AU697395B2 - Urinary test strip for determining calcium loss - Google Patents

Description

URINARY TEST STRIP FOR DETERMINING CALCIUM LOSS

TECHNICAL FIELD OF THE INVENTION

This invention relates to indicator or test devices for urine, particularly those used in the detection of urinary calcium excretion. More specifically, the present invention relates to dry reagent test strips, the method of making such and their use in the early diagnosis of osteoporosis. BACKGROUND TO THE INVENTION

Osteoporosis is a disease characterised by a decrease in bone density and mass. While multiple compression fractures of the spine ('Dowager's Hump') due to the decreased bone mass is a common symptom among geriatrics, the most serious manifestation of osteoporosis is hip fracture. Osteoporetic hip fractures cause significant morbidity and mortality within the aged population. At least 50% of those people who have suffered such a hip fracture are thereafter confined to a wheelchair, unable to walk, their lifestyles dramatically changed. Further, within 12 months from the fracture event, at least 20% of sufferers will die.

Alarmingly, this disease is becoming increasingly common in our aging population, with as many as 60% of women and 30% of men over the age of sixty years, suffering at least one osteoporetic fracture. The increasing prevalence of this disease, particularly among women, and the fact that if it is identified at an early stage, further bone deterioration can be prevented by treatment, has resulted in research into the early detection or prediction of osteoporosis in perimenopausal adults becoming of major scientific importance.

Due to the fact that osteoporosis is clearly preventable but only partially treatable, the early detection of osteoporosis is crucial if Bone Mineral Content is to be preserved in menopausal adults and further bone deterioration prevented. Studies have shown that the rate of bone loss after menopause is such that many women are losing bone at a rate of greater than 3% and up to 7% per year. Further, in the majority of cases presenting with osteoporosis, 20%-40% of Bone Mineral Content has already been lost before diagnosis.

Therefore, while a variety of scientific tests have been developed to assist in predicting the likelihood of bone fractures, such as bone densitometry, which provides a highly specialised means for directly measuring bone mineral loss, these are generally expensive procedures which are not performed routinely due to the cost. Hence, when these tests are actually performed, in the majority of cases they only confirm that the patient has lost significant quantities of Bone Mineral Content. Such tests are not helpful in actually diagnosing those perimenopausal adults who are likely to become osteoporetic.

Far more successful detection of the onset of osteoporosis is currently obtained by measuring the serum or urinary concentrations of key biochemical markers. As it is commonly known that the amount of calcium appearing in the urine is directly related to the rate of bone loss in menopausal adults, it is well established that measurement of urinary calcium acts as an early indicator of rapid bone loss, providing a clear measure of bone resorption tendency.

However, due to the fact that any estimation of total urinary calcium is only meaningful when a 24 hour urine sample is used, the difficulties associated with collecting such a large sample size have resulted in the current laboratory practice of taking a random urinary sample after a 12 hour fast, measuring the calcium content and matching it against the known constant excretion product of urinary creatinine. The ratio between calcium and creatinine which is obtained, can then be used to detect those patients at risk of osteoporosis. However, the current experimental practice as described above, has a number of inherent limitations. The most serious limitation stems from the fact that the nature of urinary calcium excretion is such that it is highly variable over a 24 hour period, being dependent upon many factors such as diet, general health, exercise and salt intake. These factors vary daily in an individual and therefore will not be accounted for by one isolated urinary calcium test.

A further disadvantage of the current experimental practice is that it involves liquid reagents and more importantly, is laboratory based which means that it is totally impractical to carry out the frequency of tests required in order to achieve an average calcium loss over a period of days or weeks. While AU-B- 27015/92 to Miles Inc. discloses a dry reagent test device for measuring urinary creatinine, it does not envisage any semi-quantitative analysis of urinary calcium loss. Being laboratory based tests up until now the use of the ratio has been substantially limited to researchers in the field of bone metabolism, rather than a wider clinical application, the present invention overcomes the problem of restricted availability of monitoring of urinary calcium loss, by providing a rapid dry reagent system which combines the two test functions, calcium and creatinine into the one device, convenient for personal use.

The device consists of a test strip with two reagent containing pads, one for calcium, the other for creatinine attached side by side to one end of the test strip, the pads react on contact with urine to product a colour change proportional to the concentration of calcium and creatinine in the urine sample. The intensity of colour may read visually by comparison with a colour chart or by a reflectance meter which calculates the ration between the tow parameters. The colour chart is two dimensional with calcium standard colours on the X axis and creatinine standards on the Y axis allowing a table of rations to be read simply.

The test therefore offers the novel advantages of economy, speed and simplicity over the current laboratory based method for measuring the calcium/creatinine ratio.

The use of such a test should permit more frequent monitoring by the clinician or self monitoring on a daily or weekly basis, all of which with current laboratory methods would be impracticable and expensive.

Thus, it is the object of this invention to provide a simple, fast and low cost test which can be used at home to carry out frequent urinary calcium estimates such that constant surveillance of calcium loss and hence monitoring of osteoporosis is achieved. A test which allows both home and clinical monitoring and provides a rapid, inexpensive, mass screening technique for rapid bone loss is therefore desired. Prior Art

A multilayered slide device (Ektachem, Eastman Kodak) is available for measuring Calcium in a laboratory setting. It appears to be the only example of a dry chemical reagent system for detecting Calcium. It is not intended for the convenient rapid test strip type of technology and cannot be read by visual comparison with a standard colour chart.

