WO2000056769A2

WO2000056769A2 - COMPOSITIONS, TEST KITS AND METHODS FOR DETECTING $i(HELICOBACTER PYLORI)

Info

Publication number: WO2000056769A2
Application number: PCT/EP2000/002419
Authority: WO
Inventors: George Sachs; Petra Voland
Original assignee: Cambridge Life Sciences; George Sachs; Petra Voland
Priority date: 1999-03-18
Filing date: 2000-03-18
Publication date: 2000-09-28
Also published as: AU4289200A; WO2000056769A3

Abstract

A composition of specific antigens from Helicobacter pylori for the detection of an infection with this human pathogen and the determination of eradication of the infection after eradication treatment in a biological sample is disclosed. Comparison with other tests proving the infectious status and the eradication of the tested and treated individuals is included to show the useful application of this test.

Description

COMPOSITIONS, TEST KITS AND METHODS FOR DETECTING HELICOBACTER PYLORI

TECHNICAL FIELD

The invention relates to an antigen composition that can detect the presence of antibodies specific to Helicobacter pylori The invention also relates to a method for the preparation of the antigens and the composition and a method and kit for detecting the presence of the Helicobacter py/oπ-specιfιc antibodies The method also is able to detect eradication or the organism, providing novel methodology

BACKGROUND OF THE INVENTION

Helicobacter pylon (formerly Campylobacter pylori) hereinafter also referred to as H pylori, was discovered by B J Marshall et al in 1983 It is a gram-negative, spiral shaped, motile bacterium that colonizes the human stomach that more than 50% of the world's adult population in industrial countries and almost 100% in developing countries are infected with In association with the infection, gastric disorders like chronic gastritis gastric and duodenal ulcer disease as well as gastric carcinoma occur

The diagnosis of an infection with H pylori is usually achieved in two ways Directly (invasive) by endo- scopic examination with biopsy, followed by histology and culture of the bacterium and indirectly (non invasive) by testing peripheral blood or serum samples for antibodies against H pylori or performing a ¹³C urea breath test (UBT)

Seroiogical tests and the ¹³C UBT are the two non-invasive techniques, used in the management of H pylori infection and eradication The accuracy of a seroiogical test is dependent on the nature of the antιgen(s) Most of the seroiogical tests are ELISA based and use whole cell lysates of H pylori as the antigen, often in combination with a more purified antigen preparation like recombinant vacA, cagA and/or iceA protein Using a crude lysate preparation of the whole organism can cause problems with the specificity of the test via nonspecific binding of antibodies not specific for H pylori to components of the antigen preparation that might be present in other H pylori related organisms (false positives) On the other hand crude antigen preparation might cause false negative results because unwanted components in the preparation might dilute specific antigens or interfere with the presentation of those required to determine infection The use of a total protein isolate also prevents serology from detecting loss of the organism and therefore is not suitable for evaluating success of eradication therapy The UBT gives false negatives when patients are taking PPI s due to inhibition of urease activity by neutral pH Currently, the role of serology in managing H pylori infection is as a screening procedure and for diagnosis of infection but not for determination the success of eradication That is because the tests are not designed to detect reductions in tne antibody titer during the post eradication period In contrast the UBT is highly sensitive and specific but expensive and not available to all general physicians and is not an office procedure There is an unsatisfied need for an easy non-invasive and sensitive test to both diagnose the infection and to determine eradication of H pylori infection after treatment available as an office procedure to gastroenterologists

The accuracy of IgG serology, and therefore the usetulness of that approacn in monitoring therapy (Hirschl, 1993) and to confirm H pylori eradication has already been pointed out and shown by other authors (Lerang et al , 1998, Cullen et al 1992 Kosunen et al , 1992) In a recent report an immuno- dominant outer membrane protein of H pylori has been successfully used to asses the early response to eradication therapy in patients on a seroiogical basis (Nishizono et al , 1998)

The identification of unique H pylori proteins/antigens others than cagA, vacA and iceA that can be used for diagnosis of H pylori infection and for monitoring the success of eradication therapy in patients using a Western blot based method is tnerefore highly desirable

