AU644121B2 - Rapid in vitro test for helicobacter pylori using saliva - Google Patents

Description

64 '1 2 1 COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952 COMPLETE SPECIFICATION (Original) FOR OFFICE USE Class Int. Class Application Number: Lodged: Complete Specification Lodged: Accepted: Published: Priority: Related Art: 0 oO* *o 0 0

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.".Name of Applicant: *o0* AUSPHARM INTERNATIONAL LIMITED Address of Applicant: Level 15, 99 Mount Street North Sydney 2060 New South Wales

AUSTRALIA

31-~90 0 Actual Inventor(s): ~id Address for Service: DAVIES COLLISON, Patent Attorneys, 1 Little Collins Street, Melbourne, 3000.

0 Complete specification for the invention entitled: "RAPID IN VITRO TEST FOR HELICOBACTER PYLORI USING SALIVA" The following statement is a full description of this invention, including the best method of performing it known to 1 -1A- RAPID IN VITRO TEST FOR HELICOBACTER PYLORI USING SALIVA The present invention relates generally to a method which permits the rapid in vitro detection of Helicobacter pylori infection in mammals. More particularly, the present invention contemplates a method for the detection of IgG antibodies against H. pylori in mucous secretions and thereby provides a means to monitor contemporary infection by the microbe in mammals.

Gut infections in mammals, and in particular humans, o* o 15 stimulate an immune response in mucous secretions, such as saliva, through activation of the common mucosal immune system. This response often initially parallels an antibody response in serum although is generally characterised by the presence of IgA antibodies. However, I 20 the immune response in secretion, including saliva, rapidly diminishes following elimination of the antigen bacteria or virus) from the body. Accordingly, the presence of antibody in mucous secretions reflects current, contemporary, infection. In the case of a microbial 25 infection, for example, antibodies in mucous secretions, hereinafter referred to as secretious antibodies, reflect the current status of colonisation of the microbe, such as o in the gut, and thus is a useful monitor of contemporary infection. Serum antibody, on the other hand, persists for o* 30 some time after the microbe is eliminated from the body.

A positive serum antibody test, therefore, reflects both past and present exposure to antigen which is less helpful to the clinician. A positive secretious antibody test, on the other hand, indicr present or contemporary infection by the microbe.

The present invention arose following an investigation into Helicobacter pylori (also known as Campylobacter pylori) infection in the gut of mammals. The diagnosis of H. pylori infection can be made by microscopy, microbiological culture or urease detection in gastric mucosal biopsies, urea breath test or by the presence of specific antibodies in serum ELIZAs. It might be predicted that H. pylori infection, being an infection of the gastric mucosa, would elicit an IgA antibody response in gastric secretion. However during work leading up to the present invention, it has been surprisingly discovered that the H.

pylori specific antibody in mucous secretions is of the IgG class and not IgA as might have been expected. Little IgA S* 15 antibody, if any, is detected. Accordingly, the present invention is directed to the detection of IgG in mucous secretion specific to H. pylori antigen and thereby provides a means of monitoring current, i.e. contempory infection by that microorganism in mammals.

A test currently available is the CLOtest (registered trademark of Delta West, Ltd., Perth, Western Australia) which detects the presence of urease in biopsy specimens.

0 Although CLOtest is an effective monitor of 25 H. pylori infection, it requires an invasive procedure, the collection of a biopsy.

•In accordance with the present invention, there is provided a rapid in vitro test for contemporary H. pylori S* 30 infection by determining the presence of specific antibodies, and in particular IgG antibodies, to the microbe in mucous secretions and thereby obviating the need for an invasive procedure.

-3- In one embodiment, the present invention contemplates a method for detecting contempory infection by H. pylori in a mammal comprising contacting a mucous secretion from said mammal with an antigen component from H. pylori for a time and under conditions sufficient for an IgG antibody in said mucous secretion specific to said antigen component to form a complex therewith and then subjecting said complex to a detecting means. Preferably, the antigen component is immobilized onto a solid support.

Another embodiment of the present invention provides a test kit for detecting H. pylori-specific IgG antibody in mucous secretions in a mammal, said test kit comprising a 15 solid support having an antigen component of H. pylori immobilized thcreon, an antibody conjugated with a reporter .molecule capable of producing a signal, said antibody *o specific against IgG antibody and optionally, when said reporter molecule is an enzyme, a substrate for said 20 enzyme.