Reference to patent literature discloses no other dry reagent test devices able to give a calcium specific detactable response on contact with a calcium containing urine. This also appears to be the case for other biological fluids such as blood.

It seems that a carrier matrix incorporating a reagent composition able to give detectable response on contacting with urine may not exist other than in the form of the Ektachemmultilayered slide system. U.S. patent application number 93-19668 discloses a method for a dry test device measuring specific gravity by giving a detectable response to the total divalent cation content of a urine sample.

Despite reference to an embodiment which would permit the invention with suitable modification to be used for detecting urinary calcium or other calcium containing fluids, this was merely incidental to the experiment purpose of the invention namely to determine the total divalent cation content. The total divalent cations were thought to be able to indirectly give a measure of the urine specific gravity.

The present invention as opposed to the "Specific Gravity" patent is concerned solely with Calcium ion and takes some trouble to avoid measuring the other main divalent cation of urine namely Magnesium.

In contrast with calcium the literature discloses many dry chemical reagent systems for creatinine detection. The majority either take the form of multilayered slide devices for use in formal laboratory settings or the more conventional strip device with an absorbent carrier matrix attached to one end of the inert support. The detection means are either enzymatic or various refinements of the Jaffe reaction. Japanese patent JP87287261 discloses that carboxylate polymers offer protection to paper matrix from alkaline conditions of the Jaffe reaction. Similarly AU-B-27015/92 discloses another means of alkaline protection for matrix materials when using the Jaffe reaction, but neither envisage a means for quantifying urinary calcium loss. SUMMARY OF THF INVENTION

Accordingly, the present invention provides a dry reagent test device which includes means adapted to indicate the calcium concentration of a urine test sample and means adapted to indicate the creatinine concentration of the test sample, such that the ratio of calcium to creatinine can be directly determined.

Thus, as not one of the previous described testing methods is suited to determining the urinary calcium excretion rate outside a medical laboratory, perimenopausal adults who wish to routinely monitor their urinary calcium loss in order to detect the early clinical symptoms of osteoporosis, and so take preventative action, will now be able to perform urinary calcium assays at home using the reagent impregnated test strips of the instant invention.

The previous disadvantages of the above mentioned laboratory 'wet tests' such as their restricted availability and expense, gave rise to infrequency of use of these tests by perimenopausal adults. This consequently resulted in the detection of osteoporosis occurring in the majority of cases only after significant bone loss had occurred. However, the above disadvantages will no longer be impediments to the early diagnosis of osteoporosis, as test devices incorporating the instant invention offer the advantages of routine calcium excretion monitoring, as well as availability, economy, speed and simplicity over the current laboratory based method for measuring the calcium/creatinine ratio.

Preferably, the dry reagent test device includes two reagent containing pads, wherein one of said pads is adapted to indicate the calcium concentration of a urine test sample, while the second of said pads is adapted to indicate the creatinine concentration of the test sample, such that the ratio of calcium to creatinine can be directly determined. It is to be understood that the term 'pad' can include any reagent containing region on a dry reagent test device and can be inherent in the device and not necessarily attached separately to a substrate to form the device as a whole. Preferably, the test device is in the form of a strip, with the two reagent impregnated pads adjacent each other and located at one end of said strip. Also preferably, the pads consist of an absorbent or bibulous carrier matrix.

More preferably, the pads consist of latex.

Preferably, the pad adapted to indicate the creatinine concentration of the test sample is impregnated with a reagent composition comprising a creatinine specific dye, a buffer, a fixative agent and a carrier (which can also act as a water imbibing agent).

Further preferably, the creatinine specific dye is Dinitro benzoic acid.

Further preferably, the buffer used in the creatinine sensitive reagent composition is Sodium Metasilicate

Also preferably, the pad adapted to indicate the calcium concentration of the test sample is impregnated with a reagent composition comprising a Calcium sensitive dye, a means of fixing that dye, a buffer, and a Magnesium specific binding agent. If necessary, optional agents can be added. Further preferably, the Calcium sensitive dye is an MSTPM dye such as

Thymolphthalein complexone mono sodium salt.

The present invention further provides a method for determining the calcium: creatinine ratio in urine which comprises contacting a urine test sample with said dry reagent test device, and determining the degree of colour development of each of the creatinine specific and calcium specific dyes .

It is preferable that the ratio of calcium:creatinine can be semi- quantitatively determined by the visual comparison of the results of the colourmetric reactions of the two reagent containing indicator pads with a standard colour chart. It is also preferable that the ratio of calcium: creatine can be quantitively determined by comparing of the results of the colourmetric reactions of the two reagent containing indicator pads with the urine test sample, using a reflectance meter.

A further aspect of the invention is that a method for the diagnosis of the potential for osteoporosis is also provided which comprises contacting a urine test sample with said dry reagent test device, determining the degree of colour development of each of the creatinine specific and calcium specific dyes, then determining the calcium : creatinine ratio.

