CA2131317C

CA2131317C - Test for helicobacter pylori

Info

Publication number: CA2131317C
Application number: CA002131317A
Authority: CA
Inventors: Robert C. Boguslaski; Robert J. Carrico
Original assignee: Serim Research Corp
Current assignee: Serim Research Corp
Priority date: 1992-03-24
Filing date: 1993-03-03
Publication date: 1998-02-24
Anticipated expiration: 2013-03-03
Also published as: EP0633946B1; US5314804A; WO1993019200A1; AU3736193A; JPH07505279A; EP0633946A4; CA2131317A1; DK0633946T3; EP0633946A1; DE69330515T2; DE69330515D1; US5420016A; JP2638682B2

Abstract

A rapid method and easy to use unitized test device is disclosed for determining the presence of Helicobacter pylori in a biological tissue specimen by detecting the presence of urease in the tissue. The system basically utilizes a multilayer test device for separating and optimizing the various reactions involved, i.e. the urease in the specimen with a substrate and the ammonia generated thereby with an indicator element.

Description

TEST FOR HELICOBACTER PYLORI

RAC~ OUND OF THE lNv~.N-llON

FIELD OF THE INVENTION
The Present invention relates to a rapid dry reagent chemistry test for Helicobacter Pylori. More specifically, it relates to a test for determining the presence of a urease producing microorganism such as Helicobacter pylori in h~man gastric mucosa biopsy specimens.

BACKGROUND INCLUDING A DESCRIPTION OF THE RELATED ART

In the diagnosis and management of gastrointestinal disorders, the determination of Helicobacter pylori (formerly known as CamPylobacter) is becoming increasingly important. The presence of spiral or curved microorganisms in biopsy specimens of human gastric mucosa has been recognized for several decades [Freedberg, A. S. and Barron, L. E., The presence of spirochetes in human gastric mucosa, A~l. J. Dis. Dis., 7:
443-5, (1940)]. A bacterium with curved morphology was cultured from gastric-biopsy specimens from patients with gastritis in 1982 [Warren, J. R., Unidentified curved bacilli on gastric epithelium in active chronic gastritis, Lancet, 1;1273, (1983)] and has recently been named Helicobacter pylori. Clinical research since 1982 indicates that H. PYlori has a role in causing some forms of gastritis and might also be involved in the WO93/19200 ~J~ ~ 1 7 PCT/US93/01819 pathogenesis of peptic ulcers [Peterson, W. L., Helicobacter pylori and peptic ulcer disease, New Eng. J.
Med., 324: 1042-8, (l99l)].
H. pylori grows on the gastric epithelium and does not penetrate the tissues. It is also found on tissue from the esophagus and duodenum. The bacterium appears to be protected from stomach acid by the action of a urease which the bacterium produces. The urease has very high specific activity [Dunn, B. E., et al., Purification and characterization of urease from Helicobacter pylori, J. Biol. Chem., 265: 9464-9, (l990)] and hydrolyzes endogenous urea from the host tissues to form ammonia which neutralizes stomach acid.
A recent publication suggests that the best means to diagnose H. Pvlori infections in gastric mucosa biopsies is a combination of culture and direct microscopic observation of the microorganism on tissue following histologic staining (Peterson, W. L., op. cit.).
However, the value of culture for this purpose has been questioned [Martinez, E. and Marcos, A., Helicobacter pylori and peptic ulcer disease, Lancet, 325: 737, (1991)]-An alternative test for H. pylori utilizes its urease activity. A mucosal biopsy specimen is incubated in a medium containing urea and a pH sensitive dye [Owen, R. J., et al., Rapid urea hydrolysis by gastric Campylobacters, Lancet, l: lll, (1985)]. Urease produced by H. pylori releases ammonia through hydrolysis of urea and when enough ammonia is produced to raise the pH of the medium the dye changes color. Human tissues do not produce urease and H. pylori is the principal urease producing microorganism that inhabits the stomach.
Occasionally biopsy specimens contain other urease producing bacteria that grow in the assay medium during the incubation and produce a false positive result [Marshall, B. J., et al., Rapid urease tests in the management of CamPYlobacter PYlori]. The urease test for WO93/19200 ~1 31 ~17 PCT/US93/01819 H. pylori relies on preformed urease and cell growth is not required; therefore, the specificity of the test is improved by including a bactericide in the medium. This inhibits the growth of other microorganisms that might contaminate the specimen (loc. cit.).
A commercial test for H. pylori, named CLOtest, described and claimed in U. S. Patent No. 4,748,113, detects urease activity on biopsy specimens. The incubation medium is solidified by including a gelling agent. This medium also contains phenol red which turns pink when ammonia released from urea raises the pH above 6Ø The system is buffered so that specimens cont~m;n~ted by fluids from the intestine do not raise the pH and cause a false positive result. When specimens are first inserted into the CLOtest gel they may have a slight pin~ tinge if blood or alkaline bile is present.
The analyst is required to record the initial appearance of the specimen. The test is positive only if the pink color increases in intensity or area.
CLOtest requires three hours incubation at 30~C and up to 21 hours additional incubation at room temperature.
About 75% of biopsy specimens infected with H. pylori give positive results in 20 minutes and 90% are positive by 3 hours. Twenty-four hours are required to verify negative results because 5% of infected specimens become positive between 3 and 24 hours.
The traditional liquid urease tests use a few hundred microliters of medium and ammonia produced by a positive specimen becomes mixed with the medium by diffusion and mechanical stirring. The CLOtest reduces mechanical stirring by using gelled medium and specimens with high urease activity will produce a red color near the specimen in a short time. However, ammonia produced slowly by weakly positive specimens has time to diffuse throughout the gel and a larger amount of ammonia must be produced to give a color change. Thus, the incubation time required for a color change increases 3 1 7 4 ~

