BACKGROUND OF THE INVENTION Factors adversely affecting the function of the gastrointestinal system in humans and other animals are exceedingly varied in their nature. Such disorders may arise in the upper or lower gastrointestinal tracts or both. There is a broad range of causes of gastrointestinal disorders, including genetic, physiological, environmental, and psychogenic factors. Accordingly, the diagnosis and management of these disorders can be exceptionally difficult. A detailed discussion of gastrointestinal tract functions, disorders, causes, and treatments can be found in Spiro, Clinical Gastroenterology (3d. edition 1983).

Among the chronic disorders of the upper gastrointestinal tract are those which fall under the general category of peptic ulcer. The upper gastrointestinal tract is generally defined as including the oesophagus, the stomach, the duodenum, the jejunum, and ilium. Peptic ulcers are lesions of the gastrointestinal tract lining, characterised by loss of tissue due to the action of digestive acids and pepsin. It has been generally held that peptic ulcers are caused either by gastric

hypersecretion, or more often by decreased resistance of the gastric lining to digestive acids and pepsin.

About 20 million Americans develop at least one peptic ulcer during their lifetime; ulcers affect about 4 million Americans every year; more than 40,000 Americans have surgery because of persistent symptoms or problems from ulcers every year; and about 6,000 Americans die of ulcer- related complications every year. The major symptom of a peptic ulcer is a painful burning or gnawing feeling in the stomach area that lasts between 30 minutes and 3 hours. This pain is often interpreted as heartburn, indigestion or hunger. The pain usually occurs in the upper abdomen, but sometimes it may occur below the breastbone. In some individuals the pain occurs immediately after eating. In other individuals, the pain may not occur until hours after eating. The pain frequently awakens an affected person at night. Disturbed appetite and weight loss are other symptoms of peptic ulcer; however people with duodenal ulcers may experience weight gain because they eat more to ease their discomfort. Recurrent vomiting, blood in the stool and anaemia are other symptoms of peptic ulcer.

If left untreated, peptic ulcers can affect the surrounding nerves, which become agitated and cause a great amount of pain. Furthermore, peptic ulcers can cause haemorrhage from the erosion of a major blood vessel; a tear in the wall of the stomach or intestine, with resultant peritonitis; or obstruction of the gastrointestinal tract because of spasm or swelling in the area of the ulcer. As with the management of any disorder, the rapid, precise, and accurate diagnosis of gastrointestinal disorders is of paramount importance.

However, the diagnostic methods typically employed in the art are often slow, cumbersome, costly and may yield equivocal or inaccurate results. See, eg. , Spiro, supra.

Infection with the bacterium Helicobacter pylori is known to play an important role in causing peptic ulcers.

Antibiotics are the most effective treatment for peptic ulcers caused by H. pylori. Other causes of peptic ulcer are chronic use of non-steroidal anti-inflammatory medications and drugs related to aspirin. Therefore, in addition to correctly diagnosing a peptic ulcer, it is essential to correctly determine the cause of the ulcer, as this will affect the treatment regime.

H. pylori produces urease, which breaks down urea into CO2 and NH3. Therefore, the detection of urease in a sample of gastric material is indicative of the presence of H. pylori. Tests for the detection of urease typically involve placing a sample suspected of containing urease in contact with a mixture of urea and a pH indicator. Any urease present will break down the urea, resulting in a pH change which is visible as a colour change. However, one problem with these tests is that urea is labile and deteriorates over time. Therefore the shelf life of these tests is limited.

In an attempt to increase the shelf-life of these tests, diagnostic test comprising urea as a liquid or a gel have been constructed. In some cases, the liquid form of the test is stored frozen and thawed immediately before use.

The gel form of the test is ideally kept cool, at 1 to 8°C, but not frozen as the gel separates under these conditions. The shelf-life of these tests can be increased further by providing a vapour tight seal.

Depending upon the efficiency of the seal, the shelf life of these tests can be 1 to 2 years at room temperature.