In a preferred embodiment,the mammal is a human.

The present invention is further described by 25 reference to Figures 1 and 2. Figure 1 is a graphical representation of the relationship between saliva ELISA scores and H. pylori infection. Levels of IgG antibody decrease following elimination of the bacteria from the stomach. Figure 2 is a graphical representation showing a 30 concordance of serum and saliva antibodies.

By "mucous secretion" is meant the secretion from mucous-secreting epithelial cells mucous membrane) such as those which line the canals, cavities and tracts that communicate with the external air, and in particular -4the nose, throat, respiratory tract, eyes, genital and urinary passages and the digestive system. In a preferred embodiment, the mucous secretion is saliva, sputum or nasal secretion. Even more preferably, the secretion is saliva.

The solid support contemplated by the present invention includes a nitrocellulose membrane, glass or a polymer. The most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene. The solid supports may be in the form of strips, tubes, beads, discs or microplates, or any other surface suitable for conducting an immunoassay.

15 Antigen is used in its broadest sense and includes H. pylori whole cells or homogeneous, near homogeneous or heterogeneous extract from H. pylori and which is capable of binding to specific antibody in a mucous secretion.

Antigen components contemplated by the present invention 20 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.

The antigen component of H. pylori contemplated by this invention is either covalently or passively bound to the solid surface. The binding processes are well-known in the art and generally consist of cross-linking, covalently binding or phsically adsorbing the antigen to the solid support.

The detecting means contemplated by the present invention allows the identification of an antibody-antigen complex. This is facilitated by contacting the solid support with a second antibody, conjugated with a reporter molecule, and which is specific for at least part of the class of H. pylori-specific antibody found in the secretion, which, in accordance with the invention, is IgG.

By "reporter molecule" as used in the present specification is meant a molecule which, by its chemical nature, provides an analytically identifiable signal which allows the detection of antigen-bound antibody. Detection may be either qualitative or quantitative. The most 15 commonly used reporter molecules in this type of assay are either enzymes, fluorophores or radionuclide containing molecules radioisotopes). In the case of an enzyme immunoassay, an enzyme is conjugated to the second antibody, generally by means of glutaraldehyde or 20 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, p-galactosidase and alkaline phosphatase, 25 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 colour change. For example, 5-bromo-4-chloro-3-indolyl phosphate is suitable for use with alkaline phosphatase 30 conjugates; for peroxidase conjugates, 1,2-phenylenediamine, 5-aminosalicylic acid, 3,3:5,5:tetra methyl benzidine or tolidine are commonly used. It is also possible to employ fluorogenic substrates, which yield a fluorescent product rather than the chromogenic substrates noted above. Examples of fluorogenic substrates are 21st fluorescein and rhodamine. When activated by illumination with light of a particular wave-length, the fluorochrome-labelled antibody absorbs the light energy, inducing a state of excitability in the molecule, followed by emission of the light at a characteristic colour visually detectable with a light microscope.

Immunofluorescence and EIA techniques are both well established in the art and are particularly preferred for the present method. However, other reporter molecules, such as radioisotope, chemiluminescent of bioluminescent molecules, may also be employed.

The choice of a particular reporter molecule 15 conjugated antibody will be, for the most part, determined by the intended use and user of the test kit of the present S* invention. Additionally, although the test is appropriate for all mammals, it is most applicable and useful to monitoring H. pylori infection in humans.

S* Accordingly, in a preferred embodiment, the present invention provides a method for detecting contempory infection by H. pylori in a human comprising contacting a mucous secretion from said human with an antigen component 25 of H. pylori immobilized onto a solid support for a time and under conditions sufficient for an IgG antibody in said S.mucous secretion specific to said antigen component to form a complex therewith and then contacting said complex with an effective amount of a second antibody labelled with a S. 30 reporter molecule and specific to the H. pylori specific IgG antibody and then detecting binding of said second antibody to said IgG antibody by the reporter molecule.