disproportionately as the urease content of the specimen decreases. In addition the buffer included in the test medium to consume acid or base on the specimen inhibits pH changes due to production of ammonia and this reduces assay sensitivity and increases incubation times.

SUM~ARY OF THE lNv~N~l~IoN

The present invention involves a method and device for determining the presence of Helicobacter pylori in a biological specimen by detecting the presence of urease using a system for separating and optimizing the various reactions involved. The biopsy specimen is first reacted with a urease substrate under optimized pH conditions on one side of a diffusion membrane element. The ammonia generated by the reaction of urease with the substrate, i.e. urea, permeates to the other side of such membrane where it comes into contact with an optimized indicator element to produce a detectable response.
BRIEF DESCRIPTION OF THE DRAWINGS

Figure l is a perspective view of a unitized test device showing the placement of the various layers before use.
Figure 2 is a longitudinal sectional view of the device of Figure l shown with the specimen added. The curved arrow shows how the device is folded over on itself in use.
Figure 3 is the same sectional view as in Figure 2 except that the device has been folded on itself.
Figure 4 is the reverse side of the device showing a positive result for the control and specimen.

2 1 ~

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is characterized by isolation of an optimized specimen and substrate reaction to produce ammonia from an optimized test indicator reaction to detect the generated ammonia. By doing this, the sample components do not interfere with the indicator and the sample can be incubated at a pH that is optimal for urease. Also, buffer in contact with the specimen does not have an adverse effect on assay sensitivity. This separation mechanism can be illustrated by the following detailed descriptions of the drawings.
Figure 1 is a perspective view of a device of the present invention. A strip like test device 10 is made up of a substrate element 11 consisting of an absorbent paper matrix containing the buffered dried residue of a substrate for urease, i. e. urea which paper matrix is attached to a clear plastic support member 12 using double faced adhesive tape. An indicator element 14 consisting of~an absorbent matrix containing the dried residue of a pH sensitive indicator material, the indicator element 14 being covered with a diffusion membrane element 13, is attached to the support member 12 at the edges of the indicator element 14. The substrate element 11 and the indicator element 14 are positioned on the support member 12 such that when the device is folded at perforation line 17, the two elements are contiguous to or become adjacent to one another. Figure 1 also shows a grid 16 for positioning the sample and a control 15 comprising urease attached to the diffusion element 13.
Figure 2 is a sectional view of device 10 of Figure 1 taken along the lines 2-2. A specimen 18 is shown positioned on the surface of the diffusion element 13.
The arrow above the device shows how the device is folded over on itself.