United States Patent 5,846,751 is related to a sensitive and specific antigen preparation for the detection of H pylori in biological samples The preparation uses a range of antigens derived from size exclusion chromatography of detergent-solubi zed H pylori cells United States Patent 5,459,041 discloses an antigenic composition for detecting the presence of antibodies specific for H pylori wherein said antigen is a surface structure resolving into bands migrating at 63,000, 57,000 and 31 ,000 dalton bands when electrophoresed on sodium dodecyl sulfate polyacrylamide gel United States Patent 5,859,219 relates to a purified vacuolating toxin from H pylori and methods to use same

SUMMARY OF THE INVENTION

The subject invention has several distinct aspects One aspect is a composition of antigens from H pylori present in the lysate of whole bacterial cell preparations that is capable of detecting the presence or absence of specific antibodies against H pylori with high accuracy and reliability Another aspect is a method for the preparation of such a composition A further aspect is a method for detecting the presence of antibodies resulting from Helicobacter pylori infection in a biological sample which makes use of such a composition In particular the method relates to monitoring the success of eradication treatment of Helicobacter pylori An additional aspect of the invention is a kit for determining the presence of antibodies formed in response to Helicobacter infection in a biological sample, the kit comprising such a composition BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1A and 1 B show the average titers or speciπc antibodies, expressed as percent IOD, with HP1 , HP2, HP3 and HP4 from H pylori strain Hp504 (ATCC#43504) present in sera from 9 patients diagnosed with a H pylori infection achieved in two independent experiments The serum samples from each patient were obtained before 3 months and 5 months after eradication therapy

Figure 2 shows the average titers of specific antibodies expressed as percent IOD, with HP1 , HP2, HP3 and HP4 from H pylori strain Hp08 (clinical isolate) present in sera from 9 patients diagnosed with H pylori infection The serum samples from each patient were obtained before, 3 months and 5 months after eradication therapy

Figure 3 shows the average titers of specific antibodies expressed as percent IOD, with HP1 , HP2, HP3 and HP4 from H pylori strain Hp02 (clinical isolate) present in sera from 9 patients diagnosed with H pylori infection The serum samples from each patient were obtained before, 3 months and 5 months after eradication therapy

Figure 4 summeπzes the data shown in the previous figures It shows the average titers of specific antibodies, expressed as percent IOD, with HP1 HP2, HP3 and HP4 from all three different H pylori strains Hp504, Hp08 and Hp02 present in sera from 9 patients diagnosed with a H pylori infection The serum samples of each patient were obtained before, 3 months and 5 months after eradication therapy

DETAILS

The composition according to the invention comprises at least three Helicobacter pylori derived proteins or their antigenic regions, wherein the proteins are selected from the group of Helicobacter pylori derived proteins which are identified by SDS PAGE to consist of antigens specific to Helicobacter pylori of molecular weights 32 kd, 30 kd 23 kd and 15 kd and wherein the 15 kd antigen consists of two different Helicobacter pylori derived proteins These antigens from H pylori have not been used in this combination in other available tests The antigens were characterized by SDS-PAGE as described herein and microsequencing after immunoblotting

The apparent molecular weights of the antigens (referred to as HP1 , HP2, HP3 and HP4, HP4 consists of HP4a and HP4b) calculated according to the molecular weight standards are as follows HP1 with -32 kd, HP2 with -30 kd, HP3 with -23 kd HP4a with -15 kd and HP4b with -15 kd

The antigens were identified by determining 20-21 N-terminal ammo acids and performing a blast search as follows HP1 was identified to be the response regulator from H pylori (Tomb et al , 1997), HP2 is the 26 kd antigen from H pylori (O'Toole et al 1991 ,Tomb et al , 1997), HP3 is the nonheme iron-containing ferπtin from H pylori (Doig P et al 1992 Frazier B A et al , 1993), HP4a is the thiore- doxin from H pylori (Tomb et al , 1997) and HP4b is a histone-like DNA-bindmg protein from H pylori (Tomb et al , 1997)

SDS PAGE in connection with the invention refers to SDS-Polyacryiamide Gel Electrophoresis SDS PAGE preferably is carried out as described under Materials and Method Before SDS PAGE the antigens preferably are reduced and denatured