Hence, a medical practitioner may use a nitrocellulose or other suitable solid phase support membrane strip carrying immobilized H. pylori antigens, such as soluble sonicate. The strip is then contacted with the mucous secretion. Preferably, the strip is placed under the tongue for a time and under conditions sufficient to allow potential H. pylo.i specific antibodies of the IgG class in saliva to bind to the immobilized antigens. Alternatively, the source of mucous secretion may be nasal secretion or sputum. The test strip, once exposed to mucous secretion, is then contacted with a second antibody conjugated with a reporter molecule for a time and under conditions sufficient for said second antibody to bind to the first 15 antibody. Preferably, the reporter molecule is an enzyme and even more preferably alkaline phosphatase. The test 9* strip is then washed and a substrate for the reporter molecule, or in the case where the reporter molecule is alkaline phosphatase, 5-bromo-4-chloro-3-indolyl phosphate, S. 20 is contacted with the strip. The substrate reacts with the reporter molecule giving a visual signal. For example, alkaline phosphatase hy.olyses 5-bromo-4-chloro-3-indolyl phosphate to a purple product. This entire procedure can occur in t-he medical practitioner's office.

When a more quantitive enzyme-linked immunosorbent assay (ELISA) or immunoblot is required, such as in a clinical laboratory, a microtitre tray may be used carrying immobilized H. pylori antigens in the wells of the tray.

30 In this case, samples of mucous secretion, saliva, are added to the well to allow potential H. pylori-specific IgG antibodies to bind to the immobilized antigen. Excess -8secretion is washed away and a second antibody specific to IgG conjugated to a reporter molecule is added to allow an antigen-antibody-conjugated antibody complex to form. This complex is detected by adding a substrate to the reporter molecule as described above to allow, for example, a visual signal which may then be quantitated spectrophotometrically or by other means.

The present invention, especially when in the form of the niitrocellulose strip, has many advantages over the presently available assays for H. pylori. The use of mucous secretion, and in particular saliva, to assay for H.

pylori antibody enables diagnosis of current or contemporary infection and thus enables the medical 15 practitioner to:

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0 a) Link gut symptoms with H. pylori which would enable decisions to be made with respect 06 further investigation (including invasive 20 procedures) and/or management use of specific anti-H. pylori agents). The latter may be expected to have special significance with respect to H. pylori-associated non ulcer dyspepsia, gastritis, duodenal ulceration, 25 gastric ulceration and related and other *0 conditions.

0G b) Have for the first time a convenient non-invasive

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test in the doctor's room, clinic or hospital to 30 follow patients with proven peptic ulcer to detect early recurrence. A positive test enables early diagnosis and prevention or early treatment of recurrent peptic ulcer. A negative test has a reciprocal usefulness in analysis of the diagnostic approach to dyspepsia.

Additionally, the test contemplated by the present invention provides a simple yes/no answer, not requiring, for example, the taking of blood. It is read in minutes and developed without any special preparation of specimen by the clinician. A significant advantage of the present invention is the use of mucous secretion saliva) to test for antibodies specific to H. pylori.

Hence, the present invention extonds to a kit for detecting H. pylori-specific IgG antibody in mucous secretions in a mammal, such as a human, said test kit comprising in compartment form a first compartment adapted to contain a solid support having an antigen component of 15 H. pylori immobilized thereon, a mecond compartment *0.e containing an antibody conjugated with a reporter molecule Scapable of producing a signal, said antibody specific against IgG antibody and optionally containing a third o compartment, when said reporter molecule is an enzyme, 20 containing a substrate for said enzyme. The kit may also contain additional compartments such as to receive suitable mucous material and/or for one or more diluents and/or buffers. The kit may also be packaged for sale in a suitable form.

The following examples further define the invention but should not be construed to limit the scope thereof.

a Comparative data are provided (Table 1) showing the a 5 efficacy of the present test relative to other procedures 30 such as the CLOtest, histological examination, culture and an assay for antibodies in serum in patients with no evidence of H. pylori infection (normal gastric histology) and patients with strong evidence of H. pylor, infection (active chronic inflammation of the gastric mucosa).

EXAMPLE 1 ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) 1. Preparation of Helicobacter pylori antigen Two methods of preparing H. pylori antigen are used: Formalinisation of whole organisms and a centrifuged sonicate.

a) Formalinisation of whole organisms i) Helicobacter pylori are harvested from chocolate agar plates into PBS.