, W093/192~0 ~ ~ 3 ~ 3 ~ 7 PCT/~S93/01819 Figure 3 shows the device of Figure 1 when i L is folded over on itself. A cont-ol 15 and a specimen 1&
are positioned between the substrate element 11 anG the diffusion element 13.
Figure 4 shows the reverse side of the device 10 of Figure 1 after reaction has taken place to give a positive response to urease in the indicator element 14, which is read through the clear support element 12 to indicate a positi~e result for the control 19 and the specimen 20.
The methods and devices of the present in~ention -- utilize several components which are fabricated as a multilayered multielement test device. The several components basically comprise 1. a substrale element to contain the urease substrate under optimized reaction conditions, 2. a diffusion element to separate the various reactions and allow ammonia to pass and 3. an indicator element to give an indicia or response to a positive result. Each of these components and the assembly thereof will be described below.

Diffusion Element Diffusion elements (membranes) must be hydrophobic to prevent passage of liquid water and solutes.
Diffusion of water vapor through this mem~rane is acceptable because this will not neutralize the indicator dye.
Diffusion membranes should be as thin as possible in order to m; nim; ze the length of the diffusion path for ammonia. The practical lower limit for membrane thic~ness is determined by the quantity of materiai required to maintain mechanical integrity of the membrane. Commercial membranes range from 50 to 250 ~m thic~.
Mean pore diameters for commercial membranes range from 0.05 to 10 m. Membranes with the larger pore A

~3~3~7 WO93~19200 PCT/~S93/01819 diameters offer less resistance to dif usion of ammoniz.
Thus, pore diameters of 1.O to lO ~m are preferable fo-present zpplications.
Hyàroshobi~ memDraneS can be cas~ with 2 variety of polymers. They include polyvinylidene difluoride, po~ytrifluoroethylene, polyvinyl chlorlde, polypropyiene, poly~inylidene dichloride and polytetrafluoroethylene.

Indicator Element Indicator elements can be either hydrophilic or hydrophobic. They ser~e mainly as a support or matrix for the indicato~ dye and should have 2 large surface area to m~imi ze exposure of the dye to ammonia gas diffusing through the diffusion membrane. The indicator element will be viewed from the side osposite the diffusion membrane, therefore, it is advantageous for the indicator element to be thin so color changes on the diffusion membrane side will be more readily visible to an observer. .~icroporous membranes are useful diffusion elements because they are thin. Large pore diameters favor rapid dirfusion of ammonia to the dye coating.
Woven or non-woven fabrics can also be used for the indicator element but they are generaiiy thicker than 2S microporous membranes.
The hydrophobic membranes mentioned above for diffusion membranes can be used as indicator elements.
Hydrophilic membranes are also useful and they can be fabricated from cellulose esters, nitrocellulose, nylon, polysulfone and hydroxylated polyvinylidene dif~uoride.
Indicator elements will generally be coated or impregnated with indicator by dipping them into a solution of the indicator and drying. The amoun. of indicator applied can be varied by varying the indicator 3S concentration in the dip solution.

~ 7 8 ~
Substrate Element One purpose of the substrate element is to expose the biopsy specimen to urea. A second function is to adjust the pH of the specimen to the optimum for urease activity and for formation of ammonia through the ammonia/ammonium ion equilibrium. Thus, the substrate element will contain a buffer, urea and possibly reagents that enhance the activity of urease such as EDTA.
The substrate element matrix can be cellulose based papers, the hydrophilic membranes mentioned above or a woven or nonwoven fabric. Reagents will be incorporated by dipping the matrix into an aqueous solution of the reagents and drying.
Biopsy specimens might not contain enough water to dissolve the dry reagents in the substrate element and give m~ximum enzyme activity. In this situation, the substrate element can be hydrated by applying a few drops of water. Biopsy specimens are placed on the diffusion membrane and a wet substrate element is placed on them.
The assembly is held together with a clamp.
Alternatively, a substrate element can be prepared by dipping the matrix in a urea solution and drying.
When the test is to be initiated the substrate element is hydrated with a buffer solution which will maintain the proper pH and other reaction conditions.
Devices can also be assembled with a thin membrane as substrate element mounted on the diffusion membrane.
Then biopsy specimens are placed on the substrate element and they are covered with a paper wick that does not contain reagents but is saturated with water. In this situation the substrate element should be a thin porous membrane because ammonia must diffuse through it to reach the indicator element.
The substrate element contains a buffer at pH 7.0 to 9Ø The pH optimum for H. pYlori urease is 8.2 [Mobley, et al., J. Clin. Microbiol., 26:831-6 (1988)]. The pKa :-;