Further, a method for the preparation of a dry reagent test device as claimed in claim 3 is provided, which is used in the detection of urinary calcium loss which comprises: a) preparing an absorbent pad incorporating a reagent composition which is adapted to measure the creatinine concentration of a urine test sample, wherein the creatinine sensitive reagent composition consists of:

(i) a creatinine sensitive dye which reacts with creatinine under alkaline conditions,

(ii) a fixing agent, (iii) a buffer (iv) optional agents b) preparing an absorbent pad incorporating a reagent composition which is adapted to measure the calcium concentration of a urine test sample, wherein the calcium sensitive reagent composition consists of: (i) a calcium sensitive dye which reacts with calcium ions under alkaline conditions, (ii) a fixing agent,

(iii) a buffer

(iv) a magnesium specific binding agent (v) optional agents

The invention described above thus provides a rapid, dry reagent test device, convenient for personal use, which enables the urinary calcium : creatinine ratio to be semi-quantitatively or quantitively determined as one measurement. Use of this test device permits more frequent monitoring of urinary calcium excretion by a clinician or self monitoring by perimenopausal adults on a daily or weekly basis. Beneficially, it is thus possible to achieve an early diagnosis of osteoporosis and consequently allow for preventative treatment to be initiated. Advantageously, the costs to the public health care and hospital system are therefore minimised, as osteoporetic fractures can be avoided.

BRIEF DESCRIPTION OF DRAWINGS

An illustrative embodiment of the present invention will now be described with reference to the accompanying figures, in which :

Figure 1 is a side elevation view of a dryreagent test strip incorporating the creatine sensitive pad and the calcium sensitive pad

Figure 2 is a plan view illustration of a dry reagent test strip incorporating the present invention. A preferred embodiment of the invention is shown which comprises a dry reagent test strip made of an insoluble plastic material, which includes upon it the two reagent containing indicator pads. These pads are adjacent each other and located at one end of the strip. The pads are located adjacent each other for easy comparison and visual determination of colour development. The figure further displays the development of the calcium and creatinine sensitive dyes upon their respective indicator pads after contact of the dry reagent test strip with a urine test sample.

Figure 3 is a graphical illustration of the linear relationship between the reflectance values derived from varying concentration of urinary creatinine.

Figure 4 is a graphical illustration of the non-linear relationship which results when reflectance values over a physiological range of urine calcium levels are measured. DETAILED DESCRIPTION OF THE INVENTION

Urine contains a wealth of biochemical markers which have been excreted from the body. Urine can be assayed in order to determine the concentrations of specific metabolites or electrolytes and variations of such metabolite concentrations from their normal ranges are therefore seen to be indicative of various medical conditions. The present invention provides a means for the early detection of osteoporosis by monitoring the urinary calcium excretion rate utilising a dry reagent test device which enables the ratio of calcium to creatinine in a urine test sample to be determined.

Typically, the dry reagent test device is in the form of a strip which can be made of any inert, insoluble material such as plastic. Contained at one end of the inert plastic carrier are two adjacent reagent containing indicator pads which can be made of any absorbent or bibulous material.

One of the pads is calcium sensitive, while the other pad is creatinine sensitive. These pads react on contact with urine to produce a colour change proportional to the concentration of calcium and creatinine in the urine sample. The colour may be read by a visual comparison of the dye development on the indicator pads with a colour chart, or by placing the test strip in a reflectance meter which calculates the ratio between the two parameters. The Calcium sensitive Component. Similar to the creatinine sensitive component, the calcium sensitive component of the dry reagent test device preferably takes the form of a pad contained at one end of an inert plastic carrier adjacent to the creatinine sensitive pad. All reagents are present in the dry state.

A calcium sensitive dye to effectively exhibit colour development in this system must possess a number of critical characteristics. Firstly, in the presence of calcium, it must give a suitably detectable response. The colour generated must be evenly displayed across the range of normally encountered urinary calcium values, not necessarily in a linear fashion but preferentially with a greater response in the physiological range and low pathological range but still able to continue to respond to successive increments in the high pathological range.

The chromogen or dye-precursor must be selective for calcium or able to function in conditions which minimise its binding of other divalent cations- specifically magnesium in the case of urine. Similar to the creatinine specific dye, the calcium dye must be of such a configuration that it is amenable to fixing within the confines of the absorbent carrier matrix. This is most important because on contact of the reagent containing pad with urine, the soluble dye will leach into the urine sample and part of the detectable response will be lost, causing inaccuracy. In practice, hydrophobic dyes and their precursors do not leach, it is only the hydrophilic or water soluble dyes which require fixing. Known dyes used for clinical assessment of calcium in body fluids function by forming chelates with calcium. In such dyes, the reactive groups are anionic and endow the dye with water soluble characteristics and in turn the need for fixing with a suitable mordant.

Apart from their calcium chelating ability, a further characteristic shared by these commonly accepted clinical dyes is their pH indicator function. In fact, in the presence of calcium, the pH induced colour change will occur at a lower pH level. This is the mechanism of calcium generated dye colour. Due to the fact that these dyes are pH indicators, as well as the fact that most compiexing reactions with calcium are most efficient at an alkaline pH, it is necessary to distinguish colour development caused by a high pH from that caused by calcium.