In a preferred embodiment of the invention antigens HP1 HP2, HP3 and HP4 are present in the composition according to the invention

Preferentially the antigens in the composition according to the invention are present attached to a solid phase In connection with the invention a solid phase preferably relates to a solid phase suitable for attachment of antigens, such as microtiter plates or membranes such as nitrocellulose and PVDF membranes

In a preferred embodiment of the invention the composition according to the invention can be achieved by preparing a lysate of whole bacterial cell preparations of Helicobacter pylori and subjecting the ly- sate to gel separation After separation the antigens may be transferred onto a solid phase, for example by electrotransfer to membranes In another aspect of the invention the proteins of the composition can be prepared according to recombinant methods This can be achieved by cloning the complete sequence coding for the antιgen(s) or part of it into an appropriate expression vector for an Escherichia coli expression system These systems depend on expression of the protein of interest by induction of a system integrated promoter After expression of the protein in high amounts it can be isolated and purified by affinity chromatography because it was expressed as a fusion protein or because flag has been attached to it The possibilities of isolation and purification are entirely depending on the chosen system In the case that only parts of the sequence of a protein are used for recombinant expression, an antigenicity plot has to be performed to make sure that highly antigenic regions of the protein are not lost thereby loosing the capability of immuno reactivity with the specimen to be tested

In a further embodiment the invention relates to a method for detecting the presence of antibodies resulting from Helicobacter pylori infection in a biological sample comprises the steps of

(a) contacting the sample with a composition according to the invention,

(b) permitting the sample and said composition to form an antigen-antibody complex with respect to any antibody specific for said antigens of the composition contained in the sample,

(c) detecting the presence of any formed antigen-antibody complex denoting the presence of Helicobacter pylori infection Biological sample in connection with the invention preferably relates to human sera For detection of the presence of any formed antigen-antibody complex in step (c) it is preferred to use gold label or enzyme conjugated antibody, in particular an anti-Human IgG antibody The person skilled in the art is familiar what kind of gold label or enzyme conjugated anti-Human IgG antibody can be used in connection with the detection of a said antigen-antibody complex Anti-Human IgG antibodies which are conjugated to horseradish peroxidase may be mentioned by way of example

A further embodiment of the invention is a kit for determining the presence of antibodies formed in response to Helicobacter pylori infection in a biological sample, the kit comprising a composition according to the invention preferably attached to a solid support Optionally the kit may comprise additional components such as a positive control (human serum containing antibodies against H pylori), buffer solutions, suitable gold label antibody or an enzyme conjugated anti-Human IgG antibody and a suitable enzyme substrate In a preferred embodiment of the invention the kit comprises a test strip wherein a composition according to the invention is attached to a nitrocellulose membrane and a suitable gold label antibody is used for detection of the presence of any formed antigen-antibody complex After contacting the test strip with the biological sample the formation of a coloured line will denote the presence of Helicobacter pylori infection The person skilled in the art is familiar with such type of test strip This format of test strip is for example widley used is pregnancy hCG tests

The method for detecting the presence of antibodies resulting from Helicobacter pylori infection is particularly suitable for determination of the eradication of Helicobacter pylori during and after eradication treatment as it allows to detect reductions in the antibody titer during the post eradication period This method comprises the steps of

(a) diagnosis of infection with H pylori,

(b) monitoring antibody titers during eradication treatment,

(c) determination the eradication of the infection after eradication therapy,

wherein at least in steps (b) and (c) the presence or absence of antibodies resulting from H pylori infection is determined by a method according to the invention

Further subjects of the invention will be evident from the description and the patent claims

Materials and Methods

Materials: All materials used were of highest purity grade available Bacterial strains:

H pylori strain ATCC#43504 (Hp504)(Amerιcan Type Culture Collection, Rockville, Maryland) and two clinical isolates, Hp08 and Hp02, were used as the source of H pylori proteins As a control for the specificity of the seroiogical reactivities Campylobacter jejuni strain ATCC#29428 was included into the experiments