21313~7 for ammonium ion is 9.3 and higher pH favors formation of ammonia from ammonium ion. A pH between 8.0 and 8.5 would be optimal for m~X;mum urease activity and ammonia diffusion.
Urease from H. pylori has a Michaelis constant of 0.3 mM urea [Dunn, et al., J. Biol. Chem., 265:9464-9 (l990)]. Thus, urea concentrations of 5 mM or higher in the substrate element should provide nearly maximum enzyme activity.
Positive Control A positive control can be included in a test device by introducing a small amount of urease onto the device at a location away from the specimens being tested.
Ureases from various sources have properties similar to H. pylori urease; therefore, jack bean urease for instance can be used to make a positive control. The enzyme can be impregnated into a matrix such as cotton string and dried. The string can be mounted at the desired location on the diffusion membrane and when the wet substrate element is placed on the diffusion membrane the positive control urease will become active and generate ammonia. The time required to give a visible response with the positive control can be adjusted by adjusting the amount of urease incorporated into the string. An alternative method for including the positive control into the test device would be to incorporate the urease into a suitable composition and simply applying the composition to the diffusion element and drying.

E~ Indicators An ideal pH indicator for inclusion in the indicator element would change from a colorless to a colored form when a proton is removed by ammonia. However, most indicators change from one color in the acid form to W093/19200 ~ PCT/US93/01819 another color in the deprotonated form. To obtain reasonable sensitivity with the present test the indicator should change from a weak color such as yellow in the protonated form to a strong color such as red or blue in the basic form.
An additional function of indicator dyes is,to trap ammonia as ammonium ion and this is achieved more efficiently when the pKa of the dye is low compared to that for ammonium ion, pKa = 9.3.
Indicator pKa Cresol Red 8.3 Phenol Red 7.9 Bromocresol Purple 6.3 Chlorophenol Red 6.0 Bromocresol Green 4.5 Bromophenol Blue 4.0 Cresol red and phenol red dried in membranes are not acidic enough to give a color change in the presence of low levels of ammonia. Bromocresol purple and chlorophenol red give satisfactory responses to ammonia if they are dried into a membrane with about 40%
glycerol. The glycerol acts as a humectant and/or solvent. Bromophenol blue responds to ammonia without glycerol present and the color remains stable for at least a few hours if the test device is not opened to the atmosphere. Preferably the pKa of the indicator dye should be less than 8.0 and still more preferably should be within a range of about from 2.0 to 6Ø