From the above, it can be seen that it is important to select a dye as well as other non-specific ingredients which materially minimise the tendency for pH induced background colour, which is manifest as a high blank value for the un reacted pad. The dyes which most suitably fulfil the above requirements are: Alazarin Complexone, Eriochrome Blue, Cresolphthalein Complexone, Thymolphtha.ein Complexone and other conventional compiexing agents. Cresolphthalein Complexone was first described in 1954 (A. Flaschka et al, Helv Chim Acta 37, 113) and is the most well accepted by clinical laboratories. Thymolphthalein Complexone is considered advantageous for the purposes of this invention because of is additional hydrophobic groupings in the form of isopropyl substituents which facilitate fixing of the dye to the pad.

As discussed above, in such calcium dyes, particularly those favoured for clinical applications, the reactive groups which interact with and complex the metal ion as a chelate, are anionic and endow the dye with water soluble characteristics. This property is in turn responsible for the great tendency of these dyes to solubilise and leach from the carrier matrix at the time of testing on contacting with an aqueous sample such as urine.

As leaching and loss of the newly formed dye-calcium complex will prevent the test from working, the need for immobilising or 'fixing' the calcium- sensitive dye within the carrier matrix with a suitable mordant is apparent. Common mordants include tannin acid, lactic acid or oleic acid. All these mordants are acidic and are used with basic dyes. Salts of chromium, iron or aluminium are basic or metallic mordants and combine with acidic dyes. Generally mordants carry a complementary charge to that found on the dye they fix. More effective mordants take the form of polyelectrolytes: either poly acids or poly bases and these are able to bind to and fix some highly soluble dye species. Even greater strength of dye binding will be found if the mordant is bound to or is part of an insoluble resin or matrix. In this case, the slight solubility of some dye-mordant complexes will be obviated. As the calcium sensitive indicators preferred in this invention, take the form of acid dyes, the present invention can utilise a wide range of basically charged polyelectrolytes able to function as mordants. Further, as any polyelectrolytes to be effective in complex formation between calcium and its chelating dye, must retain their charge in the alkaline range of about pH9.0 to pH12.0, the polyelectrolytes employing a quaternary ammonium species are preferred.

While the mordants of the instant invention are not to be taken as being limited to polymers containing quaternary ammonium salts with a monomer number exceeding twelve, some examples of effective mordants in the present invention are: polyvinylbenzyltrimethylammoniumchloride, polyvinylmβthylpyridine- chloride, polytrimethylaminoethylmethacrylatechloride, polytrimethyl- aminopropylamidomethacrylate, polydiallyldimethylammoniumchloride and polymonoallyltrimethylammoniumchloride. A number of commercially available mordants containing suitable quaternary ammonium salts such as MATEXIL (ICI) or ALCOFIX (DOW), also proved highly effective for the purposes of the invention. Equally effective were the quaternary ammonia containing polyelectrolyte species available commercially as flocculants and used for example in water purification. A further source of suitable quaternary ammonium bound polymer was found in commercial ion exchange resins (eg DOWEX from Dow and Asahi Chemicals). When such resins were added in powder form to the calcium sensitive reagent composition used in this invention, all the acidic dyes were mordanted.

Finally it has been found quite surprisingly and unexpectedly that the cationic charge introduced to the latex emulsion of this system solely for the purpose of providing a compatible matrix for Calcium ions, also was able to act as a mordant for the various dyes. This was found to be a unique and specific characteristic of cationic latex and did not occur with amnionic or non ionic stabilised latex emulsions. Furthermore other cationic monomer species could be introduced as alternative polymers in the latex system. In all cases the cation was a quaternary ammonium salt. Thus a latex polymeric system of styrene and Tri methyl amino ethyl methacrylate chloride could function equally as well as Styrene and vinyl benzyl tri methyl ammonium chloride. It follows that both matrix films will be compatible with Calcium and function as a mordant for the dye. A further mordant system quite distinct from cationic polymers has unexpectedly been found when including starch gels as a component of the reagent composition in amounts between 1% and 5%. This feature of starch seems to be general for all the acidic dyes used to detect Calcium in this invention. The reagent composition also includes a buffer to provide a stable pH environment. Several buffers have been found suitable in the reagent composition. Amino methyl Propanol, (AMP) or 3- (Cyclo hexylamino) 1 propane sulphonic acid (CAPS) and Sodium Borate but others are well known in the art. It is an important requirement that the buffer does not bind Calcium ions in the competition with the dye.

The choice of buffer will be influenced partly by the particular dye used in the system and its optimal pH but also by the need to select a high pH range at which Magnesium is not bound by the dye.

The purpose of the buffer is also to closely maintain the PH of the medium at the selected level to ensure that changes in the dye colour are due to complex formation with Calcium and not to any uncontrolled variations in the reagent composition pH causing the dye to function as a pH indicator. The buffer must be of significant strength or overcome the influence on pH of the urine sample with which the matrix is contacted. The concentration of the buffer in the reagent composition is about 0.1 M to about 0.5M. The buffer is required to maintain the pH of the reagent composition in a range of about pH 10 to about pH 12. At the higher pH levels Cresolphthalein Complexone and Thymolophthalein Complexone respond to Calcium ions specifically however it is possible to include a magnesium compiexing agent in the reagent composition and employ a lower pH.