Bacteria were grown on blood agar plates (BBL TSA 5% sheep blood, Becton Dickinson, Cockeysville, MD) for 24 hr or in brain heart infusion (BHI) supplemented with 0 25% yeast extract (Difco Laboratories, Detroit, Ml) and 6% horse serum (Gibco BRL, Grand Island, NY) until reaching an OD₆₀₀ of 0 8-1 0 at 37° C in a microaerobic atmosphere (5% 0₂, 10% C0₂, 85% N₂) Bacteria grown in broth culture were collected by spinning for 10 mm at 5000 x g, washed once with phosphate buffered saline (PBS) pH 7 5 and then suspended in 1 ml ice cold deionized H₂0 Cells grown on up to three blood agar plates were harvested directly into 1 ml ice cold deionized H₂0 Lysis of the cells was obtained by three cycles in a French pressure cell with 20,000 psi at 4° C The lysates were always kept on ice or at -20° C

Human sera and antibodies specific for H. pylori proteins:

Human sera were provided by Dr D Vaira (S Orsola Hospital, Bologna, Italy) We tested sera from nine with H pylori infected patients (mean age 62 2, 5 female, 4 male, table 1 ) obtained before, 3 and 5 months after eradication therapy and sera from ten non-infected patients, (mean age 42 6, 5 female, 5 male, table 2) obtained before therapy The H pylori infection and the status of the gastrointestinal damage of all these individuals had been confirmed and examined by several assays (endoscopy, Clo, Colt, Histology, ELISA ¹³C UBT, table 1 and 2) The ELISA used for this purpose is described in Literature (Vaira et al , 1988a, 1988b, 1989, 1991 , 1994a, 1994b, Oderda et al , 1989a, 1989b, 1991 , 1992, Menegatti et al , 1995, 1996, 1998) According to these tests H pylori infection has been eradicated after treatment in all patients

As controls well characterized and speciπc polyclonal antibodies against a synthetic peptide of the urease B subunit from H pylori (αUreB#744, Byk Gulden, Konstanz, Germany), the urease A subunit from H pylori (αUreA#30588, Dr H Mobley, Univ of Maryland, Baltimore, MD) and a commercially available antiserum against the Hsp60 from Synechococcus sp strain PCC 7942, (StressGen Biotechnologies Corp , Victoria, BC, Canada) (αHsp) were used The latter detects specifically HspB of H pylori. H. pylori eradication therapy:

Substance Dose

Amoxicillin 1g2xday

Clarithromycin: 500mg1xday

Omeprazole: 20mg 1x day

Total duration of treatment: 7 days

Table 1 : List of H. pylori infected patients

N=normal; AG=antral gastritis; AE=antral erosions; ED=erosιve duodenitis; SAG=severe antral gastritis; DU=duodenal ulcer, GU=gastrιc ulcer atr=atrophy; IM=intestinal metaplasia; D=dysplasia; MIAG=mild active gastritis; MICG=mild inactive gastritis m=male; f=female, A=antrum, C=corpus, n d.=not done; u.e =under evaluation

Table 2: List of non-infected patients

N=πormal, AG=antral gastritis, AE=antral erosions, ED=erosιve duodenitis, SAG=severe antral gastritis, DU=duodenal ulcer, GU=gastrιc ulcer atr=atrophy, IM=mtestιnal metaplasia, D=dysplasιa, MIAG=mιld active gastritis, MICG=mιld inactive gastritis m=male, f=female, A=antαιm, C=corpus, n d =not done, u e =ιιnder evaluation

SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Western-blot Analysis:

The protein content of the bacterial lysates was determined by the method accorαing to Lowry (Lowry et al., 1951 ) with bovine serum albumin as a standard. The lysates were dried in a speedVac concentrator. Afterwards the pellets were suspended in gel sample buffer (4% SDS, 12% glycerin, 4% β-mercaptoethanol, 0.01 % Serva Blue G250 in 50 mM Tris/HCI pH 6.8) and boiled for 10 min. H. pylon and C.jejuni lysates were separated on 1.0 or 1.5 mm 7.5-16.5% SDS-tricine gradient gels (Schaegger and v. Jagow, 1987). Therefore a 7.5% and a 16.5% acrylamide solution for the separating gel and a 4% acrylamide solution for the stacking gel were prepared according to the scheme presented in table 3. Using a gradient-mixer the gel was poured slowly, but with continuous flow between the glass plates of the gel-sandwich. The separating gel was overlaid with deonized H₂0 and allowed to polymerize for 1 hr. Afterwards the water was removed, APS and TEMED was added to the stacking gel solution that was poured on top of the separating gel. A comb with the appropriate number of sample pockets was inserted and removed after polymerisation for 1 hr The gel-sandwich was placed in an electrophoresis chamber half filled with bottom running buffer (0.2 M Tπs-HCI, pH 8.9). The upper compartment was filled with top running buffer (0.1 M Tris base, 0.1 M Tπcine, pH 8.25). The protein samples (preparation see above) were loaded into the pockets and electrophoresis was performed with 15 mA constant current over night. Low or broad range prestained molecular mass standards (BioRad, Hercules, CA, USA) were separated in parallel on each gel. The protein pattern after electrophoresis was determined by Silver staining (Heukeshoven, 1985) or Coomassie blue staining. For calculation of molecular weights RFPL scan software (Version 2.01 , Scanalytics) was used.

Table 3: Composition of the Tricine gradient gel:

Stacking gel Separating gel 4% 7.5% 16.5%

1.0 mm/1.5 mm 1.0 mm 1.5 mm 1.0 mm 1.5 mm

Acrylamide solution 3.05 mT ^"5Mml

Acrylamide solution II 6.66 ml 10.0 ml

Gel buffer 3 ml 6.66 ml 10.00 ml 6.66 ml 10.0 ml Glycerol ^{" ~}l T33 g ^" ^~~Υry-

8.4 ml ^""10 3^~ ml 15.0 ml 5.3 ml 10.0 ml

10% APSln H₂O 100 μ[ 40 ^"uT 50 μl ^" 40 μl ~5δ^~μT TEMED ιδ^"T-r ^"3.757.1^" 5 μl ^"375 ul ^~~5^~μT^"

Acrylamide solution I: 48% (w/v) acrylamide, 1.5 (w/v) bisacrylamide (BioRad, Hercules, CA, USA) in H2O Acrylamide solution II: 46.5% (w/v) acrylamide, 3 0% (v v) N,N'-methylene-bιs-acrylamιde (BioRad, Hercules, CA, USA) in H₂0 Gel buffer: 3M Tπs-HCI, pH 8.45: 0.3% SDS APS=ammonιumpersulfate TEMED=N, N N , N tetramethylendiamin, SDS=sodιumdodecylsulphate, * freshly prepared,

** added before the gel is poured

Subsequently the proteins were transferred to nitrocellulose or PVDF membranes (BioRad, Hercules, CA, USA) using 192 mM glycιne/25 mM Tπs/20% methanol as the transfer buffer After the electro- transfer onto the membrane, proteins were visualized with Ponceau S solution (0 2% Ponceau S in 3% trichloπcacid) to be able to cut the membrane into individual strips for incubation with the different human sera and antibodies Unspecific binding sites were blocked by incubating for 1 hr in 5% nonfat milk in PBS/0 1% Tween20 Incubation with the human sera (1 500), αUreB#744 (1 1 85,000 and 1 150,000), αUreA#30588 (1 400,000) and αHsp60 (1 150,000) in 5% nonfat milk in PBS/0 1% Tween20 was performed over night at 4° C After 4x washing with PBS/0 1% Tween20 the membranes were incubated with a peroxidase labeled anti human/rabbit IgG antibody (1 20,000, American Qualex, San Clemente, CA) After washing 4x with PBS/0 1 % Tween20, the reaction was visualized by using the ECL™-system or the ECL^TWPlus-system (Amersham Life Science Inc , Arlington Heights, IL,) for nitrocellulose membranes or PVDF membranes respectively according to the manufacturers manual The membranes were exposed to X-ray films (Fuji Medical Systems USA Inc , Stamford, CT,) for time periods between 30 sec and 30 mm and developed with a film processor (Konica SRX-101A, Konica Corporation, Japan)

Quantitative Western-blot Analysis

The reactivities of the antibodies in the sera with the antigens of interest were evaluated by imaging the autoradiographs with a Radioanalytic Imaging System (Ambis QuantProbe™ Software, version 4 31 ) and using RFPLScan⁰ (version 2 01 , Scanalytics) for determining the integrated optical density (IOD) of the protein bands of interested using a Gaussian calculation method provided with the program The IOD of the particular protein band before treatment was set at 100% The change of the reactivity, reflecting the titer of soecific antibodies in the sera, during the post treatment period was calculated compared to the 100% level

Protein Sequencing.