Assembly of Devices and Method of Use The devices of the present invention can be assembled and used in several ways. One advantageous way has been described above and shown in the drawings.
Basically, the diffusion element is positioned between WO93/19200 2 1 3 ~ 3 ~ 7 PCT/US93/01819 the substrate element and the indicator element.
However, it must be appreciated that the urease in the specimen must be reacted with the substrate to generate ammonia and the ammonia must reach the indicator element.
One utilitarian method to achieve this result is to place the specimen on the diffusion element which is held in contiguous relationship with the indicator element and bring the substrate into contact with the side of the diffusion element opposite the indicator element. This can be achieved most conveniently by positioning both the substrate element and diffusion element (contiguous with the indicator element) on a clear plastic support in an end to end relationship such that the specimen can be added directly to the diffusion element, water added to the substrate element and the device folded over on itself to form a multilayered multielement device where the specimen is sandwiched between the substrate element and the diffusion element. Moreover, means, such as a clamp which exerts an even overall pressure,"may be utilized to keep the substrate element and the diffusion element in intimate contact with the tissue specimen included therebetween.
~ nder certain circumstances it may be necessary to adjust the sensitivity of the test devices of the present invention. For example, there may be ammonia or other ammonia producing substances in or accompanying the fluids associated with the tissue specimens undergoing testing for urease. Such sensitivity adjustments may be accomplished by adding to the diffusion element and/or indicator element, predetermined amounts of chemicals or adsorbants which react quickly with or tie up the initial surge of ammonia through the diffusion element. By doing this, only the ammonia generated by the urease in the tissue specimen will react with the indicator in the indicator element to give a positive response.
A particularily useful embodiment of the present involves assembling various system components into a test ~ ~ 3 ~ ~ ~ 7 WO93/19200 PCT/~S93/01819 kit which makes the use of the test system more con~enient and facile for the test operator. Such a test kit advantageously would comprise the test device shown in Figures l - 4 as well as a rehydrating solu~ on for the substrate pad and a holder for ret~ining the test device in a folded position during the reaction of the urease in the test specLmen with the various test reagent components. The rehydrating solution may simply be purified water or a ouffer solution for optimizing the reaction of the urease in the test sample with the urea in the substrate element. Placement of the buffer in the - rehydrating solution o~viates the need for including the buffer in the substrate element along with the urea.

The following examples are illustrati~e of the present invention.
Example 1 Pre~aration of S~nthetic Biops~ SPecLmen Jac~bean urease (from GDS Technology, Inc., Elkhart, IN) was dissolved at 2.0 mg/mL in 0.02M potassium phosphate buffer, pH 7.3, l mM ethylenediaminatetracetic acid (EDTA). Dilutions of this stoc~ enz~me solution were assayed for activity as follows:
The assay was conducted at 30~C in 0.l5M sodium phosphate buffer, pH 7.7. One milliliter reactions contained 1.O mM ADP, 0.25 mM NADH, 1.O mM
a-ketoglutarate and 15 units/mL glutamic dehydrogenase (bovine liver, Sigma, St. Louis, MO).
The mixture was incubated at 30~C for about four minutes until the absorbance at 340 nm stabilized.
Then urease was added and the change in absorbance from 200 to 260 seconds later was recorded. One 3S unit of urease acti~ity hydrolyzes one micromole of urea per minute.

~A

3 ~ ~
WO93/19200 PCT/~S93/01819 Cotton wound po~yester ~hread was submerge~ ~n z solution containing 0.43, l.33 and 3.83 units of urease/mL for -~~ve minutes. Then the threads wer_ removed and driec in an oven at 60~C for five minutes.

Substrate Element Rayon fabr _, grade No. 9343732 manufactured by Veratec, Inc., Walpole, MA, was dipped into a solution containing lO0 mM sodium phosphate buffer, pH 8.0, 1.0 m~
EDTA and lO0 ~M urea. Then the fabric was dried at 50~C
for lO minutes.

Diffusion Element Hydrophobic Versa~el lO000 membrane obtained ~~m Gelman Sciences, Ann Ar~or, MI was employed.

Indicator Element A solution of reagent alcohol-water (l:g) contain ng l.5 mg bromophenol blue/mL (water soluble form, A1Gr~ C:~
Chemical Co., Milwaukee, wisconsin) and lS m~ sulfam~-acid was impregnated into Versapo- lO000 mem~rane available from Gelman Sciences anà then the mem~rane was dried at 60~C for seven minutes. (Reagent alcohol was 90 ethanol, 5% methanol and 5% isopropanol.) A solution of ethanol-water (l:9) containing l.5 mg bromophenol blue/mL and 2.0 mM sulfamic acid was impregnated into hydrophilic Durapore membrane (Mll1ipore Corp., Bedford, ,M~ ) . Then the membrane was dried 2t 60~C
for five minutes.