15 ii) The bacteria are washed 3 times in PBS (see 3.

below) by centrifugation for 15 minutes at 2000xg.

iii) Washed bacteria are resuspended in l%(v/v) formalin in PBS.

iv) The suspension is incubated at ambient 20 temperature for 30 minutes.

v) The bacteria are washed with PBS by centrifugation.

b) Centrifuqed sonicate 25 i) Helicobacter pylori are harvested fro'- chocolate agar plates into PBS.

ii) The bacteria are washed 3 times in PBS by centrifugation for 15 minutes at 2000xg.

iii) Washed bacteria are resuspended in 5ml of PBS and 30 subject to 5 cycles of sonication (30 seconds at 6u followed by 60 seconds rest which constitutes one cycle).

iv) Sonicated organisms are centrifuged for minutes at 10,000xg.

v) The supernatant is harvested and used as the antigen preparation.

-11 Antigen prepared by method is slightly superior in its ability to distinguish between saliva from infected and non-infected subjects.

2. Coating of ELISA plates with antigen i) Polystyrene ELISA plates are used (Polysorb, Nunc, Denmark).

ii) Antigen is optimally diluted (highest dilution giving maximum sensitivity for antibody positive saliva without increasing reactivity of antibody negative saliva) in coating buffer (see 3. below) iii) An aliquot of 100ul of diluted antigen is added 15 to "antigen" wells of an ELISA plate and lOul of coating buffer (without antigen) is added to "buffer" wells.

a ce iv) Plates are incubated overnight at 4 0 C temperature.

v) Incubated plates are emptied, an aliquot of 100 S* 20 pl of 5% w/v dried skim milk powder/coating buffer (see 3.'ii below) added for 30 minutes at ambient temperature. Plates are again emptied and aliquot of 100 pl of coating buffer added and emptied immediately by flicking the contents out.

e 3. Buffers i) Phosphate buffered saline (PBS): 0.14M NaC1, 0.003M Na 2

HPO

4 0.001M NaH 2

PO

4 .2H 2 0 in 1 litre of 3* 30 deionized water adjusted to pH 7.2.

ii) Substrate buffer: 10.lg citric acid, 14.2g disodium hydrogen orthophosphate (Na 2

HPO

4 150 pl H 2 0 2 w/v in 1 litre of deionized water adjusted to pH iii) Coating buffer: 2.42 TRIS[tris(hydroxymethyl) amino methane], 58.44g NaC1, in 1 litre of deionized water adjusted to pH -12- 4. Treatment of plates after antigen coating For long term storage (6 months) of antigen coated plates, plates are dried after coating and blocking and stored at 40C with a dessicant. This procedure is necessary for long term preservation of the antigen coated plates.

5. ELISA Procedure a) Horse radish peroxidase method i) An aliquot of 100ul of saliva diluted 1/2 in 0.05% dried skim milk powder/PBST (PBSTM) or 100ul of saliva diluted 1/2 in PBSTM is added to an antigen well and to a buffer well of the ELISA plate.

ii) Plates are incubated for 90 minutes at ambient temperature.

iii) Plates are washed 5 times by immersion in PBST.

20 iv) An aliquot of 100ul of horse radish peroxidase *oo anti-human IgG diluted optimally (highest dilution g'o' giving maximum sensitivity for antibody positive saliva without increasing reactivity of antibody negative saliva) in PBSTM is added to antigen and 25 buffer wells.

v) Plates are incubated for 90 minutes at ambient 'temperature.

vi) Plates are washed 5 times by immersion in PBST.

3 vii) An aliquot of l0ul of horse radish peroxidase 30 substrate (Product T-2885, Sigma, USA) in substrate buffer is added to antigen and buffer wells.

viii) Plates are incubated for 30 minutes at ambient temperature.

-13ix) l0ul of 1M H 2

SO

4 is added to antigen and buffer wells.

x) Optical densities (OD) are read using an ELISA plate reader (Titertek Multiscan MCC/340, Flow Labs, Australia).

xi) For each serum or saliva sample, the OD of the buffer well is subtracted from the OD of the antigen well and the resultant OD is converted to ELISA units using a standard curve (constructed from doubling dilutions of a standard antibody positive serum).

xii) A survey of 100 saliva from patients who had been demonstrated by biopsy to be infected (or not infected) is used to determine the number of ELISA units corresponding to infection.

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-14- EXAMPLE 2 IMMUNOBLOTTING ASSAY.

1. Preparation of Helicobacter pylori antigen The procedure for the preparation of H. pylori antigen for the immunoblotting assay is identical to that for the ELISA assay (Example 1).