For microsequencing proteins were transferred to PVDF membrane (0 2 μm pore size) and visualized by Coomassie blue staining (0 1 % Coomassie Brilliant Blue R250 in 45% methanol, 10% acidic acid) and destainmg (45% methanol, 10% acidic acid) of the membrane The protein bands of interest were cut out of the membrane and sequenced with a gas phase sequencer at the UCLA Protein Microsequencing Facility using the Applied Biosystems 475 A system composed of a 470 A sequencer, a 120 A phenyl thiohydantom analyzer and a 900 A data module 20-21 sequential ammo acids of each antigen could be determined and this sequence was used afterwards to perform a homology search in the database to identify the protein (table 5)

IDENTIFICATION OF THE H. PYLORI ANTIGENS.

After microsequencing 20-21 ammo acids of the three antigens of H. pylori a blast search in different data bases was performed All proteins could be identified with 85-100% identity with H. pylori proteins. Protein HP4 was a mixture of two components that both could be identified Table 5 shows the results of that search

in an 16 - 21 aa overlap

Table 5: H pylori antigens identified by microsequencing

Examples

SDS-PAGE AND IMMUNOBLOT ANALYSIS

The separation of whole cell lysates of three H pylori strains and one C jejuni strain on tπcine gradient gels was performed to show the protein patterns by Coomassie blue or silverstaining of the gels.

The following immunoblot analysis probing the membranes with the ten human sera from non-infected individuals showed that there were only a few proteins of H pylori reacting with these sera All the proteins reacting with the negative sera were mainly found in the higher molecular weight range and are probably proteins being homologous to proteins from other bacterial species and therefore causing cross reactivities with antibodies generated during infection with H pylori A similar result was seen with C. jejuni proteins supporting that these reactions are supposed to be considered as non specific

Probing the membranes with the nine human sera from H pylori infected patients obtained before eradication treatment showed again some cross reactivities with C jejuni proteins being either in the high molecular weight range or definitely different from proteins recognized in the tested H. pylori strains by these sera

Using specific sera against both subunits of urease from H pylori, urease A and urease B, and one of the heat shock proteins, HspB, showed that the antigens according the invention are different from these H. pylori proteins

All of the five antigens that are subject of this invention were clearly recognized by eight of the investigated sera from H pylori infected patients in all three tested H pylori strains (Hp504, Hp08, Hp02) One of the sera (HS #012, table 1 ) did not react with one of the antigens (HP4)

QUANTIFICATION OF THE REACTIVITIES WITH THE H PYLORI ANTIGENS

The five immuno-reactive antigens described in this invention were visualized by using the ECL™ detection system The reactivities of the five antigens of interest were evaluated by imaging the autoradiographs with a Radioanalytic Imaging System and using specialized software (RFPLScan^® version 2 01 ) for determining the integrated optical density (IOD) of each single antigen at the different time points (before treatment, 3 months and 5 months after treatment) from the three different H pylori strains The IOD of each antigen before eradication therapy was set as 100% on each immunoblot that was evaluated The changes in the reactivities of the sera with these antigens could also be looked at showing the changes in titers of specific serum IgG antibodies against HP1 , HP2, HP3, HP4a and HP4b The following figures show the serial changes in titers of serum IgG expressed in % of integrated optical density that is left 3 months and 5 months after eradication treatment in comparison to the amount before treatment The nine sera from with H pylori infected and treated patients were tested on whole cell lysate separations of H pylori strain ATCC#43504 (Hp504), Hp08 and Hp02 (clinical isolates) The results are shown for each tested bacterial strain separately to demonstrate that the accuracy of the test is independent of the source of the antigen