The tests are typically stored below room temperature in order to maintain their stability and extend their shelf- life.

The requirement to maintain these tests below room temperature causes distributors to incur major shipping costs as there must be a continuous line of refrigeration from the manufacturer of the tests to the end user. In fact, because of this problem the tests cannot easily be distributed to tropical countries, as the cost is prohibitive.

Several strategies have been devised to avoid the need for refrigeration. These have included the precipitation of the various test components onto a dry paper strip so that the addition of a biological specimen can wet the strip and cause a reaction. A disadvantage with these dry format'tests is that they dry out over several hours and thus need to be observed for reading at a critical time point, as after this time point has passed the colour becomes unstable and may fade or intensify thereby giving false results.

Alternatively, all of the test components may be dried onto a test paper and water added to the test paper before the test is performed on a sample. Furthermore, all of the test components may be dried in a tablet form which is added to water at the time that the test is performed on a sample. In these cases, because a liquid must be added, the liquid component might itself have a need for refrigeration and might have a shelf life problem. In addition persons doing the test need to ensure that the second component of the test is at hand. Therefore, if tests are being performed in two areas simultaneously separate liquid additives are required to be stored in each area.

The inventors of the present invention have now surprisingly found a simple, robust, more stable test for detecting urease in a sample.

All references, including any patents or patent applications, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country.

For the purposes of this specification it will be clearly understood that the word"comprising"means"including but not limited to", and that the word"comprises"has a corresponding meaning.

SUMMARY OF THE INVENTION The present invention provides a more stable test for detecting urease in a sample which is provided by separating the labile reagent of the test from the stable reagents of the test, and providing the labile reagents in a dried form, thereby increasing the stability of the test and therefore the shelf-life of the test. Furthermore, the test of the present invention has broad application, as it can be used to detect any agent using a chemical reaction in which the agent is known to be part and which comprises a labile reagent.

Accordingly, in a first aspect the present invention provides a kit for the detection of an agent in a sample, comprising: a). dried labile reagent (s); b). non-labile reagent (s); and c). indicator;

wherein the labile reagent (s) are provided separately to the non-labile reagent (s).

In a second aspect the present invention provides a kit for the detection of urease in a sample, comprising: a). dried labile reagent (s); b). non-labile reagent (s); and c). indicator; wherein the labile reagent (s) are provided separately to the non-labile reagent (s).

In one embodiment of the invention the labile reagent is an amide such as urea. In another embodiment, the labile reagent (s) is/are provided on or in a strip, such as a paper strip or diskette. Alternatively, the labile reagent (s) is/are provided as a tablet. The non-labile reagent (s) for use in the invention may also be provided as an aqueous solution. In another preferred embodiment, the indicator is a pH indicator and may be provided with the non-labile reagent (s). Alternatively, the indicator may be provided with the labile reagent (s). The pH indicator may be an effective concentration of phenolsulfonphthalein. The kit of the present invention may also comprise a positive control, for example urease, which may be provided at a concentration of 0.001 Units to 1000 Units per square millimetre of a paper strip or diskettes, preferably at a concentration of 2.0 Units per square milimeter of paper strip or diskette. The present invention further provides a paper strip or diskette with a single labile reagent dried thereon, preferably the labile reagent is urea at a concentration of 0.001 grams per litre to 4000 grams per litre.

In another aspect of the invention, the kit of the invention may be used to detect an agent in a biological sample, such as a stomach biopsy. The presence of urease in a stomach biopsy indicates the presence of urease-

producing Helicobacter, for example Heliobacter pylori, in the stomach biopsy.

In another aspect, the present invention provides a method of detecting the presence of Helicobacter pylori in a stomach biopsy, comprising the steps of adding the stomach biopsy to a combination of reagents a) and b) and indicator c) of a kit of the invention, and detecting the presence of H. pylori by virtue of the colour change of the said indicator.

The present invention further provides a method of detection of an agent in a sample, comprising adding a sample to a combination of reagents a) and b) of a kit of the invention, and detecting a change in the indicator c).