The tendency of the above dyes to bind Magnesium as well as Calcium is increased at lower pH levels of about 7 to about 8. However even then the association constant is much greater for Calcium than for Magnesium also the molar extinction for the Calcium dye complex is about double that for the Magnesium complex. Complex formation with Magnesium progressively declines as the pH is raised from about 7 to about 12. However the inclusion of a specific binding agent for Magnesium at any pH will minimise interference from this ion.

In practice a combination of high pH and the inclusion of a Magnesium binding agent in the reagent composition of the present invention will effectively eliminate magnesium as found in physiological fluids as a source of interference with the Calcium essay.

Examples of suitable masking agent are 8-Hydroxyquinoline or it's -5- sulphonic acid salt or N-Benzoyl-N-Phenyl-Hydroxylamine other suitable compiexing agents for Magnesium would readily be known to those skilled in the art. It has also been found that other non reactive materials may be included in the reagent composition to benefit such features of the present invention as the hue and depth of colour of the developed dye, the dispersion of colour across the reactive pad, the surface smoothness of the matrix and the speed of colour development and other desirable characteristics. Surface active agents have been found to speed the rate of matrix wetting as well as improving the brightness of the developed dye. Preferred agents (in amounts between about 0.1 % and about 2%) are Sodium Monyl benzyl (ethoxy)₅ Sulphate or Sodium Lauryi Sulphate but many other would be known to those skilled in the art of dry reagent strip manufacture. Additional salts, either organic or inorganic have been found to increase the sensitivity and range of reaction. Some preferred ones being Ammonium Sulphate and Sodium Acetate in amounts up to about 10% by weight of the final reagent composition.

Additional high molecular weight non ionic hydrophytic polymers can optionally be included in the reagent composition to improve surface characteristics of the reagent containing film which in turn assists a smooth non abrasive wipe off of excess urine or biological fluid sample.

Inclusion of this form of hyrophylic polymer can also be used to adjust the viscosity and other physiological parameters in order to facilitate the casting technology used to form a film from the reagent composition.

The present invention has used Poly vinyl pyrrolidine or Poly vinyl alcohol or methyl cellulose in amounts up to about 1 % in the reagent composition. Many other similar hydrophytic polymers would be suitable and well known to those familiar with the art.

It is envisaged that the reagent composition be used in the dry form contained within an absorbent carrier matrix. The carrier functions as a non reactive water stable porous housing and support for the dry reagents.

Generally - the absorbent carrier takes two forms. One, a pre existing porous pad into which the reagent composition is incorporated by a dipping process - single or multiple to impure sequentially reagents requiring different solvents aqueous to organic. The pad can take the form of a porous, granular, or micro porous medium composed of any number of inert materials such as cellulose, glass, diatomaceous earth or polymeric substances such as poly acids or polysulphane and others known in the art.

The second type of absorbent carrier takes the form of a polymeric substance usually but not always a film forming water stable emulsion known as a latex. This is included in a soluble form within the reagent composition and the total is cast onto an inert substrate as a film or membrane. On drying this film solidifies as a water insoluble but porous matrix containing the reagent composition.

However the use of absorbent carries such as filter paper and other polymeric substances presents a problem of compatibility with the strongly alkaline conditions of the reagent composition of the present invention. Australian patent Au-B 27015/92 to Miles Inc. addresses this problem and disclosed that substances known as moderating agents can be included in the reagent composition to moderate the averse effects of alkaline on an absorbent carrier.

Surprisingly and unexpectedly during the process of developing a suitable polymeric Latex for use as a film forming substance in the present invention, it was found that copolymers substantially of Styrene, that is 50% by weight or greater, were impervious and resistant to the destructive effects of the strong alkaline used within the confines of the present invention. Copolymers of 50% or greater of Styrene protected even known alkaline sensitive bonds such as esters. Thus copolymers of Styrene Butylacrylate or Styrene and Amino ethyl methacrylate or Styrene and Acrylic acid were all found to provide water stable emulsions giving rise to films resistant to the effects of strong alkaline.

A prefered embodiment therefore of the present invention includes in the reagent composition, an alkaline resistant Latex based on predominantly Styrene copolymers.

A further preferred embodiment includes as an absorbent carrier either cellulose filter type papers or microporus materials or standard (non Styrene) polymeric Latex films and optionally within the reagent composition an alkaline moderating agent as disclosed in the "Merc patent". According to the present invention a reagent composition is provided which includes a Calcium sensitive dye, a method of fixing that dye, a buffer and a porous or bibulous carrier matrix.

On contacting the dried composition in its matrix with Calcium containing urine or a physiological fluid, a detectable colour response develops. The intensity of this response is determined by the Calcium concentration of the sample fluid and it is sufficiently differentiated to allow quantitation either by direct visual colour comparison against a standard chart - or by measurement in a reflectance photometer at an appropriate wave length. The Creatinine sensitive Component.

The creatinine sensitive chromogen (and reagent composition) of the present invention must possess a number of critical characteristics. Primarily, it must be compatible with the adjacent calcium reagent composition which operates in a medium of high pH. In the presence of creatinine it must give a suitably detectable response. The response must be displayed across the range of normally encountered creatinine values, preferably in a linear fashion. Further, the dye must be specific for creatinine and be non-responsive to any interfering substances normally found in urine. The dye must also be amenable to fixing within the confines of the porous carrier matrix and on contact with the urine samples, no soluble dye should leach from the matrix.