Figure 1A and B shows the data for the sera being tested on Hp504 antigen preparations in two independent experiments Differences between the two data sets obtained with antigens from strain Hp504 show that the out come could be depending on the antigen preparation itself and/or the performance of the Western-blot analysis This points out that those parameters need to be standardized when applying this approach for the development of a commercial available kit Figure 2 and 3 show the results for the sera being tested on Hp08 and Hp02 antigen preparations respectively

In all cases there was a significant decrease detected in the reactivities of the nine sera with the five antigens 3 months after therapy that increased further 5 months after treatment The detected decrease in titers of H pylori specific antibodies shows eradication of the infection what is supported by the results of the other tests that were performed on the patients (table 1 )

Figure 4 summarizes the data of the previous experiments and shows the average titers of specific antibodies against HP1 , HP2, HP3, HP4a and HP4b from all three different H pylori strains in the patient's sera As shown here the average titer of antι-HP1 antibodies at 3 months after eradication treatment decreased to 38 5% (= 61 5% reduction) and to 18 03% (= 81 97% reduction) at 5 months after end of treatment respectively The average titer of antι-HP2 antibodies found at 3 months is down to 49 73% (= 50 27% reduction) and to 31 08% (= 68 92% reduction) at 5 months after therapy respectively For antι-HP3 antibodies there is a decrease in the average titer to 36 29% (=63 71 % reduction) at 3 months and a further decrease to 28 87% (= 79 13% reduction) Finally the average titer of anti- HP4 antibodies at 3 months is down to 31 61 % (= 68 39% reduction) and to 14 12% (= 85 88% reduction) at 5 months after therapy

ACCURACY OF A COMBINATION OF HP1 , HP2, HP3 AND HP4 IN A TEST SET

A combination of the described five antigens from H pylori on a Western-blot test strip applying the correct cut-off setting for each of the antigens provides a sensitive test for the diagnosis of an infection with H pylori, for monitoring the early response to eradication therapy and for determining the eradication of the infection It is preferred to provide a test strip that contains all of the investigated antigens because the study showed that one or the other of the antigens is recognized differently by the different sera Providing the combination and not a mixture of HP1 , HP2, HP3 and HP4 on a strip also decreases drop-outs if a serum fails to react with one of the antigens Table 4 shows the cut-off setting for each of the antigens Antigen cut-off at 3 months post therapy cut-off at 5 months post therapy

HP1 58% titer decrease 78% titer decrease

HP2 44% titer decrease 63% titer decrease

HP3 58% titer decrease 74% titer decrease

HP4 62% titer decrease 84% decrease

Table 4: Cut-off setting for H pylori antigens used in test kit

References cited

Cullen D J E et al The Lancet, Nov 7, 1992, 340 1162-1163

Doig P , J Bacteriology, 174(8), 2539-2547, 1992

Frazier B A J Bacteriology, 175(4), 966-972, 1993

Heukeshoven J and Dernick R , 1985, Electrophoresis, 6 103-1 12

Hirschl A M et al , The J of Infect Diseases, 1993, 168 763-766

Kosunen U T, et al The Lancet, April 11 ,339 1992, 893-895

Lerang F et al , Scand J Gastroenterol , 33(7) 710-715, 1998,

Lowry et al , J Biol Chem , 193 265-275, 1951

Marshall B J et al Microbiol Lett , 25 517-525

Menegatti M et al , Clinical Science, 91 219-223, 1996

Menegatti M et al , Digestive Desease and Sciences, 43 2542-48, 199

Menegatti M et al , The American Journal of Gastroenterology, 90 1278-1281 , 1995

Nishzono A et al , Clin and Diagn Lab Immunology, 1998, 5 56-861

O'Toole et al , J Bacteπol , 173(2) 505-513, 1991

Oderda G et al , Digestive Diseases and Sciences, 36 572-576, 1991

Oderda G et al , Gut 33 1328-30, 1992

Oderda G et al , Gut, 30 912-916, 1989b

Oderda G et al , Lancet, 690-692, 1989a

Schaegger H and v Jagow G , Anal Biochem , 1987, 166 368-379

Tomb J-F et al , Nature, 388, 539-5471997

Vaira D et al , British Medical Journal, 297 397, 1988b

Vaira D et al , Digestive Disease and Sciences, 40 1622-1626, 1994b

Vaira D et al , Gastroenterology, 97 1069-1070, 1989

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Claims

1 Composition comprising at least three Helicobacter pylori derived proteins, wherein the proteins are selected from the group of Helicobacter pylori derived proteins which are characterized by SDS PAGE to consist of antigens specific to Helicobacter pylori of molecular weights 32 kd, 30 kd, 23 kd, and 15 kd and wherein the 15 kd antigen consists of two different Helicobacter pylori derived proteins