The present invention further provides a method a method of detecting a predisposition to developing a peptic ulcer or predicting the probable development of a peptic ulcer in a patient when tested with a kit of the present invention, the method comprising the steps of providing a stomach biopsy to a combination of reagents a) and b) and indicator c) of a kit of the invention, and detecting a change by the indicator.

The present invention further provides the use of a kit of the invention in the detection of an agent in a sample, in which the sample is added to the combination of reagents a) and b) and indicator c) and a change is detected by the indicator.

The invention will now be described by way of a non- limiting example with reference to the following drawings.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows a kit according to a preferred embodiment

of the invention.

Figure 2 shows a chemical reaction applicable to the detection of urease.

DETAILED DESCRIPTION OF THE INVENTION In one embodiment, the invention provides a kit for the detection of urease in a biological sample, for example a stomach or duodenal biopsy. This has application to the detection of Helicobacter pylori in the stomach biopsy and or duodenal biopsy and therefore the diagnosis of gastric ulcer or duodenal ulcer.

Referring to Figure 1, the illustrated kit comprises a) a first component 4 as a salable plastic vial containing a pH indicator such as phenolsulfonphthalien (phenol-red) in an aqueous solution 8, b) a second component 6 as a paper strip comprising dried urea, and c) a third component 10 as a paper strip or diskette comprising impregnated urease for use as a positive control. The phenolsulfonphthalien is at an effective concentration of 0.10 milligrams/mL, although the concentration may range from 0.1 mg per mL to 500 mg per L; the urea is at a concentration of 2.5mg/square millimitre, although the concentration may range from 0.01 to 50mg per square millimitre, and the urease is at a concentration of 2.0 Units per diskette or 0.04 Units per square millimitre, although the concentration may range from 0.001 to 1000 Units per square millimitre. This provides a positive reaction within 5 minutes after insertion of the positive control strip or diskette into the test vial fluid. As used herein one unit of urease will liberate one micromole of ammonia from urea per min at pH 7.0 at 25° Celcius.

The second component of the kit is prepared using a solution of urea at the aforementioned concentration,

which is employed to impregnate grade #1 filterpaper strips of 25mm X 6mm. In a preferred embodiment of the kit, each second component will have between 0. 001mg urea to 500mg urea impregnated onto it.

As used herein, "an effective concentration"of an indicator is a concentration of indicator which effects a readily discernible colour and an oberservable change in the original colour, in response to a positive urease presence in a biopsy or in response to insertion of the positive control diskette when used in the kit of the present invention at ambient temperature.

Typically, the indicator is present at a level of from about 0.1 mg per litre to about 500 mg per litre.

Indicators among those useful herein have pKa values of from about 6.0 to 8.5, and include p-nitrophenol, bromothymol blue (dibromothymol sulfonphthalein), phenol red (phenolsulfonphthalein) neutral red (2-methyl-3-amino- 6-dimethylaminophenazine), quinoline blue (cyanine), cresol red (o-cresolsulfonphthalein), matacresol purple (m-cresolsulfonphthalein), and thymol blue (thymolsulfonphthalein). Bromothymol blue, phenol red, neutral red and cresol red are preferred indicators for use in the compositions of this invention. Further indicators may be readily identified by the person skilled in the art.

It will be clearly understood that the invention is not limited to the detection of urease in a sample of gastric material, as the detection of urease is useful as a surrogate marker for several bacterial infections caused by urease producing organisms. Urease is also found in various plants and seeds at various times in their growth cycle. It will be further understood that the present invention may be used to detect an agent other than urease, provided that the chemical reaction used to detect

the agent comprises a labile reagent. It will also be understood that the invention is not limited to the detection of an agent in a stomach biopsy, or indeed a biological sample. For example, the present invention may be used to detect an agent in a soil sample.

