WO1996012965A1

WO1996012965A1 - Helicobacter pylori diagnostic methods and kits

Info

Publication number: WO1996012965A1
Application number: PCT/IB1995/001028
Authority: WO
Inventors: Lily Chan; Randolph Moeckli; Daria Foong Yun Chin
Original assignee: Genelabs Diagnostics Pte Ltd.
Priority date: 1994-10-20
Filing date: 1995-10-19
Publication date: 1996-05-02
Also published as: AU3814395A

Abstract

The invention describes an assay for detecting Helicobacter pylori infection. The assay is intended for the detection of infection with Helicobacter pylori and for the monitoring of the status of infection following treatment. The assay involves an immunoblot for biological fluid samples and includes a kit in which Helicobacter pylori antigen is immobilized on a membrane support. Also provided is a method for diagnosing disease associated with Helicobacter pylori infection.

Description

HELICOBACTER PYLORI DIAGNOSTIC METHODS AND KITS

I. Field of the Invention

T e present invention relates to methods and kits for rapid in vitro detection of Helicobacter pylori infection in mammals. More particularly, the present invention relates to methods of detecting antibodies which are associated with Helicobacter pylori infection and related disease.

II. Background of the Invention Helicobacter pylori is a curved Gram-negative bacterium that is commonly present in the human stomach; once acquired, this organism persists for decades (Blaser, M.J.,

(1990) J. Infect. Pis. 161 :626-633). Multiple lines of evidence now indicate that H. pylori infection nearly universally results in chronic gastritis (Dixon, M.F. (1991) J. Gastroenterol. and Hepatol. 6: 125-130). Although most persons with H. pylori-m^' άuceά gastritis remain asymptomatic, this condition is a significant risk factor for the development of both peptide ulceration and gastric adenocarcinoma (Peterson, W.L.

(1991) N. Engl. J. Med. 324: 1043-1048, and Nomura, A., Perez-Perez, G.I., and Blaser, M.J., (1991 ) N. Enel. J. Med. 325:1 132-6).

During the past 10 years, since the successful culture of H. pylori many diagnostic tests have been used to determine if patients are infected with this bacteria. The "gold standard" is direct identification of the organism by culture and/or histology. Although accuracy rates have been reported to be between 77 and 94%. this test has the disadvantage of being particularly invasive requiring endoscopic biopsy of gut material. Another commonly used test is the Urease Assay. The Urease Assay, however, also requires endoscopic biopsy.

A non-invasive test is the "Carbon-Labeled Urea Breath Test". Although this test is non-invasive it can be impractical because it utilizes urea labeled with ^l3C and ^l4C, and reading the test requires either a mass spectre-photometer or a gamma counter, respectively. Serological testing presents an alternative to the invasive tests and urea breath test described above. Individuals infected with H. pylori will mount a significant humoral response to H. pylori proteins (Jones. D.M., et al., J Med Micro (1986) 22:57-62). Experimental infection in humans has revealed measurable antibody within four weeks after ingestion of the bacteria (Morris, A.J., et al., Ann Intern Med (1991) 1 14:662-3). IgA antibodies are found locally in gastric fluid, while IgG antibodies are the most common circulating antibody. The rapid development of antibodies post infection with H. pylori provides a useful tool for the development of non-invasive diagnostic tests. Serology can be useful to determine the presence of the bacteria in gastric related diseases as well as to monitor the success of treatment. A measurable drop in titer in

ELISAs, for example, has been a useful measure of the success of antibacterial treatment (Kosunen, T.U., et al.. Lancet (1992) 339:893-5).

The recent NIΗ Consensus statement on H. pylori and Peptic Ulcer disease reached the following conclusions: There is an immediate need for the development of non-invasive tests for diagnosing H. pylori infection; these tests should be able to monitor the success of treatment; and all individuals with Peptic Ulcers should be diagnosed and treated with an antibacterial regimen.