The traditional assay method for the determination of creatinine is the Jaffe method. This involves the use of picric acid as the creatinine sensitive dye and entails a complex formation (charge transfer) between creatinine and picric acid in alkaline conditions to form a red coloured dye. While this invention exclusively assays creatinine in urine, it is well accepted that nominal changes to the traditional test need to be made, due to the fact that negligible amounts of Jaffe positive, non-creatinine materials are found in urine.

Due to the explosive nature of picric acid, it is no longer used in dry chemistry systems and current standard assays for creatinine instead utilise the dinitro derivatives. 3, 5-dinitrobenzoic acid was first described as a replacement for picrate as a creatinine sensitive dye by Langley and Eveans in 1936. However, 3,5-dinitrobenzamide, 2,4-dinitrobenzoic acid, 3,5-dinitrobenzoyl- phenyl glycine, 3,5-dinitrohydroxyphenylpropionic acid, 3, 5- dinitrobenzotrifluoride and others can also be used in this invention. These dye precursors are similar to calcium dyes because they are acidic or transform to acid. Similar to the calcium system discussed above, in order that the reaction of a creatinine sensitive dye, preferably 3, 5-dinitrobenzoic acid dye, with the creatinine results in a distinct colour change, it is crucial that the reagent system in which this reaction is occurring, be maintained at a high pH. That both the calcium and creatinine systems require strongly alkaline conditions is convenient because the similarity in the reaction pH conditions reduces the possibility of either pad interfering with the other and because both systems employ acidic dyes, the mordants used are similar.

Thus the Jaffe reaction appears to be an ideal partner for the calcium dye system of the present invention and one embodiment of this employs 3,5 dinitrobenzoic acid in the reagent composition.

AU-B-27015/92 to Miles Inc. discusses the use of a strong base, for example one of the alkali and alkaline metal hydroxides, to maintain the pH to a range of about 12-13 in order that the creatinine reacts readily with the creatinine reactive dye. It then goes onto discuss the deleterious effect of such strong bases upon the absorbent carrier and the consequential need for a moderating agent to minimise these effects. While the intensity of colour and the rate of reaction are intimately dependent on the pH value of the reagent composition, it has been found that a small increase of pH of the order of 0.1 units will change the kinetics of the reaction sufficient to introduce an error of +5% in the determined value. For this reason, one difficulty inherent in dry reagent technology with the Jaffe reaction has been the limitations imposed by the poor performance of the commonly used buffers in the region of about pH 11 to about ρH13 and particularly in the present invention about pH12 to 13. Dry chemical technology also imposes additional difficulties over the conventional wet system because the intended pH must develop after wetting the dry reagent composition. Having tried and rejected many of the common buffers because of poor performance (usually a downward drift over time), it was surprisingly and unexpectedly found that Sodium Metasilicate solution was able to function effectively as a buffer in the present invention in the desired pH range of about 11 to about 13.5. Most importantly, its buffering capacity was more than twice the strength of Di-Sodium Hydrogen Phosphate buffer in its buffer range. This buffer was unique amongst all of those buffers tried in the present system because within the reagent composition, it showed no downward drift of pH over time. Furthermore, it appeared to promote the even colour development of the reagent composition after being contacted with creatinine containing fluids. Sodium metasilicate further seemed to induce a desirable hardening effect on the absorbent carrier preventing any loss of substance on vigorous wiping and further still, it was compatible with the styrene based cationic latex preferred in some embodiments of the present invention.

Therefore, in contrast to the above mentioned Miles Patent, the instant invention avoids the use of strong bases to set the pH (thereby avoiding any possibility that the dye can exhibit colour development even though there may be no creatinine in the system). Instead, the reagent composition of the creatinine sensitive pad of the instant invention therefore employs a buffer to localise the pH of the reagent system to a precise 12.5. In this way, the fixed colour change can be attributed to the reaction of the dye with creatinine. The most preferred buffer is that of Sodium Metasilicate (water glass), as this is an extremely strong buffer which augments the colour development. However, other suitable buffers include Sodium Aluminium Hydroxide, Sodium Borate, Potassium Carbonate and Guanine Hydrochloride.

In addition, as both acidic creatinine sensitive dyes and acidic calcium sensitive dyes (as discussed above) are highly soluble and prone to leaching from their respective absorbent carriers, the creatinine sensitive reagent composition also preferably includes a fixing agent. The fixing agent is desirable, as the coloured form of the dinitrobenzoic acid creatinine dye, is water-soluble. Therefore, in the absence of a fixing agent, upon colour development, the colour immediately leaches off the test strip into the sample destroying any detectable results.

Not surprisingly, the same polyelectrolyte species employed as mordants for the calcium sensitive dyes, namely, polymerised quaternary ammonium salts, are also highly effective in fixing the creatinine sensitive dyes. Furthermore, use of the same quaternary ammonium cationic latex systems, will readily fix dyes such as 3,5 dinitrobenzoic acid or 2,4 dinitrobenzoic acid or 3,5- dinitrobenzamide in the absorbent carrier matrix. Poly diallyldimethylammonium chloride (Poly DADMAC) is the preferred species of fixative and can be obtained from ICI Aust Pty Ltd under the name of Matexil FC- ER.