2. Coating of the membrane with antigen i) Nitrocellulose membrane is used. Nylon based membranes can also be used.

ii) The membrane is immersed in tris-buffered saline (20mM Tris, 500mM NaCI, pH 7.5) (TBS) for 60 seconds.

iii) After blotting dry, the membrane is soaked for minutes in an optimal dilution of antigen (highest dilution of antigen giving maximum sensitivity with 20 antibody positive samples without producing positive reactions in antibody negative samples).

iv) The membrane is then incubated for 30 minutes in skim milk powder/TBS.

v) The membrane is washed twice for 5 minutes in 25 0.05%(w/v) polyoxyethylene sorbitan monolaurate/TBS

(TBST).

vi) The membrane is immersed for 5 minutes in undiluted test saliva in a test bottle or under the 06 tongue.

vii) The membrane is washed for 30 seconds under running tap water.

0 viii) The membrane is immersed in alkaline phosphatase-conjugated anti-human IgG optimally diluted (to enable distinction between antibody positive and antibody negative saliva) in PBSTM for minutes.

ix) The membrane is washed for 30 seconds under running tap water.

x) The membrane is immersed for 5 minutes in substrate (0.3mg nitro blue tetrazolium, 0.15mg 5-brono-4-chloro-3-indolyl phosphate in iml 0.1M NaHC03, 1.0mM MgC12, pH 9.8).

xi) Antibody positive samples produce a mauve colour change in the membrane whereas antibody negative samples do not alter the colour of the membrane.

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04 o o o D n 16- EXAMPLE 3 In this experiment, the levels of IgG antibody in saliva were monitored following elimination of bacteria from the stomach.

The relationship between saliva ELISA scores and H.

pylori infection as reflected by the degree of inflammation persisting in stomach biopsies of patients with healed gastric ulcers is shown in Figure 1.

The results clearly show that levels of IgG antibody are directly related to the level of H. pylori infection as indicated by the degree of gastric inflammation.

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S CC CC p 0 o r -17- EXAMPLE 4 This example shows that the saliva ELISA is more sensitive than conventional tests for H. pylori infection whilst retaining the high degree of specificity of the conventional tests. The results are shown in Table 1.

Subjects have been classified on the basis of microscopy of gastric biopsies as having active chronic inflammation, the hallmark of H. pylori infection, or normal gastric histology. All tests react infrequently with subjects who have normal gastric histology. However, the ELISA is more frequently positive with subjects who have active chronic inflammation than are the other tests i.e. the ELISA has a greater sensitivity.

o e -18- EXAMPLE In another experiment, a total of 138 patients referred to the Gastroenterology Department, Royal North Shore Hospital, NSW, for investigation of upper gastrointestinal symptoms were studied. Microbiological culture, microscopy for the detection of H. pylori, urease tests, and histology of gastric biopsies and ELISAs were performed on all patients where possible. A total of 133 were tested for urease, 131 by culture, 138 by microscopy, 123 by saliva ELISA and 138 by serum ELISA.

Immunoglobulin class of H. pylori antibodies in saliva.

Salivary IgA antibodies to H. pylori were found in tooo 1 only a minority of patients [3/18] and at very low titre.

In the 3 positive salivas optical densities in wells coated with H. pylori antigen were only 0.1 OD units above those 20 obtained in wells coated with coating buffer alone.

Attempts to increase the sensitivity of the assay using a biotin/streptavidin system were unsuccessful. In contrast, IgG antibodies were readily detectable and their presence correlated well with the presence of IgG antibodies in 25 serum. Using a cutoff of 0.08 units for saliva and 5 units for serum a concordance of 97% was found between 169 serum/saliva pairs (Figure 2).

Correlation of salivary antibodies with biopsy tests for H. pylori.

The relationships between gastric biopsy histology and saliva ELISA results together with other tests for H.

pylori infection are shown in Table 2. The serum and -19saliva ELISAs were rarely positive in subjects with normal histology, positive in 30% of subjects with only chronic inflammation and over 86% of subjects with active inflammation. The other biopsy tests for H. pylori were very similar to one another; invariably negative in subjects with normal histology, positive in 4-10% of subjects with only chronic inflammation and positive in approximately 70-72% of those with active inflammation. In subjects with only chronic inflammation, the frequency of positive ELISA's increased with the severity of the chronic inflammation. Minimal/mild and moderate/severe chronic inflammation were associated with positive serum ELISAs in 2/9 and 6/8 subjects, respectively, and positive saliva ELISAs in 2/0 and 6/9 subjects, respectively.

o 00 r 9 0. 20 TABLE 1 TEST: DEGREE OF GASTRIC INFLAMMATION ta S S *5@O

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Normal Active Chronic CLO -31 22 (Urease 0 58 test) %Positive 0 73 Culture -30 24 0 49 %Positive 0 67 Micro- 33 22 scopy 0 58 %Positive 0 73 Serum -38 14 Elisa 1 86 %Positive 2.5 86 Saliva 26 9 Elisa 1 %Positive 3.8 87 5* 0 0S 0S 0S0.