III. Summary of the Invention It would be use ul, therefore, to provide a highly specific and sensitive non- invasive diagnostic test for Helicobacter pylori infection and related disease. Such test should be simple, rapid and relatively inexpensive. It is one general object of the invention to provide a method and kit for diagnosing Helicobacter pylori infection which is specific and sensitive as well as simple, rapid and relatively inexpensive. Another object of the invention is to provide a non-invasive method for diagnosing disease associated with Helicobacter pylori infection.

Specific embodiments include a kit for detecting Helicobacter pylori infection, produced by a process of providing a Helicobacter pylori antigen, separating the antigen components, and transferring said antigen components to a solid support. Another embodiment of the invention is a method for detecting infection with

Helicobacter pylori. The method includes, providing a solid support having Helicobacter pylori proteins bound thereto, reacting biological fluid with the solid support, examining the solid support for the presence of bound antibody and positively identifying infection when bound antibody is found at any two positions on the solid support corresponding to antigens having molecular weights of 19.5kDa, 26.5kDa. and 30kDa, or alternatively, when bound antibody is found at any position on the solid support corresponding to an antigen having a molecular weight of 35kDa, 89kDa, 1 16kDa. or 180kDa. A further embodiment of the invention includes a method for diagnosing disease associated with Helicobacter pylori infection; e.g., gastritis, peptic ulcers, and gastric carcinoma. The method includes providing a solid support having electrophoretically separated Helicobacter pylori antigen bound thereto, reacting biological fluid from the patient with the solid support, examining the solid support for the presence of bound antibody, and comparing the profile of bound antibody to consensus profiles obtained from other patients with confirmed disease conditions.

This and other objects and features of the invention will become more fully understood when the following detailed description of the invention is read in conjunction with the accompanying figures.

IV. Brief Description of the Figures

Fig. 1 is a representative Western Blot strip containing separated H. pylori antigen components reacted with a seropositive serum. Molecular weights (in kD) of the various major reactive antigen components are marked, and the putative identification of the major antigen components is provided. Band labelled "Control" is the serum addition control band.

Fig. 2 shows Reactivity with Clinical Samples - a Western Blot strip containing separated H. pylori antigen components reacted with four serum samples from patients which were histology, culture, and ELISA positive (Lanes 1-4) and four serum samples from patients which were histology, culture, and ELISA negative (Lanes 5-8).

V. Detailed Description of the Invention A. Definitions The terms defined below have the following meaning herein:

1. The term "Antigen" is used in its broadest sense and includes Helicobacter pylori whole cells or homogeneous, near homogeneous or heterogeneous extracts from H. pylori and which is capable of binding to specific antibody in a biological fluid. Antigen components contemplated by the present invention include protein, polysaccharide or lipid or any combination thereof. Preferably, the antigen is protein, lipopolysaccharide or cell extract of H. pylori prepared by, for example, sonication, pressure disintegration, detergent extraction or fractionation.

2. A "biological fluid" is any fluid derived from the body of a mammal. Biological fluids may be, for example, blood, serum, urine, mucous, gastric secretions or saliva.

3. A "reporter molecule" means a molecule which, by its chemical nature, provides an analytically identifiable signal which allows the detection of antigen- bound antibody.

4. The term "Helicobacter pylori related disease" means any disease caused in whole or in part by infection with H. pylori. Such disease may be, for example, gastritis, peptic ulceration, and gastric adenocarcinoma.

5. An individual's "antibody profile" is the pattern created by antibodies contained in the individual's bodily fluid that are reactive with separated H. pylori antigen components. Thus, an individual's antibody profile may be represented, for example, by the pattern of antigen component bands which are detected in a western blot assay.

6. A "consensus profile" is an artificially created antibody response profile based a compilation of the most common antibody responses within a particular H. pylori associated disease.