Similarly to the calcium sensitive reagent composition, it has been found for creatinine that a number of non reactive substances can be optionally added in order to modify aspects of the creatinine pad that might be desirable. Addition of organic or inorganic salts will increase the intensity and range of the reaction. The addition of hydrophilic polymers as described for the calcium sensitive reagent conposition will refine the rheological properties of the casting mix and benefit such user characteristics as 'ease of wipe off. Other additives are well known to those skilled in the art of strip manufacture.

An absorbent carrier matrix for the creatinine is subject to the same destructive alkaline conditions described for the calcium reagent composition but because of the higher pH range, the conditions are more severe. The creatinine analyte however is uncharged and unlike calcium will be equally suited by an anionic or cationic latex. Thus, one embodiment of the invention is a reagent composition containing an alkali stable Styrene-acrylic copolymer latex as the absorbent carrier, but also with a polycationic soluble polymer as a mordant to fix the dye.

Another embodiment iincludes in the reagent composition, a latex copolymer of Styrene and a quaternary ammonium monomer which is able to function as a mordant as well as an alkaline stable latex.

Another embodiment includes as an absorbent carrier, either cellulose filter type paer or microporous polymer materials or standard (non Styrene) polymeric latex film and optionally within the reagent composition an alkaline moderating agent as disclosed in the US patent application 27015/92.

According to the present invention, a reagent composition is provided which includes a creatinine sensitive dye, a method for fixing that dye, a buffer and a porous or bibulous matrix. On contacting the dried compsition in its matrix with creatinine containing fluid, a detectable response occurs. The intensity of this response is determined by the creatinine concentration of the sample fluid and it is sufficiently differentiated to allow quantitation either by direct visual colour comparison against a standard chart or by measurerment in a reflectance photometer at an appropriate wavelength. WORKED EXAMPLES

The present invention is further illustrated in the following worked examples: Example 1

The creatinine sensitive indicator pad of the test device was prepared using the following reagents:

1. A latex emulsion employing a polymeric system of Styrene and Acrylic acid in a ratio of 70:30 with a non-volatile solids content of 42%. 2. 0.2M 3, 5 Dinitro benzoic acid in 0.2M NaOH.

3. 50% (gm/gm) Poly diallyldimethylammonium chloride (Poly DADMAC)

4. 25% (gm/gm) Sodium Metasilicate buffer pH12.8

5. Kaolin

These reagents were combined in the following order: 10g of the kaolin and 10g of the Dinitrobenzoate solution were blended.

To this was added an equal weight of the Latex emulsion. When evenly dispersed, 12g of Poly DADMAC and 7.5g of Sodium Metasilicate were added and the whole mixed well.

This composition was passed through a 40 micron exclusion filter and then spread onto a stable plastic substrate (such as Melinex from ICI) with a wet film thickness of 100 microns. The film was dried in a forced air oven at a temperature of about 60 degrees C, following which it was sectioned into 4mm wide ribbons which were applied along one edge of a plastic card adjacent to a similar Calcium sensitive ribbon, both being attached using a tranfere adhesive. On further sectioning the cards and ribbons were reduced to strips with the Calcium and Creatinine sensitive pads at one end.

The creatinine sensitive indicator pad in test strips made according to the above formulation, will, upon contact with a solution containing creatinine, exhibit a mauve colour change. On testing with a reflectance photometer (such as Betachek Lynx from

National Diagnostics Australia), it can be demonstrated (as shown in FIG 1 ) that the reflectance values obtained across the physiological range of urinary creatinine is essentially a linear function. Example 2

The calcium sensitive indicator pad of the test device was prepared using the following reagents:

1. A latex emulsion employing a polymeric system of Styrene and Diallyldimethylammonium chloride in a ratio of 80:20 with a non-volatile solids content of 40%.

2. Thymolphthalein Complexone mono sodium salt 3. 1.0M Sodium Borate buffer pH 10.0

4. 8-Hydroxy quinoline sodium sulphonate.

These reagents were combined in the following way: 1.0g of Thymolphthalein Complexone was dissolved in 15ml of Borate buffer. 4.0g of 8-Hydroxy quinoline sodium sulphonate was dispersed in 50g of the latex. Finally, the dye in the buffer and the 8-Hydroxy quinoline sodium sulphonate in latex were combined. This composition was spread onto a stable plastic substrate (such as Melinex from ICI) with a wet film thickness of 100 microns and driedand processed in the same fashion as the creatinine sensitive film. These are finally stored in a dessicator as dual pad (Calcium and

Creatinine) strips.

The calcium sensitive indicator pad in test strips made according to the above formulation, will, upon contact with a solution containing calcium ions, exhibit a blue colour change. On testing with a reflectance photometer (such as Betachek Lynx from

National Diagnostics Australia), it can be demonstrated (as shown in FIG 3) that the reflectance values obtained across the physiological range of urinary calcium levels, show regular incremental increases but not a linear function.

Claims (22)

1. A dry reagent test device which includes means adapted to indicate the calcium concentration of a urine test sample and means adapted to indicate the creatinine concentration of the test sample, such that the ratio of calcium to creatinine can be directly determined.