- Tests with Urease Impreqnated Threads Test devices were assem~led by placing a l.0 x l.0 cm square of Versapor indicator mem~rane on a 2 x ~ cm A; * Trade-mark ~ ~ 3 ~
WO93/19200 PCT/~S93/01819 piece of PP2500 transparency film (3M Corp., Minneapc is, MN). The indicator membrane was covered with 1.5 x 1.5 cm square of Versapel l0000 membrane (diffusion membrane).
The membrane stac~ was fastened in place with o~erlaminating tape with a 6 mm diameter hole cut to provide access to the diffusion membrane. A 5 mm segment of thread impregnated with urease was placed on the diffusion mem~rane and covered with a l.0 x l.0 cm piece of substrate element that was saturated with water. This element was fastened in place with tape.
Threads impregnated with solutions containing various levels of urease were tested in devices. The indicator element was yellow due to the protonated for~
of the indicator dye and positive results with urease impregnated threads appeared as blue areas roughly outlining the shape of the underlying thread. The time required to obtain positive results with threads impregnated with different levels of urease are presented below along with the times that identical pieces of thread gave to positive results in CLOtest.

Urease in Time for Positive Result Impregnating Solution Diffusion 25~units/mL) Device CLOtest 0 No pos. result No pos. result 0.43 60 min No pos. result l.3 20 min 24 hrs 3.8 ll min l.5 hrs It is apparent that the diffusion device gives positive results much more quic~ly than the CLOtest.

Another diffusion device was made with tHe hydrophilic Durapore membrane (Millipore Corp., Bedforà, A'-' * Trade-mark W093/19200 ~ 7 PCT/~S93/Ot819 MA) impregnated with bromophenol blue as indic2tor element. Thread impregnated with 3.8 units urease/mL
gave a positive result in seven minutes.

E~AMPLE 3 A test device for detecting Helicobacter Pvlori by assaying for urease acti~ity was prepared as follows:

Substrate Element Filter paper ~237 manufactured by Ahlstrom Filtration, Inc., Mt. Holly, PA was dipped in a solution composed of 250 mM sodium phosphate, pH 8.0, 250 mM urea and l.O mM EDTA. Then the paper was dried at 60~ C fo-lO min.

Diffusion Element Versapel lOOOO membrane.

Indicator Element Bromophenol blue (sultone form - Aldricn Chemica1) was dissolved at 2.0 mg/mL in reagent alcohol. Versapei 10000 membrane was dipped in this solution and then d~ied at 60~ C for 5 min.

Assembly of DeYices A 2 X 2 cm piece of Scotch double coated polyester film tape ~90729 (3~ Corp.) was attached to a piece of PP2500 transparency film and a l X l cm square of indicator membrane was fastened at the center of the tape. A 2 X 2 cm piece of untreated Versapel lOOOO
diffusion membrane was placed o~er the indicator membrane so the outer edges of the diffusion membrane were held in * Trade-mark WO93/19200 ~ J A 31 7 PCT/US93/01819 place by the tape. A 2 X 2 cm piece of substrate element was fastened to transparency film with Scotch double coated polyester film tape.

T with Urease Con~i n i nq Matrix A 5 mm segment of urease impregnated thread was placed on the diffusion membrane over the yellow indicator membrane. A few drops of water were applied to the substrate element and it was placed on top of the thread on the diffusion membrane. The assembly was held together with a clamp and the indicator membrane was observed for a color change. Blue areas outlining the shape of the urease impregnated threads become visible at the following times:

Urease in Impregnating Time of Appearance of Solutions Blue Color (units/mL) (min) 0.43 50 1.3 ll This example represents the use of a device of the present invention to determine Helicobacter pylori in an actual biopsy specimen.