B. Immunoblot Preparation 1. Antigen

Helicobacter pylori, may be obtained from the American Type Culture Collection (ATCC, Rockville, Md.). The preferred embodiment contemplates the use of strain designation NCTC11916. This strain is an ulcer producing strain which contains all the proteins associated with pathogenesis and immunologically important antigen components, including CagA, vacuolating protein, and the various Urease subunits. Other strains which contain these proteins are known in the art and can be utilized as well. The antigen preparation was obtained as follows and as detailed in

Example 1. Briefly, bacteria were cultured in broth base medium to a suitable density. These were harvested by centrifugation and washed several times in PBS. The pellet was weighed, gently sonicated and resuspended in PBS to a concentration of 100 mg/ml of wet weight pellet. At this point the sonicate may be stored in small aliquots at -70 degrees C for future use. Alternatively, an H. pylori antigen preparation is available commercially from Microbix Biosystems (Ontario, Canada).

2. Separation of Antigen Components

Antigen components are separated by size according to the present invention. Such size fractionation is preferentially accomplished electrophoretically but may be accomplished by other methods known to those skilled in the art; e.g., chromatography.

H. pylori antigenic components are separated as follows, and detailed in Example 1. The H. pylori antigen preparation is loaded on a poly acrylam ide gel which may or may not contain individual wells. Preferably about .66-10 ug antigen preparation is loaded on the gel per centimeter of well width. More preferably about 5 ug cm well width is loaded. Prior to loading the antigen preparation is boiled in order to denature the antigen components, preferably for about 5 minutes. The poly aery lam ide gel can be any concentration determined to give adequate resolution of the desired antigen components described infra. The polyacrylamide gel consists of two parts; a resolving gel, preferably between 10 and 13% acrylamide and a stacking gel, preferably between 4 and 8% acrylamide. In the most preferable embodiment the polyacrylamide gel has an 11.5% resolving section and a 6% stacking section. Electrophoresis is conducted until the antigen components have separated sufficiently to discriminate between them. It is preferable to start electrophoresis with a relatively low current until the pyronin Y had migrated into the resolving gel at which point the current is increased until the Pyronin Y has migrated to between about 9 and 12 cm into the resolving gel. Following electrophoresis the gel is removed from the glass plates and the upper gel and the bottom portion of the resolving gel from the tracking dye downward is cut off. Separation conducted under the preferred conditions described above provides optimum separation of particularly useful antigen components as will be seen below.

Molecular weights of antigen components useful in the present invention are of necessity approximate figures, because of the limitations of current molecular weight determination procedures. In the present invention, molecular weight markers can be run adjacent to the H. pylori antigen. After electrophoresis, the position of the molecular weight markers may be determined by techniques known in the art for example, coomassie blue staining the polyacrylamide gel or ponceau S stain after transfer to the solid support.

3. Transfer of Antigen Components to Solid Support The antigen preparation will for convenience and preference be bound to a solid support. Suitable solid supports include a nitrocellulose membrane, glass or a polymer. The most commonly used polymers for this purpose are cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene, but the invention is not limited to them. The solid support is preferentially in the form of strips, but may also be tubes, beads, discs, or microplates, or any other surface suitable for conducting an immunoassay.

Antigen components of H. pylori useful in this invention may be either covalently or non-covalently ("passively") bound to the solid surface. Suitable binding processes are well known in the art and generally consist of crosslinking, covalently binding or physically adsorbing the antigen to the solid support.

As detailed in Example 1, a piece of nitrocellulose membrane which has been presoaked in a Tris-based buffer, is layered onto a piece of wet blotting paper, and the trimmed gel is laid onto the nitrocellulose paper. Another piece of wet blotting paper is laid over the trimmed gel and this sandwich is placed into a transfer tank and transferred by applying a voltage. These techniques are known to those of skill in the art. The membrane is removed from the transfer apparatus after the transfer process and placed in IX PBS until optionally slotting on of additional proteins such as anti-human IgG, anti-human IgA, or recombinantly or synthetically produced peptide Helicobacter pylori antigens. Such additional proteins are useful, for example, as positive or negative controls suitable for the type of biological fluid being tested. H. pylori peptide antigens which encompass specific eptiopes can be useful as positive controls and for confirming infection. Slotting techniques are well known to those of skill in the art.