2 Composition according to claim 1 , wherein proteins were isolated and purified

3 Composition according to claim 1 wherein all said Helicobacter pylon derived proteins are present

4 Composition according to claim 1 , which is a combination and not a mixture of said Helicobacter pylori derived proteins

5 Composition according to claim 1 wherein the 23 kd antigen is a nonheme iron-containing ferntin from Helicobacter pylori, one of the 15 kd antigens is a thioredoxm from Helicobacter pylori and the other 15 kd antigen is a histone-like DNA-bindmg protein from Helicobacter pylori

6 Composition according to claim 1 , wherein the antigen specific to Helicobacter pylori of molecular weight 32 kd represents a response regulator from Helicobacter pylori

7 Composition according to claim 1 wherein two proteins forming the 15 kd antigen are present

8. Composition according to claim 1 wherein the 30 kd, the 23 kd and at least one of the 15 kd antigens is present

9 Composition according to claim 1 , wherein the antigens are present attached to a suitable solid phase

10. Composition according to claim 9, wherein the solid phase is a microtiter plate

11. Composition according to claim 1 wherein the antigens are present attached to membranes

12 Composition according to claim 11 wherein the membranes are nitrocellulose or PVDF membranes.

13. Composition according to claim 9, wherein a combination and not a mixture of all said antigens is provided on a test strip

14. Method for the preparation of a composition according to claim 12 by preparing a lysate of whole bacterial cell preparations of Helicobacter pylori, subjecting the lysate to gel separation and transferring the proteins to the membranes

15. Method according to claim 14 wherein the Helicobacter pylori is Helicobacter pylori strain ATCC#43504

16. Method according to claim 14 wherein the gel separation is carried out in 7.5-16.5% SDS-tricine gradient gels.

17. A method for detecting the presence of antibodies resulting from Helicobacter pylori infection in a biological sample, the method comprising

(a) contacting the sample with a composition according to claim 1 ;

(b) permitting the sample and said composition to form an antigen-antibody complex with respect to any antibody specific for said antigens of the composition contained in the sample;

(c) detecting the presence of any formed antigen-antibody complex denoting the presence of Helicobacter pylori infection

18. A method according to claim 17 wherein in step (c) an enzyme-conjugated anti-Human IgG antibody is used for detection of any formed antigen-antibody complex.

19. A method according to claim 18 wherein the anti-Human IgG antibody is conjugated to horseradish peroxidase.

20. A method according to claim 17 wherein in step (c) gold labled antibody is used for detection of any formed antigen-antibody complex

21. A method according to claim 17 wherein the biological sample is human serum.

22. A kit for determining the presence of antibodies formed in response to Helicobacter pylori infection in a biological sample, the kit comprising a composition according to claim 1

23. A kit according to claim 22 wherein a combination and not a mixture of all said antigens is provided on a test strip.

24. A kit according to claim 22 additionally comprising a positive control, an enzyme-conjugated anti- Human IgG antibody optionally a suitable enzyme substrate and buffer solution. A kit according to claim 22 comprising a test strip, wherein a composition according to claim 1 is attached to a nitrocellulose membrane and a gold labled antibody is used for detection

In a method for determination the eradication of Helicobacter pylori the improvement consisting in the detection of the presence or absence of antibodies resulting from Helicobacter infection by a method according to claim 17, before, during and after eradication treatment

Use of a combination of antigens from Helicobacter pylori for detecting the presence or absence of antibodies resulting from Helicobacter pylori infection wherein the antigens are selected from the group of Helicobacter pylori derived proteins which are characterized by SDS PAGE to consist of antigens specific to Helicobacter pylori of molecular weights 32 kd, 30 kd, 23 kd, and 15 kd and wherein the 15 kd antigen consists of two different Helicobacter pylori derived proteins