Assembly of Devices A l X l.25 inch piece of substrate element as prepared in Example 3 above was fastened with polyester film tape to one end of a l X 4.8 inch piece WO93~19200 PCT/~S93/01819 of 0.01 inch thic~ clea~ polystyrene film. ?n indica.~-element was made by impregnating Versapel 5000 memDrane with bromphenol blue 2S àesc~bed in Example 3 above. A 0.75 X 1 cm piece of incicator element was placed 0.75 inch from the substrate element on the polystyrene film. The indicator element was covereà with a 1 X 1.25 inch piece of Versapel 5000 mem~rane (diffusion element) and the overl2?ping edges fastened to the polystyrene film with strips of polyester film tape.
The following materials were mixeà in a mortar and pestle to obtain a viscous solution:

1.4 g Polyvinyl Alcohol (2000 ~, 7 hydrolyzed) 2.4 ml 0.02 M Potassium Phosphate Buffer, p~ 7.2, 1.0 m~ EDTA
0.4 ml 4 mg Congo RedJ ml water 0.026 ml 385 Units Urease/ml buffer One end of a 2 mm diameter stainless steel -od was dipped into the viscous solution and touched a~ains~ the diffusion element near one corner. This provided a positive control spot and was allowed to dry.

2~ Test with Biopsy SPecimens Three biopsy specimens were taken from the stomach of a patient suffering from a gastric disorder and were placed about 3 mm apart on the diffusion eiement of the test device. The substrate element was rehydrated with five drops of water cont~ining 1 mg of sodium azide per ml. The device was folded on itself as described herein such that the substrate element came into contact wit..
the specimen and the diffusion element and device was fastened with a clamp to ensure continued contact.
Within three minutes blue spots appeared on the opposite side of the polystyrene support where the A
* Trade-mark WO93/19200 J~ 3 ~ 7 PCT/~Sg3/01819 specimen was placed incicating that urease was presen.-.
By 6 to 7 minutes 2 blue spot ap~eared in the area adjacent to tne positive control.

E~AMP~E 5 Substrate Element Ahlstrom 237 paper was dipped into lO m~' urea ir.
water and dried at 60~C for l to l5 minutes.

_ Indicator Element A lO0 mM sulfamic acid solution was prepared by dissolving 242 mg of the acid in 5 ml of water and then adding 20 ml reagent alc3hol. Two dip solutions we-e prepared. The first used 2.5 ml of the sulfamic 2Ci d solution, 2.5 ml reagent alcohol and lO mg bromphenoi blue. The second used l.25 ml of the sulfamic acid solution, 3.75 ml reagent alcohol and lO mg brompr.e~.oi blue. Versapel 5000 membranes were impregnated with these solutions and dried at 60~C for 5 minutes.

Construction of Devices The indicator membranes described above were made with dips cont~;ning 2S and 50 mM sulfamic acid. Test devices were constructed with each of these membranes 25 described in Example 4. The substrate paper desc~ibed in the present ~xample and Versapel 5000 membrane were the substrate and diffusion elements, respectively.

Tests with Urease Impreqnated Thread Segments of thread, 5 mm, impregnated with l.33 units urease/ml (Example l) were placed on the dif~usion membranes of test devices. Then the substrate eLement A * Trade-mark ~1317 ~093/19200 PCT/US93/01819 - was hydrated with lO0 mM TRIS-HCl, pH 8.2, l.0 mM EDTA.
0.1% sodium azide. The substrate elements were folded over onto the diffusion membranes and clamped in place.
The times required for distinct blue images of the threads to appear were recorded. The indicator element made with dip containing 25 mM sulfamic acid required 25 to 35 minutes for the image to appear and the one made with dip containing 50 mM sulfamic acid required 55 to 65 minutes.

Claims

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A method for detecting urease in a biological tissue specimen comprising:
a) positioning the tissue specimen on one side of a diffusion element permeable to ammonia;
b) contacting the specimen with a pH optimized urease substrate comprising a solution of urea and a buffer having a pH of about from 7.0 to 9.0, said contacting producing ammonia;
c) allowing the ammonia to permeate through the diffusion element to contact an indicator element positioned to the opposite side of and in contiguous relationship to the diffusion element, said indicator element comprising a matrix containing a pH indicator having a pKa of about 2.0 to 6.0; and d) observing the reaction of the ammonia with the indicator element.

2. The method of claim 1 wherein the diffusion element is a membrane having a pore size of about from 0.05 to 10 µm.

3. The method of claim 1 wherein the said solution of urea and a buffer is contained in a matrix to form the urease substrate.