In the present invention, commercially prepared anti-human IgG is slotted near the bottom of the membrane, which is the area of the low molecular weight proteins. The anti-human IgG will act as a positive control; i.e., reactive with both infected and uninfected samples. The membrane is removed from the slotting apparatus and blocked, preferably in PBS buffer containing about 5% non-fat dry milk at room temperature on a rocker platform. The nitrocellulose is rinsed in PBS-Tween buffer and the sheets are then air dried on paper towels, and left in a 37 degree C incubator overnight for drying. Sheets are cut into convenient sized strips, preferably about 3mm wide and kept in dry containers until used in an assay.

C. Immunoassay Method In a related aspect, the invention is directed to a method of diagnosing individuals with Helicobacter pylori infection wherein a solid support with separated antigen components bound thereto is used to examine an individual's biological fluid for the presence of H. /> /øri-specific antibodies. Biological fluid to be tested for H. pylori-specific antibodies are contacted with the solid support described above. The formation of antigen/antibody complex is then detected by conventional techniques, generally by reaction with a reporter-labelled secondary antibody which is specific for the type of biological fluid tested.

The most commonly used reporter molecules in this assay are either enzymes, fluorophores or radionuclide containing molecules (i.e., radioisotopes). In the case of an enzyme immunoassay, an enzyme is conjugated to the secondary antibody, generally by means of glutaraldehyde or periodate. As will be readily recognized, however, a wide variety of different conjugation techniques exist, which are readily available to those skilled in the art. Commonly used enzymes include horseradish peroxidase, glucose oxidase, Beta-galactosidase and alkaline phosphatase, among others. The substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable color change. 1. Human Serum Samples

Fifty-one (51) human serum samples which were positive for //, pylori by histology, culture, and ELISA (GAP Test, BioRad, USA), were collected. Patient serum was considered positive if the patient was culture, histology, and ELISA positive, with or without an ulcer. One hundred and ten (110) human serum samples which were negative for H. pylori were also collected. Patient serum was considered negative if the patient was culture, histology, and ELISA negative and did not have an ulcer. 2. Assay

As described below, and in Example 2, the serum samples described above were reacted with the solid support prepared above by western blot assay. Briefly, the strips with electrophoretically separated H. pylori antigenic components bound thereto were placed into reaction trays, one strip per well. The strips were pre- wetted in wash buffer (Tris buffer containing "Tween 20™" detergent). After 10 minutes, the buffer was aspirated, and blotting buffer, containing Tris, inactivated goat serum, and 5% non-fat dry milk, was added. Human serum samples were added to individual wells to a final dilution of 1/100 and incubated for one hour at room temperature. The fluid was removed by aspiration and the strips were washed 3 times with wash buffer for 5 minutes per wash, while on the rocking platform. Anti-human IgG alkaline phosphatase conjugate diluted with blotting buffer at an appropriate dilution (1/1000) was added to the strips and incubated for one hour at room temperature. The strips were washed again 3 times as described previously. Helicobacter pylori antigen bands which were reactive with patient sera were visualized after the addition of a solution of nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate. The presence of a purplish-blue band indicates the presence of specific antibodies in the test fluid to that antigen on the strip. The enzymatic reaction is stopped by rinsing the strips in distilled water. The results are summarized in Table 1, below.

Table 1 Helicobacter Western Blot Results

Number of Serum Samples Reactive with Specific Antigen Components

(Molecular Weights in kDa)

Serum Type Positives

19.5 26.5 30 35 89 116 180

Positive 36 46 43 24 14 31 4 49/51

Negative 4 12 4 1 1 2 0 6/110

It can be seen from Table 1 that antigen components which typically react have approximate molecular weights of 116kDa (possibly the CagA protein), 89kDa (possibly the Vacuolating protein), 60kDa (possibly the ureB protein or the hsp60 heat shock protein), 35kDa (unknown), 30kDa (possibly the ureH protein), 26.5kDa (possibly the ureA protein), and 19.5kDa (possibly the ferritin like protein). Figure 1 is a Western Blot strip which shows a reactivity profile including all of the above mentioned antigen components.

