Figure 1: Size exclusion chromatography of Helicobacter pylori solubilisate and purification of the 120 kDa antigen. Positions containing the proteins of interest are indicated (dashed box). Fractions containing the 120 kDA, 88 kDa and 86 kDa polypeptides were further analyzed by SDS-PAGE (insert). Relative molecular weights arc indicated.
598
Eur. J. Clin. Microbiol. Infect. Dis.
SB-12 (data not shown). Protein purification was performed by size exclusion chromatography in the presence of low concentrations of SDS. A representative chromatograph is shown in Figure 1, Only a minor fraction (below 1%) of the cellular. protein represents the 120 kDa protein. The polypeptide appears in fraction 46 (Figure 1, insert). Whereas fractions 44 to 47 are nearly homogeneous 120 kDa preparations, fractions 48 and 49 are contaminated mainly with two other species-specific antigens with relative molecular weights of 88 kDa and 86 kDa, respectively. For further purification, fractions from 44 to 47 were pooled, concentrated by ultrafiltration and subjected again to chromatography under identical conditions. Repeat chromatography using a Superose 12 matrix with a separation range of 3 x 105-103 Da (Superose 6:5 x 106-5 x 103 Da) did not improve the results. The repeat chromatography step resulted in further purification of the 120 kDa protein (Figure 2, lane 2). Silver staining of SDS-PAGE gels revealed approximately 8090 % homogeneity.
2,5 ¸
I ! !
2 ,O -
1 | I
E C
a
!
to O
I-,
---.__
Vol. 11. No. 7
595
Serodiagnosis of Helicobacter pylori Infections with an Enzyme Immunoassay Using the Chromatographically Purified 120 Kilodalton Protein B. G e r s t e n e c k e r 1, B. Eschweiler 1, H. V/3gele I, H.K. Koch 2, U. Hellerich 3, M. Kist I*
A membrane-associated 120 kDa protein of Helicobacterpylori with known speciesspecificity was isolated and used in an enzyme immunoassay (EIA) for the detection of ttelicobacterpylori-specific IgG antibodies in patient sera. The EIA was compared with two other methods used for serodiagnosis of HeHcobacterpylori infections: an EIA using sonicated whole Helicobacterpylori cell antigen and Western immunoblot. In a prospective study 127 unselected patients (76 patients with antrum gastritis, 51 patients without gastritis) who underwent gastroscopy were studied histologically and serologically. The EIA using the purified 120 kDa protein had the highest specificity (92 %) compared with the EIA using a whole cell sonicate of a single Helicobacter pylori strain as antigen (60.7 %) and the immunoblot (90.2 %). The sensitivity was 96 %, 100 % and 92 %, respectively. Sera of three control patients reacted strongly in all three methods, indicating possible Helicobacterpylori infection with negative histological findings. The EIA using the 120 kDa protein as antigen was shown to be a specific and sensitive technique for the serodiagnosis of Helicobacter pylori infections.
Helicobacter pylori, a spiral-shaped gram-negative microaerophilic organism, can be isolated from gastrointestinal biopsies in more than 50 % of patients undergoing endoscopy (1, 2). Various Studies have demonstrated a strong association between colonization of the gastric mucosa with Helicobacterpylori and the occurrence of chronic antrum gastritis and peptic ulceration (3-6). Helicobacterpylori infections are predominantly diagnosed by histological examination of gastric biopsies (7), furthermore by detection of urease activity in biopsy material and by culture of the microorganism. The 13C-urea or 14C-urea breath test is used as a non-invasive method for diagnosis of Helicobacter pylori infections (8-10). Because they are costly or time-consuming or involve expOSUre to radiation, the diagnostic methods mentioned are less suitable for screening of patients prior to endoscopy and for follow-up studies after treatment of the infection. Therefore, inexpensive, non-invasive, sensitive and specific methods 2Institute of Medical Microbiology, and 3Institute of Pathology, University of Freiburg Hermann-Herder-Str. 211.. 7800 Fre~bmg, Germany ' rman . Pt lvate Institute for Pathology,7800 Frelburg, Ge y
such as enzyme immunoassays (E/As) are desirable. Most cases of Helicobacter pylori infection in adults seem to be chronic (11). Antigens persisting in the gastrointestinal tract obviously provoke high titers of circulating Helicobacter pylorispecific antibodies (12, 13). Various crude antigen preparations such as sonicated whole bacterial cells, acidic glycine extracts and formalin-treated cells have been used as antigen for the detection of specific antibodies in patient sera. Furthermore, mixtures of whole cell antigen from different strains of Helicobacter pylori have been tested (14-16). However, due to cross-reacting proteins such as the flagellin (17) and other possibly membrane-associated proteins (18) included in these antigen preparations, the sensitivity and specificity of such tests vary considerably. Therefore, efforts have been made to prepare more defined protein antigens. Recently, individual proteins of molecular weights between 24.7 kDa and 128.8 kDa of Helicobacter pylori were purified by SDS-PAGE and electroelution and tested as solid-phase immobilized antigens with sera from infected and non-infected patients (19). Good discrimination between positive and
596
E u r , J. Clin. M i c r o b i o l . Infect. Dis.