S 0 -21 Detection of H. pylori by the urease test, culture, microscopy, serum ELISA and saliva ELISA in patients with no evidence of H. pylori infection (normal gastric histology), and patients with evidence of active H. pylori infection (active chronic inflammation of the gastric mucosa).

TABLE 2 Relationship between ELISAs and biopsy histology.

tests with gastric q *o S

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0 9 Gastric Serum Saliva CLO Culture Microscopy Inflammation NONE 26 1 23 1 25 0 25 0 27 0 CHRONIC 39 17 35 14 48 6 53 2 52 4 ACTIVE 7 48 7 43 15 39 15 36 16 39 Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred tn or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

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Claims (16)

1. A method for detecting contempory infection by Helicobacter pylori in a mammal comprising contacting a mucous secretion from said mammal with an antigen component fiom H. pylori for a time and under conditions sufficient for an IgG antibody in said mucous secretion specific to said antigen component to form a complex therewith and then subjecting said complex to a detecting means.

2. The method according to claim 1 wherein the antigen component is immobilized onto a solid support.

3. The method according to claim 2 wherein the solid support comprises nitocellulose membrane, glass and/or a polymer.

4. The method according to claim 3 wherein the polymer comprises cellulose, polyacrylamide, nylon, polystyrene, Ve, polyvinyl chloride and polypropylene or any combination of two or more thereof. The method according to any one of the preceeding claims wherein the antigen component of H. pylori comprises whole cell extract and/or one or more isolated components thereof.

6. The method according to claim 5 wherein the isolated component comprises a protein, polysaccharide or lipid or any combination thereof. *i

7. The method according to claim wherein the isolated component is protein or a lipopolysaccharide. -23-

8. The method according to claim 1 wherein the mucous secretion is isolated from the nose, throat, respiratory tract, eyes, genital and urinary passages or the digestive system or any combination thereof.

9. The method according to claim 8 wherein the mucous secretion is saliva, sputum and/or nasal secretion. The method according to claim 9 wherein the mucous secretion is saliva.

11. The method according to claim 1 wherein the detecting means comprises an antibody conjugated with a reporter molecule and which is specific for the H. pylori-specific IgG antibody in the mucous secretion.

12. The method according to claim 11 wherein the reporter molecule comprises an enzyme, fluoronhore or radionuclide.

13. The method according to any one of the preceeding claims wherein the mammal is human.

14. A method for detecting contempory infection by H. pylori in a human comprising contacting a mucous secretion from said human with an antigen component of H. pylori 6. immobilised onto a solid support for a time and under conditions sufficient for an IgG antibody in said mucous secretion specific to said antigen component to form a complex therewith and then contacting said complex with an effective amount of a second antibody labelled with a reporter molecule and specific to the H. pylori-specific IgG antibody and then detecting binding of said second antibody to said IgG antibody by the reporter molecule. -24- The method according to claim 14 wherein the mucous secretion is saliva, sputum or nasal secretion.

16. The method according to claim 15 wherein the mucous secretion is saliva.

17. The method according to claim 14 wherein the reporter molecule is an enzye, 1luorophore or radionuclide.

18. A kit for detecting H. pylori-specific IgG antibody in mucous secretions in a mammal, such as a human, said test kit comprising in compartment form a first compartment adapted to contain a solid support having an antigen component of H. pylori immobilized thereon, a second compartment containing an antibody conjugated with a reporter molecule capable of producing a signal, said antibody specific against IgG antibody and optionally containing a third compartment, when said reporter molecule is an enzyme, containing a substrate for said enzyme.

19. A method according to claim 1 or 14 or a test kit according to claim 18 substantially as described herein with reference to the examples and accompanying figures. DATED this 28th day of September, 1993 AUSPHARM INTERNATIONAL LIMITED e By Its Patent Attorneys I* C DAVIES COLLISON CAVE 930928,q:\oper\eb,67676.279,24