From the results obtained, it is evident that serum taken from individuals with Helicobacter pylori infections does not always react with each antigen component while negative serum samples can show considerable reactivity to some antigen components. Thus, it is not useful to simply characterize serum as reactive or non-reactive for purposes of predicting whether any particular sample is from an infected or an uninfected individual. It has been unexpectedly discovered that Helicobacter pylori infection can be diagnosed with sensitivity and specificity by selecting antigen specific criteria for making a positive diagnosis. According to the present invention a serum sample is positive for H. pylori infection if it is reactive with any two antigen components corresponding to molecular weights of 19.5kDa, 26.5kDa, or 30kDa, or alternatively, if it is reactive with any one antigen component corresponding to a molecular weight of 35kDa, 89kDa, 116kDa, or 180kDa. When this criteria is applied to the data in Table 1, it can be seen from the column labelled "Positives" that the test criteria correctly identifies 49/51 positive samples as positive and 104/110 negative samples as negative. Sensitivity of the assay may be defined as a fraction equal to the number of positive serum samples which were actually western blot positive divided by the number of patients which were expected to be positive. Likewise, specificity of the assay may be defined as a fraction equal to the number of serum samples which were accurately diagnosed divided by the total number of samples tested. Thus, by these criteria, the above described test is 96% sensitive (49/51) and 95% specific (153/161) for Helicobacter pylori infection.

It will be appreciated by those skilled in the art that as more data is received, the antigen specific criteria for a positive diagnosis may be altered; e.g., additional antigen components may be added to the assay, currently evaluated antigen components may be deleted from the assay, and different combinations may be required for a positive test result.

C. Diagnosis of H. pylori Associated Disease

In another related aspect, the invention is directed to a method of diagnosing a Helicobacter pylori associated disease by examining that individual's biological fluids for antibodies to Helicobacter pylori antigen components and comparing the profile of antibody reactivity to a consensus profile.

As described above, individuals infected with H. pylori may have different clinical outcomes ranging from gastritis to peptic ulcers and gastric cancer. It is as yet unclear why a particular individual's only clinical outcome is gastritis while another individual will develop more serious disease. Without being bound to a particular theory, these clinical outcomes may represent progressive stages of disease, certain individuals may be predisposed to peptic ulcers and gastric carcinoma, and/or environmental factors may influence disease progression. It is further possible that predisposition to a clinical outcome may be related to immunologic factors.

Results obtained with the solid phase assay, described above and shown in Figure 2, indicate that there is a heterogeneous antibody response to infection with H. pylori; i.e., different infected individuals generate antibodies to different antigen components. It has been discovered that while the infected population produces a heterogeneous antibody response, subpopulations have homogeneous antibody responses. Furthermore, these subpopulations of reactivity profiles may be linked to clinical outcome. For example, individuals who suffer from H. pylori induced gastritis are likely to show a similar antibody responses which would be different than an individual suffering from H. pylori induced gastric adenocarcinoma.

A library of antibody response profiles is produced, according to the invention, by first diagnosing individuals for H. pylori related disease with standard methods known in the art (e.g., culture, histology, and ELISA) and subsequently obtaining antibody response profiles from them. The library of antibody response profiles is then used to determine the highest correlation between disease and antibody response, thereby forming a consensus profile for each disease.

Diagnosis of a test individual is then made by testing the individual's biological fluid in order to obtain an antibody response profile and then comparing the proflle to the consensus profiles for each disease. If criteria for a match is met for any one of the diseases, a positive diagnosis is made.