In one embodiment of the invention, the first component of the kit of the invention comprises an indicator in an aqueous solution 8 contained in a salable plastic tube 4; however, the container may be any salable container 4 able to hold a solution 8. The solution may comprise 1% w/v phenol red, 0.145% w/v citric acid. 20% methy hydroxy benzoate (MBH). However, the solution 8 will depend upon the chemical reaction which is to be performed and is limited only in comprising the non-labile reagents 8 of the chemical reaction which is to be performed. Moreover, the indicator need not be present in the solution 8 but may, for example, be present in or on the second component 6 of the kit. In a preferred embodiment of the invention the indicator is a pH indicator; however, it will be clearly understood that the type of indicator will depend upon the chemical reaction which occurs.

The second component 6 of the kit preferably comprises dried urea, because this is the labile reagent of the chemical reaction of the preferred embodiment. However, it will be clearly understood that the reagent of the second component 6 will depend upon the chemical reaction which will occur, provided that it is a labile reagent.

Moreover, it will be understood that the method of drying the labile reagent into or onto the second component 6 will depend upon the labile reagent. For example, the reagent may be in solution, for example 30% w/v urea, which impregnates the second component 6 and then is allowed to evaporate, thereby leaving dried labile reagent in the second component 6. The method of drying the labile reagent will also depend upon the nature of the

second component 6. For example, the second component 6 may be a paper or material strip, which may be impregnated with a solution comprising the labile reagent then dried at 37°C. Alternatively, the second component 6 may be a tablet comprising crystals of labile reagent, optionally in combination with an inert carrier.

The third component 10 of the kit is to be used as a positive control, and is preferably provided separately to the first and second components of the kit. Therefore, it will comprise the agent which the kit is being used to detect. Therefore, in a preferred embodiment, the third component 10 comprises 2 Units of dried urease (available, for example, from Sigma Aldrich product #UI500). The third component 10 may be a paper or material strip.

Alternatively, the third component 10 may be a tablet comprising crystals of the agent which the kit is being used to detect, optionally in combination with an inert carrier.

In a method of the invention, the kit of the preferred embodiment is used for detecting the presence of urease in a stomach biopsy by combining the first 4 and second 6 components of the kit and adding a stomach biopsy. This provides urea which the urease, if present in the stomach biopsy, breaks down to two molecules of ammonia and one bicarbonte ion and two molecules of water, thereby increasing the pH. The increase in pH is detected by the change in colour of the pH indicator from yellow to red in the case of phenolsulfonphthalein. If no change in colour is detected, the third component 10 of the kit may be used as a positive control. The order in which the first 4 and second 6 components of the kit and the stomach biopsy are combined is unimportant; however, in a preferred embodiment of the invention the first 4 and second 6 components are combined before the stomach biopsy is added to the kit. The urea of the second component 6 may

dissolve into the aqueous solution 8 of the first component 4, or it may not. To hasten the speed with which the urea dissolves into the aqueous solution 8 of the first component 4, the kit may be shaken. The kit may also be shaken when it contains the stomach biopsy.

The concentration of the reagents of the kit will effect the rate at which the chemical reaction occurs. Suitable concentrations may readily be determined by a person skilled in the art. The temperature at which the kit is used will also effect the rate at which the chemical reaction occurs. Again, a suitable temperature may readily be determined by a person skilled in the art.

The chemical reaction shown in Figure 2 has application in the detection of urease; however, a solution which comprises urea is unstable if it is kept above 37°C for more than a few hours. The detection of urease is useful as a surrogate marker for several bacterial infections caused by urease producing organisms, for example Helicobacter pylori. Urease is also found in various plants and seeds at differing times in their growth cycle.

Therefore, a test which simply detects urease in a sample is very useful. Moreover, a test which rapidly detects the present of a microorganism is very useful as delays in detecting a microorganism can effect the ability to successfully detect the microorganism. Furthermore, delays in diagnosis of a condition may affect the prognosis of the patient. Therefore, a simple, rapid test could be used performed outside large hospitals to accurately diagnose medical conditions.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.