n e g a t i v e s e r a was a c h i e v e d w i t h h i g h - m o l e c u l a r w e i g h t p r o t e i n s a b o v e 80 k D a . E v a n s e t al. (20) r e p o r t e d t h e d e v e l o p m e n t o f a n E I A using a p a r t i a l l y p u r i f i e d a d h e s i n - l i k e p r o t e i n (Nacetylneuraminyllactose-binding hemagglutinin ( N L B H ) ) . T h e a p p a r e n t m o l e c u l a r w e i g h t o f the N L B H r a n g e s b e t w e e n 50 k D a a n d 150 k D a ; t h e p r o t e i n s e e m s to b e o r g a n i z e d in f i l a m e n t o u s structures. P u r i f i e d u r e a s e c o a t i n g m i c r o t i t r e p l a t e s was also t e s t e d as a n t i g e n f o r d e t e c t i o n of Helicobacter pylori-specific s e r u m a n t i b o d i e s (21), b u t o n l y 25 % o f t h e p o s i t i v e s e r a t e s t e d r e a c t e d s t r o n g e r t h a n n e g a t i v e sera. Species-specificity of the membrane-associated 120 k D a p r o t e i n has b e e n d e m o n s t r a t e d (22) so t h a t this p r o t e i n m i g h t b e s u i t a b l e f o r s e r o d i a g nosis o f Helicobacter pylori i n f e c t i o n . In this p a p e r w e d e s c r i b e t h e d e v e l o p m e n t o f an E I A using t h e p u r i f i e d 120 k D a p r o t e i n i m m o b i l i z e d on microtitre plates, and the comparative evaluation o f t h e E I A in a p r o s p e c t i v e s t u d y using 127 s e r a f r o m h i s t o l o g i c a l l y d e f i n e d cases.
Material and M e t h o d s
Organisms, Helicobacter pylori strain 151 (Hp 151) was grown in liquid cultures in Brucella Broth supplemented with 3 % foetal calf serum under micro-aerophilic conditions (5 % oxygen). After incubation (48 h at 37 °C) bacteria were harvested by centrifugation and washed twice with sterile phosphate-buffered saline (PBS) o f p H 7.4. Cell sediments were stored at -20 °C. Patient Sera. The sera were collected by two of the authors (H.K. Koch and U. Hellerich). The patients underwent gastroscopy; histopathological examination of biopsies was performed by the same investigators to detect gastritis and Helicobacter pylori. Sera from 76 patients with histologically confirmed Helicobacter pylori-associatcd antrum gastritis and sera from 51 patients without positive histological findings (control patients) were examined for Helicobacter pylori specific IgG antibodies. The sera were taken after gastroscopy. The age of the patients ranged from 20 to 88 years (mean
age 49.5 years). The gastric biopsies were evaluated using the gastritis classification system of Whitehead which distinguishes in the pyloric and fundie region between active and inactive superficial gastritis and active and inactive chronic gastritis. Table 1 summarizes data on the histological classification of gastritis and the associated microscopic detection of Helicobacter pylori in the biopsies.
Solubilization. Five to six bacterial cell sediments (containing up to 500 mg protein) were resuspended and pooled in 30 ml homogenization buffer containing 1% (w/v) of the zwitterionic detergent (3-[(3-cholamidopropyl)-dimethylammonio]-l-propanesulfonate (CHAPS)) (23). The suspension was homogenized by two cycles of pulsed sonication (5 min for each cycle at 4 °C). Unbroken ceils and unsolubilized material were removed by centrifugation (17,000 x g, 40 min, 4 °C). The cleared supernatant was collected and stored in aliquots (5 mg protein/ml) at -20"C. Purification of the 120 kDa Protein. The 120 kDa protein was purified by size exclusion chromatography in the presence of low concentrations of sodium dodecylsulfate (SDS). Briefl}; 1 ml aliquots of the CHAPS-solubilisate were applied to the column (Superose 6 prep grade HR 16/50, Pharmacia/LKB, Sweden; separation range 5 x 106 Da to 5 x 103 Da) equilibrated with 100 mM sodium phosphate (pH 6.8) containing 0.1% SDS. Chromatography was performed at a flow rate of 0.2 ml/min. Eluted material was detected at a wavelength of 280 nm. The corresponding fractions were analyzed by SDS-PAGE (24) and Coomassie brilliant blue (25) or silver staining (26). Fractions 44 to 47 of up to 15 runs containing the 120 kDa polypeptide were pooled and concentrated to a volume of 4-5 ml by ultrafiltration (Centricon-10, Amicon, USA). The concentrate was subjected again to chromatography using identical conditions as described above. The identity of the purified protein was confirmed on immunoblot analysis (22) using a positive human control serum containing specific IgG antibodies against the 120 kDa protein and the serum of a hyperimmune rabbit immunized with the purified 120 kDa protein. Enzyme bumunoassays. For the 120 kDa E1A the purified 120 kDa antigen (2 tag/ml) was coated onto microtitre plates (100 pA/well, 48 h at 4 °C) in the presence of the chromatography buffer with the pH adjusted to 9.6. After washing the plates with PBS/1% Tween-20 (PBS-T), unspecific protein binding sites were blocked with 1% human serum albumin (HSA, fraction V; Sigma, USA) in PBS (1 h at 37 °C). Patient sera were diluted
Table I: Association of histologicallyconfirmed gastritis and bacteriological detection of Hellcobacterpylori in gastric biopsies of 127 patients taken from the antral area which were classified according to the gastritis classification system of Whitehead.