The following examples, which illustrate various methods and compositions in the invention, are intended to illustrate, but not limit the scope of the invention.

Materials

Helicobacter pylori strain NCTC 11916 is obtained from the American Type Culture Collection (Rockville, MD).

All reagents and chemicals can be obtained from Sigma Chemical Co. (St Louis, MO). Culture reagents may be obtained from Difco (Detroit, MI).

Anti-human IgG can be obtained from the Berkeley Antibody Co. (Berkeley, CA). The "Mighty Small™" transfer apparatus may be obtained from Hoeffer Scientific Instruments (San Francisco, CA). EXAMPLE 1 Preparation of Helicobacter pylori Antigen A. Antigen Preparation

The Helicobacter pylori strain (NCTC 11916) obtained from the ATCC is grown in broth-based media to a suitable density. Specific ingredients are listed in Table 2 below.

Table 2 Helicobacter pylori Culture Media Ingredients

Brain Heart Infusion Broth 500 ml

Yeast Extract 2 grams

Horse Serum 50 ml

The bacteria are harvested by centrifugation and washed several times in Phosphate Buffered Saline (PBS). The pellet is weighed, gently sonicated and resuspended in PBS to a concentration of 100 mg/ml of wet pellet.

B. SDS-PAGE

The protein concentration of the antigen is determined by the Bradford protein assay. Approximately 80 ug of the antigen preparation is mixed in a volume of 800 ul with sample buffer containing glycerol, 2-mercaptoethanol, and pyronin Y. This mixture is boiled for 5 minutes and then layered in the well of a polyacrylamide gel having a 6% stacking portion and an 11.5% resolving portion. In addition, molecular weight markers comprised of: Myosin 200 kD, Phosphorylase b 97.4 kD, Bovin Serum Albumin 69 kD, Ovalbumin 46 kD, Carbonic Anhydrase 30 kD, Trypsin inhibitor 21.5 kD, and Lysozyme 14.3 kD; are added in an adjacent well. Polyacrylamide gel electrophoresis (PAGE) was conducted at 15 mAmps overnight and was increased to 30 mAmps. Pyronin Y runs at the buffer front and electrophoresis is allowed to continue until the pyronin Y has migrated 10.5 cm into the resolving gel.

C. Transfer of Antigen to Solid Support

The polyacrylamide gel is removed from the glass plates and the stacking gel and lower portion of the resolving gel below the pyronin Y is cut off. A piece of nitrocellulose measuring 12.8 cm by 10.5 cm which has been presoaked in a Tris-based buffer (pH 8.3), is layered onto a piece of wet blotting paper, and the trimmed polyacrylamide gel is laid onto the nitrocellulose paper. Another piece of wet nitrocellulose blotting paper is laid over the trimmed gel and this sandwich is placed into the transfer apparatus (Mighty Small™) and transblotted by applying a voltage of 30 V for 30 minutes and 100 V for 1.4 hours.

The membrane is removed from the transfer apparatus and stained with ponceau S. The position of each molecular weight marker is marked on the filter with indelible ink. Commercially prepared anti-human IgG (Berkeley Antibody Co.) is diluted to a concentration of about 10 ug/ml in 0.1 M Carbonate buffer, pH 9.6. and slotted at approximately 1.6 cm from the bottom of the membrane, which is the area of the low molecular weight proteins. The membrane is placed in blocking solution containing 5% non-fat dry milk in PBS pH 7.5 for 45 minutes on a platform rocker. The nitrocellulose is then washed for 15 minutes in PBS-0.05% polyoxyethylene (20) monolaurate "Tween 20™" (Sigma). The sheets are then air dried on paper towels, and left in a 37 degree C incubator overnight for drying. Sheets are cut into strips according to the width of each lane and the length of the gel from top to bottom and kept in dry containers for use.