2. A dry reagent test device according to Claim 1 or claim 2 wherein the means adapted to indicate the calcium concentration of a urine test sample and the means adapted to indicate the creatinine concentration of the test sample are both in the form of reagent containing pads.

3. A dry reagent test device according to Claim 2 wherein the two reagent containing pads are adjacent each other and located at one end of said device.

4. A dry reagent test device according to claim 3 wherein the pads consist of an absorbent or bibulous carrier matrix.

5. A dry reagent test device according to claim 4 wherein the pads consist of an inert plastic material.

6. A dry reagent test device according to any one of claims 1 to 5 wherein the pad adapted to indicate the creatinine concentration of the test sample contains a reagent composition comprising a creatinine specific dye, a buffer, a fixative agent and a dye dispersant or carrier.

7. A dry reagent test device according to claim 6 wherein the creatinine specific dye is 3, 5-dinitro benzoic acid.

8. A dry reagent test device according to claim 7 wherein the buffer used in the creatinine sensitive reagent composition is Sodium Metasilicate.

9. A dry reagent test device according to any one of claims 1 to 5 wherein the pad adapted to indicate the calcium concentration of the test sample incorporates a reagent composition comprising a Calcium sensitive dye, a means of fixing that dye, a buffer, and a Magnesium specific binding agent.

10. A dry reagent test device according to claim 9 wherein the Calcium sensitive dye is an MSTPM dye.

11. A dry reagent test device according to claim 10 wherein the Calcium sensitive dye is Thymolphthalein complexone mono sodium salt.

12. A method for determining the calcium : creatinine ratio in urine which comprises contacting a urine test sample with the dry reagent test device of claim 1 , and determining the degree of colour development of each of the creatinine specific and calcium specific dyes .

13. A method according to claim 12, wherein the ratio of calcium -creatinine can be semi-quantitatively determined by the visual comparison of the results of the colourmetric reactions of the two reagent containing indicator pads with a urine test sample.

14. A method according to claim 12, wherein the ratio of calcium: creatine can be quantitively determined by comparing the results of the colourmetric reactions of the two reagent containing indicator pads with a -urine test sample, using a reflectance meter.

15. A method for the diagnosis of osteoporosis which comprises contacting a urine test sample with the dry reagent test device of claim 1 and comparing the degree of colour development of each of the creatinine specific and calcium specific dyes such that the urinary calcium : creatinine ratio can be determined.

16. A method for the preparation of a dry reagent test device as claimed in claim 3 which is used in the detection of urinary calcium loss which comprises: a) preparing an absorbent pad incorporating a reagent composition which is adapted to measure the creatinine concentration of a urine test sample, wherein the creatinine sensitive reagent composition consists of:

(i) a creatinine sensitive dye which reacts with creatinine under alkaline conditions,

(ii) a fixing agent,

(iii) a buffer

(iv) optional agents b) preparing an absorbent pad incorporating a reagent composition which is adapted to measure the calcium concentration of a urine test sample, wherein the calcium sensitive reagent composition consists of:

(i) a calcium sensitive dye which reacts with calcium ions under alkaline conditions,

(ii) a fixing agent,

(iii) a buffer

(iv) a magnesium specific binding agent

(v) optional agents

(c) incorporating the above pads onto a stable substrate.

17. A method for the preparation of a dry reagent test device as claimed in claim 3 which is used in the detection of urinary calcium loss which comprises: a) preparing an absorbent pad incorporating a reagent composition which is adapted to measure the creatinine concentration of a urine test sample, wherein the creatinine sensitive reagent composition consists of:

(i) 3,5-dinitrobenzoic acid

(ii) a polymer containing quaternary ammonium salts

(iii) sodium metasilicate

(iv a dispersant/water imbibing agent b) preparing an absorbent pad incorporating a reagent composition which is adapted to measure the calcium concentration of a urine test sample, wherein the calcium sensitive reagent composition consists of:

(i) a dye of the MSTPM group

(ii) a polymer containing quaternary ammonium salts

(iii) a buffer which maintains the pH of the composition in a range of about pH10-12

(iv) a magnesium specific binding agent

(c) incorporating the above pads onto a stable substrate.

18. The method of claim 18 wherein the creatinine sensitive reagent composition consists of:

(i) 3,5-dinitrobenzoic acid

(ii) polydiallyldimethylammonium chloride

(iii) sodium metasilicate

(iv) kaolin and wherein the calcium sensitive reagent composition consists of:

(i) Thymolphthalein complexone

(ii) Polydiallyldimethylammonium chloride

(iii) Sodium borate

(iv) 8-Hydroxyquinolinesodium sulphonate.

19. A dry reagent composition for determining the creatinine concentration in a urine test sample comprising:

(i) a creatinine sensitive dye which reacts with creatinine under alkaline conditions,

(ii) a fixing agent,

(iii) sodium metasilicate

(iv) optional agents.

20. A method for maintaining the pH of a dry reagent composition from about pH11 to about pH13.5 by using sodium metasilicate buffer.

21. An absorbent carrier matrix for a dry reagent composition comprising a polymeric latex emulsion using a majority styrene copolymer with a polycationic quaternary ammonium.

22. An absorbent matrix as claimed in claim 22 which incorporates a mordant.