WO1996004554A1 - Bandelette reactive permettant de determiner une perte de calcium avec les urines - Google Patents

Bandelette reactive permettant de determiner une perte de calcium avec les urines Download PDF

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Publication number
WO1996004554A1
WO1996004554A1 PCT/AU1995/000465 AU9500465W WO9604554A1 WO 1996004554 A1 WO1996004554 A1 WO 1996004554A1 AU 9500465 W AU9500465 W AU 9500465W WO 9604554 A1 WO9604554 A1 WO 9604554A1
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Prior art keywords
calcium
creatinine
reagent
dye
reagent composition
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PCT/AU1995/000465
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English (en)
Inventor
Anthony Brandon Bransgrove
Brandon Stephen Bransgrove
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S.A.N.D. Institute (Aust) Pty. Limited
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Application filed by S.A.N.D. Institute (Aust) Pty. Limited filed Critical S.A.N.D. Institute (Aust) Pty. Limited
Priority to AU30727/95A priority Critical patent/AU697395B2/en
Priority to EP95926337A priority patent/EP0775312A4/fr
Priority to NZ290093A priority patent/NZ290093A/xx
Publication of WO1996004554A1 publication Critical patent/WO1996004554A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/84—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving inorganic compounds or pH
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L25/00—Compositions of, homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by an aromatic carbocyclic ring; Compositions of derivatives of such polymers
    • C08L25/02—Homopolymers or copolymers of hydrocarbons
    • C08L25/04—Homopolymers or copolymers of styrene
    • C08L25/06—Polystyrene
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
    • G01N33/521—Single-layer analytical elements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/70—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving creatine or creatinine
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L47/00—Compositions of homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, at least one having two or more carbon-to-carbon double bonds; Compositions of derivatives of such polymers

Definitions

  • 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.
  • 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.
  • osteoporosis 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the pads consist of an absorbent or bibulous carrier matrix.
  • the pads consist of latex.
  • 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).
  • 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).
  • the creatinine specific dye is Dinitro benzoic acid.
  • the buffer used in the creatinine sensitive reagent composition is Sodium Metasilicate
  • 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.
  • the Calcium sensitive dye is an MSTPM dye such as
  • Thymolphthalein complexone mono sodium salt 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 .
  • 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.
  • 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:
  • a fixing agent (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,
  • 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.
  • it is thus possible to achieve an early diagnosis of osteoporosis and consequently allow for preventative treatment to be initiated.
  • the costs to the public health care and hospital system are therefore minimised, as osteoporetic fractures can be avoided.
  • Figure 1 is a side elevation view of a dryreagent test strip incorporating the creatine sensitive pad and the calcium sensitive pad
  • FIG. 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the mordant is bound to or is part of an insoluble resin or matrix.
  • the slight solubility of some dye-mordant complexes will be obviated.
  • 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.
  • 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, polyvinylm ⁇ thylpyridine- chloride, polytrimethylaminoethylmethacrylatechloride, polytrimethyl- aminopropylamidomethacrylate, polydiallyldimethylammoniumchloride and polymonoallyltrimethylammoniumchloride.
  • 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.
  • 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.
  • suitable masking agent examples include 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 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.
  • 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.
  • 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.
  • 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.
  • 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".
  • a reagent composition which includes a Calcium sensitive dye, a method of fixing that dye, a buffer and a porous or bibulous carrier matrix.
  • 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.
  • 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.
  • a strong base for example one of the alkali and alkaline metal hydroxides
  • 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.
  • 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).
  • 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.
  • other suitable buffers include Sodium Aluminium Hydroxide, Sodium Borate, Potassium Carbonate and Guanine Hydrochloride.
  • 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.
  • the same polyelectrolyte species employed as mordants for the calcium sensitive dyes namely, polymerised quaternary ammonium salts
  • 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.
  • 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.
  • 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.
  • a reagent composition which includes a creatinine sensitive dye, a method for fixing that dye, a buffer and a porous or bibulous matrix.
  • a detectable response occurs on contacting the dried compsition in its matrix with creatinine containing fluid.
  • 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.
  • Example 1 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:
  • 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.
  • Latex emulsion 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.
  • a stable plastic substrate such as Melinex from ICI
  • 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.
  • a reflectance photometer such as Betachek Lynx from
  • the calcium sensitive indicator pad of the test device was prepared using the following reagents:
  • 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%.
  • Thymolphthalein Complexone mono sodium salt 3. 1.0M Sodium Borate buffer pH 10.0
  • 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.
  • a reflectance photometer such as Betachek Lynx from