EXAMPLE 2 Western Blot Assay

A. Sera

Fifty-one (51) human serum samples which were positive for H. pylori by histology, culture, and ELISA (GAP Test, BioRad, USA), were collected. Patient serum was considered positive if the patient was culture, histology, and ELISA positive, with or without an ulcer. One hundred and ten (110) human serum samples which were negative for H. pylori were also collected. Patient serum was considered negative if the patient was culture, histology, and ELISA negative and did not have an ulcer.

B. Immunoblot Assay The strips processed in Example 1 were placed into reaction trays, one strip per well, on a platform rocker. The strips are pre-wetted in wash buffer (Tris buffer containing 0.05% Tween 20™) for 10 minutes. The buffer is aspirated, and 2ml blotting buffer (Tris pH 7.4, 5% inactivated goat serum, and 5% non-fat dry milk) is added. To the blotting buffer is added 20 ul of patient serum (final dilution 1/100) and this is incubated for one hour at room temperature. The fluid is removed by aspiration and the strips are washed 3 times with wash buffer for 5 minutes each. Goat anti-human IgG (Berkeley Antibody Co., Berkeley, CA) conjugated to alkaline phosphatase diluted 1/1,000 with blotting buffer is added to the strips and incubated for one hour at room temperature. The strips are washed 3 times with wash buffer for 5 minutes each.

Helicobacter pylori antigen component bands reactive with patient serum are visualized after the addition of 2 ml of a nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate solution.

The presence of purplish-blue bands indicates the presence of specific patient antibodies in the test sera to that antigen component on the strip. The enzymatic reaction is stopped by rinsing the strips in distilled water. Results may be found in Table 1.

It is to be emphasized that the terms and descriptions used herein are preferred embodiments set forth by way of illustration only, and are not intended as limitations on the many variations which those of skill in the art will recognize to be possible in practicing the invention. Modifications and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention or defined by the scope of the following claims.

Claims

IT IS CLAIMED:

1. A method of diagnosing infection with Helicobacter pylori, comprising the steps of: a) providing a solid support having electrophoretically separated Helicobacter pylori antigen bound thereto; b) reacting biological fluid with the solid support; c) examining the solid support for the presence of bound antibody; and d) positively diagnosing infection when said bound antibody is found at any two positions on the solid support corresponding to antigens having molecular weights of 19.5kDa, 26.5kDa, and 30kDa, or alternatively, when said bound antibody is found at any single position on the solid support corresponding to antigens having a molecular weight of 35kDa, 89kDa, 116kDa, or 180kDa.

2. The method of Claim 1, wherein said biological fluid is human serum and said examining includes reacting the support and bound antibody with a reporter-labeled anti- human antibody.

3. The method of Claim 2, wherein the solid support comprises nitrocellulose membrane.

4. The method of Claim 1, wherein the Helicobacter pylori antigen has ATCC strain designation NCTC 11916

5. A kit for diagnosing infection with Helicobacter pylori, produced by a process comprising the steps of: a) providing an Helicobacter pylori antigen, b) electrophoretically separating antigen components of said Helicobacter pylori antigen; and c) transferring said separated antigen components to a solid support.

6. The kit of claim 5, wherein said Helicobacter pylori antigen is derived from ATCC strain designation NCTC 11916. 6/12965 PCMB95/01028

15

7. The kit of claim 5, wherein said electrophoretically separating is caused by the migration of said Helicobacter pylori antigen through a polyacrylamide gel such that the running front of the Helicobacter pylori antigen migrates between 9-12 cm into the polyacrylamide gel.

8. A method of diagnosing disease associated with infection by Helicobacter pylori in a patient, comprising a) providing a solid support having electrophoretically separated Helicobacter pylori antigen bound thereto; b) reacting biological fluid obtained from the patient with the solid support; c) examining the solid support for the presence of bound antibody; and d) comparing the profile of bound antibody to consensus profiles obtained from other patients with Helicobacter pylori related diseases.

9. The method of Claim 8, wherein the confirmed disease conditions include; gastritis, ulcers and gastric cancer.