H. pylori
Normal
status Positive
1
Active chronic gastritis
Inactive chronic gastritis
Active chronic gastritis + gastric ulcer
Active chronic gastritis + duodenal ulcer
44
18
7
6
Uncertain
-
2
2
2
-
Negative
32
5
8
-
-
Vo1.11,1992
597
Briefly, total cell protein of Helicobacter pylori strain Hp 151 was solubilized, separated by SDS-PAGE in an 8 % get and transferred electrophoreticalty to nitrocellulose membranes (BA 85, 0.45 ~tm; Schleicher & Schiill, Germany) (28). After blockage of unspecific binding sites with 10 % skimmed milk the diluted sera were incubated with the immobilized antigens. Bound antibodies were detected by the PAP-technique (22). Visualization of the immune complexes was achieved using diaminobenzidine as chromogen in the presence of H202. After the substrate reaction membrane strips were washed and airdried.
1/100 in the HSA-containing blocking solution and incubated for 1 b at 37 °C with the immobilized antigen. Antigen-bound IgG antibodies were detected with lkaline phosphatase-conjugated secondary antibodies goat anti-human IgG (H+L); Dianova, Germany). After a final washing substrate buffer (0.1% p-nitrophenylphosphate in diethanolamine buffer of pH 9.6) was added and the mixture incubated at 37 *C. Absorption was read at 405 rim. The cut-off value was defined arbitrarily as the duplicate value of absorption after reaction of the enzyme-conjugated secondary antibodies with the antigen and was set at OD (405 n m ) = 0.32 after a substrate reaction of 1 h at 37 °C. An EIA using sonicated whole Helicobacter pylori cells as immobilized antigen was used in parallel. Briefly, ttelicobacter pylori cells (strain 151) were suspended in coating buffer (50 mM sodium carbonate/bicarbonate pH 9.6) and disrupted by sonication (10 min, 4 °C). The sonicare was diluted to a protein concentration of 10 lag/ml and immobilized directly on microtitre plates (100 ~1/ well, 24 h, at 4 °C). The EIA was performed as described above. The cut-off value was identical to the value used in the 120 kDa EIA.
~
Results
Purification of the 120 kDa Protein. P r e l i m i n a r y experiments
had
shown
that
treatment
of
Helicobacter pylori cells with t h e z w i t t e r i o n i c detergent CHAPS w a s m o r e e f f e c t i v e in s o l u b i l i z i n g p a r t i c u l a r l y t h e 120 k D a p r o t e i n t h a n t h e n o n - i o n i c d e t e r g e n t s T r i t o n X-100, N o n i d e t P-40 o r o t h e r z w i t t e r g e n t s such as S u l f o b e t a i n
Western bnmunoblot Analysis. Sera were tested for antibodies to the dominant species-specific 120 kDa, 88 kDa and 86 kDa antigens by the immunoblot technique (27).
--0.5
120kD~ ~ 88kD~. i 86kD ~
~
,,~ • ~ ~
E
--0.4
1= O t",l
0
lip 46 48 50 52 54 56 58 fractions
--0.3
a ,Q
--0.2
%'__
--0.1
I
I
I
I
I
I
Figure 1: Size exclusion chromatography of Helicobacter pylori solubilisate and purification of the 120 kDa antigen. Positions containing the proteins of interest are indicated (dashed box). Fractions containing the 120 kDA, 88 kDa and 86 kDa polypeptides were further analyzed by SDS-PAGE (insert). Relative molecular weights arc indicated.
598
Eur. J. Clin. Microbiol. Infect. Dis.
SB-12 (data not shown). Protein purification was performed by size exclusion chromatography in the presence of low concentrations of SDS. A representative chromatograph is shown in Figure 1, Only a minor fraction (below 1%) of the cellular. protein represents the 120 kDa protein. The polypeptide appears in fraction 46 (Figure 1, insert). Whereas fractions 44 to 47 are nearly homogeneous 120 kDa preparations, fractions 48 and 49 are contaminated mainly with two other species-specific antigens with relative molecular weights of 88 kDa and 86 kDa, respectively. For further purification, fractions from 44 to 47 were pooled, concentrated by ultrafiltration and subjected again to chromatography under identical conditions. Repeat chromatography using a Superose 12 matrix with a separation range of 3 x 105-103 Da (Superose 6:5 x 106-5 x 103 Da) did not improve the results. The repeat chromatography step resulted in further purification of the 120 kDa protein (Figure 2, lane 2). Silver staining of SDS-PAGE gels revealed approximately 8090 % homogeneity.
2,5 ¸
I ! !
2 ,O -
1 | I
E C
a
!
to O
I-,