4. The method of claim 3 wherein the tissue specimen is placed on the diffusion element and the urease substrate is placed over the tissue specimen to form a multi-layered structure and a means is utilized to hold the diffusion element and the urease substrate together in intimate contact with the tissue specimen included therebetween.

5. A method as in claim 1 wherein a quantity of a material which reacts with ammonia is included in the indicator element or the diffusion element, said quantity being determined in accordance with the desired sensitivity of the test to urease in the tissue sample.

6. A method as in claim 1 wherein the pH optimized urease substrate is contained in a matrix.

7. A method as in claim 6 wherein the matrix is absorbent paper.

8. A method as in claim 6 wherein the urea is contained in the matrix and the buffer solution is added thereto as a rehydrating solution.

9. A method as in claim 6 wherein the urea and the buffer are contained in the matrix.

10. A multilayer test device for detecting urease in biological tissue specimens comprising:
a) a substrate element comprising a matrix containing a substrate for urease;
b) a diffusion element comprising a membrane permeable to ammonia and impermeable to water;
c) an indicator element comprising a matrix containing a means for detecting ammonia, the indicator element being in contiguous relationship with the diffusion element;
d) wherein a predetermined quantity of sulfamic acid sufficient to react with ammonia to produce a desired sensitivity is present in the indicator element or the diffusion element; and e) means for placing and maintaining a biological tissue specimen between the substrate element and the diffusion element.

11. The device of claim 10 wherein the substrate element contains urea.

12. The device of claim 11 wherein the substrate element matrix is absorbent paper.

13. The device of claim 10 wherein the substrate element includes a buffer to maintain the pH of the matrix in a pH range of from about 7.0 to 9Ø

14. The device of claim 10 wherein the means for placing and maintaining a biological tissue specimen between the substrate element matrix and the diffusion element is a support member comprising a clear plastic foil on which the substrate element matrix and the diffusion element are placed in a side by side relationship such that when a biological tissue specimen is placed on the diffusion element, the substrate element may be folded over including the specimen therebetween.

15. The device of claim 10 wherein the means for detecting ammonia in a pH sensitive dye having a pKa of about from 2.0 to 8Ø

16. The device of claim 10 wherein the diffusion element is a membrane having a pore size of about from 0.05 to 10 microns.

17. The device of claim 10 wherein a predetermined amount of urease is placed on the diffusion element and acts as a positive control for the test device.

18. A test kit for detecting urease in biological tissue specimens comprising:
a) an aqueous rehydrating solution;
b) a buffer having a pH of about from 7.0 to 9.0;
c) a substrate element comprising a matrix having therein a dried residue of urea;
d) a diffusion element comprising a membrane permeable to ammonia and impermeable to water;
e) an indicator element comprising a matrix containing a dried residue of a pH indicator having a pKa of about from 2.0 to 6.0 for detecting ammonia, the indicator element being in contiguous relationship with the diffusion element;
f) wherein a predetermined quantity of sulfamic acid sufficient to react with ammonia to produce a desired sensitivity is present in the indicator element or the diffusion element; and g) means for placing and maintaining a biological specimen between the substrate element and the diffusion element whereby any urease in the biological specimen will react with the urea to generate ammonia which will permeate the diffusion membrane and be detected by the indicator element.

19. A test kit as in claim 18 wherein the substrate element matrix and the indicator element are attached to a common support member in a side by side relationship allowing one to be folded over onto the other.

20. A test kit as in claim 19 wherein the kit contains a holder or clamp for the common support member to be retained in a folded position.

21. A test kit as in claim 18 wherein urease is positioned on the diffusion element apart from the biological specimen position which urease acts as a positive control for the test kit.

22. A test kit as in claim 18 wherein the aqueous rehydrating solution and the buffer are combined to form a common solution.

23. A test kit as in claim 18 wherein the buffer is contained in the substrate element.

24. A test kit as in claim 18 wherein a predetermined amount of urease is placed on the diffusion element and acts as a positive control for the test kit.