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Abstract

On décrit une bandelette réactive qui permet de déterminer directement les rapports calcium/créatinine, particulièrement ceux de l'urine. On décrit aussi un procédé de fabrication de cette bandelette et des compositions de réactifs appropriées.
PCT/AU1995/000465 1994-08-01 1995-08-01 Bandelette reactive permettant de determiner une perte de calcium avec les urines WO1996004554A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
AU30727/95A AU697395B2 (en) 1994-08-01 1995-08-01 Urinary test strip for determining calcium loss
EP95926337A EP0775312A4 (fr) 1994-08-01 1995-08-01 Bandelette reactive permettant de determiner une perte de calcium avec les urines
NZ290093A NZ290093A (en) 1994-08-01 1995-08-01 Urinary test strip; determination of calcium creatinine; manufacture of strip; reagent composition

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AUPM7176A AUPM717694A0 (en) 1994-08-01 1994-08-01 A test strip for the rapid quantification of urinary calcium loss
AUPM7176 1994-08-01

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WO1996004554A1 true WO1996004554A1 (fr) 1996-02-15

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PCT/AU1995/000465 WO1996004554A1 (fr) 1994-08-01 1995-08-01 Bandelette reactive permettant de determiner une perte de calcium avec les urines

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EP (1) EP0775312A4 (fr)
AU (1) AUPM717694A0 (fr)
CA (1) CA2196546A1 (fr)
NZ (1) NZ290093A (fr)
WO (1) WO1996004554A1 (fr)

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EP0797952A1 (fr) * 1996-03-26 1997-10-01 Hologic, Inc. Système pour déterminer les charactéristiques osseuses
AT403325B (de) * 1996-06-14 1998-01-26 Lehmann Juergen Teststreifen für urinanalyten und auswertefarbtafeln
US6436721B1 (en) 1997-07-25 2002-08-20 Bayer Corporation Device and method for obtaining clinically significant analyte ratios
WO2009050711A3 (fr) * 2007-10-18 2010-03-11 Jacob Mullerad Dispositif diagnostic pour identifier la rupture de la membrane pendant la grossesse
CN102759524A (zh) * 2011-04-28 2012-10-31 爱科来株式会社 钙测定用干式试验片
US9128168B2 (en) 2007-12-14 2015-09-08 Cornell University Method of determing excretion of sodium and other analytes
EP3640633A1 (fr) 2018-10-16 2020-04-22 UriSalt GmbH Moyens pour la détermination quantitative de la concentration d'électrolyte cationique et la concentration de créatinine et de leurs rapports
JP2020173186A (ja) * 2019-04-11 2020-10-22 株式会社ファンケル カルシウム摂取量の過不足判定方法
EP4102225A4 (fr) * 2020-04-23 2024-03-06 Yukashikado Inc Papier d'essai destiné à la mesure de minéraux dans l'urine

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Cited By (15)

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Publication number Priority date Publication date Assignee Title
US5785041A (en) * 1996-03-26 1998-07-28 Hologic Inc. System for assessing bone characteristics
AU726029B2 (en) * 1996-03-26 2000-10-26 Hologic, Inc. System for assessing bone characteristics
EP0797952A1 (fr) * 1996-03-26 1997-10-01 Hologic, Inc. Système pour déterminer les charactéristiques osseuses
AT403325B (de) * 1996-06-14 1998-01-26 Lehmann Juergen Teststreifen für urinanalyten und auswertefarbtafeln
US6436721B1 (en) 1997-07-25 2002-08-20 Bayer Corporation Device and method for obtaining clinically significant analyte ratios
WO2009050711A3 (fr) * 2007-10-18 2010-03-11 Jacob Mullerad Dispositif diagnostic pour identifier la rupture de la membrane pendant la grossesse
US9128168B2 (en) 2007-12-14 2015-09-08 Cornell University Method of determing excretion of sodium and other analytes
CN102759524A (zh) * 2011-04-28 2012-10-31 爱科来株式会社 钙测定用干式试验片
EP2538224A1 (fr) * 2011-04-28 2012-12-26 ARKRAY, Inc. Bande de test à sec pour mesurer le calcium
US8784748B2 (en) 2011-04-28 2014-07-22 Arkray, Inc. Dry test strip for measuring calcium
JP2012237745A (ja) * 2011-04-28 2012-12-06 Arkray Inc カルシウム測定用乾式試験片
EP3640633A1 (fr) 2018-10-16 2020-04-22 UriSalt GmbH Moyens pour la détermination quantitative de la concentration d'électrolyte cationique et la concentration de créatinine et de leurs rapports
WO2020078756A1 (fr) 2018-10-16 2020-04-23 Urisalt Gmbh Moyens pour la détermination quantitative d'une concentration d'électrolyte cationique et d'une concentration de créatinine et de leurs rapports
JP2020173186A (ja) * 2019-04-11 2020-10-22 株式会社ファンケル カルシウム摂取量の過不足判定方法
EP4102225A4 (fr) * 2020-04-23 2024-03-06 Yukashikado Inc Papier d'essai destiné à la mesure de minéraux dans l'urine

Also Published As

Publication number Publication date
EP0775312A1 (fr) 1997-05-28
AUPM717694A0 (en) 1994-08-25
CA2196546A1 (fr) 1996-02-15
EP0775312A4 (fr) 1998-05-06
NZ290093A (en) 1998-12-23

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