MATERIALS AND METHODS FOR DETECTING OXALATE

Abstract: The subject invention pertains to a novel assay device for detecting oxalate in a sample. The assay device comprises enzyme and dye compositions immobilized on a solid carrier matrix. The subject invention can also be used to measure the concentration of oxalate in a sample. The subject invention further pertains to a novel oxalate oxidase composition and methods of preparing the subject enzyme composition. The oxalate oxidase composition can be used in the assay device of the subject invention.

Description: DESCRIPTION MATERIALS AND METHODS FOR DETECTING OXALATE This invention was made with government support under grant number 5PO 1 DK20586 awarded by the National Institute of Health The government has certain nghts in this invention Background of the Invention Uπnary oxalate concentration is an important πsk factor in patients that are predisposed to kidney stone formation (nephrohthiasis) Oxalate concentration is also used for diagnosis of urohthiasis and primary hyperoxaluπa (Thun et al ,1991) The normal range of oxalate excretion in uπne is about 0 5 to 1 5 mM/dl (Smith, 1991) Most current methods of measuring uπnary oxalate levels rely on expensive and time-consuming techniques that involve solvent extraction, spectrophotometπc determinations or high performance chromatography and, therefore, are of little practical value in a clinical setting U S Patent No 4,455,371 (Richardson and Obzansky) descπbes an oxalate oxidase composition that is used in a method to assay for oxalate Oxalate oxidase oxidizes oxalate molecules to carbon dioxide and hydrogen peroxide An assay for oxalate using oxalate decarboxylase to convert oxalate to carbon dioxide and formate has also been descπbed (Costello et al , 1976, Ynbem et al , 1980, Santamaπa et al , 1993) Methods for detecting either the carbon dioxide, hydrogen peroxide or formate produced from the enzymatic reaction are then employed to indirectly detect the presence of the oxalate For example, formate dehydrogenase can be used to reduce NAD to NADH in the presence of formate The NADH produced can be detected spectrophotometncally However, these assays for oxalate are time-consuming and require the use of complicated equipment The isolation and puπfication of oxalate oxidase compositions from moss, barley and beet stems has been described in the art (Chiπboga, J 1966, U S Patent No 4,455,371) The methods described require extensive puπfication procedures and result in relatively low enzyme yield and specific activity In addition, these oxalate oxidase compositions can be susceptible to temperature variations and are unstable when stored for extended peπods These enzyme preparations may also require the addition of exogenous co-factors to catalyze the enzymatic reaction Oxalate oxidase compositions are also available from commercial sources (Sigma Chemical Co , St Louis, MO) but suffer from similar problems "Dip and read" or "Dipstick" assays are currently used to measure uπnary glucose levels (e g , "TES-TAPE," Eli Lilly), determine microalbuminuπa, determine uπne specific gravity, detect bacteria or viruses, determine cholinesterase activity in human plasma, and the like Urinary glucose dipsticks are categorized as enzymatic test stπps, in which the substrate (e g , glucose) is oxidized by an enzyme to yield a detectable signal Based on the signal detected on the dipstick, which is usually manifested as a color or shade of color, the concentration of an analyte in a sample can be readily determined Dipstick-based assays can provide an inexpensive and rapid method for detecting or measuring the amount of a specific analyte in a sample. Moreover, the simplicity and ease of use of dipstick-based assays makes them suitable for patient self-screening or monitoring on a routine basis. Thus, dipstick-type assays have an important clinical utility. Published Japanese patent application No. 63074500 discloses a dipstick assay that utilizes oxalate oxidase for detecting oxalate in a sample. However, this assay lacks sensitivity for oxalate concentrations below about 2 mg/dl and the oxalate oxidase enzyme used with the device may suffer from instability or degradation during its shelf life or as a result of environmental factors. In addition, the other enzyme compositions taught in the art suffer similar shortcomings and, therefore, are also unsuitable for use with a dipstick-based assay for detecting oxalate. Accordingly, there remains a need in the art for a sensitive, rapid and stable assay system to detect oxalate in a sample, the requirements of which are met by the present invention. A dipstick assay of die present invention for determining urinary oxalate levels, comprising an enzyme and dye-based assay system, is inexpensive to make and administer, would not require laboratory equipment or experience, and could be performed in significantly less time than current techniques. Summary of the Invention The subject invention concerns a novel assay device and methods for detecting oxalate in a sample. The assay device comprises enzyme and dye compositions immobilized on a carrier matrix. The assay device of the invention is contacted with a sample to be analyzed and a detectable color reaction develops on the carrier matrix if oxalate is present in the sample. The concentration of oxalate in a sample can be determined by comparing the color or color intensity that is observed on the carrier matrix with a color chart that was precalibrated using different concentrations of oxalate. The assay device of the subject invention can be used to detect oxalate in a variety of samples, including urine, blood, plasma, bile, saliva and other biological fluids. Accordingly, it is an object of the present invention to provide a sensitive, rapid, and stable assay for the detection and measurement of oxalate in a sample. The subject invention further concerns a novel oxalate oxidase enzyme composition and methods for producing the oxalate oxidase composition. The oxalate oxidase produced according to the subject invention provides an enzyme having greater specific activity and yield. The enzyme of the present invention is isolated using fewer purification steps than other oxalate oxidase compositions described in the art. Moreover, the oxalate oxidase composition of the present invention is advantageously more stable and does not require the addition of exogenous co-factors for enzymatic activity unlike other oxalate oxidase preparations. The oxalate oxidase produced and isolated according to the subject invention can be utilized with the assay device and method of the present invention to detect and measure the concentration of oxalate in a sample. Brief Description of the Drawiηgs Figure 1 shows the isoelectric focusing profile of oxalate oxidase from barley. Proteins purified by ammonium sulfate precipitation (30/60ASP), resuspended and dialyzed against 1% glycine, were introduced into a preparative isoelectric focusing column with carrier ampholines (pH 2.5 to 5.0) in a sucrose gradient. After electrophoresis at 1500 v for 22 hours at 5°C, fractions (1 ml) were removed and continuously scanned at 280 nm (line graph), then individually dialyzed, and assayed for oxalate oxidase activity (bar graph) as described in the Materials and Methods section. Only the earlier eluted fractions of the gradient profile are shown; no enzyme was detected in the later eluted fractions. Detailed Description of the Invention The subject invention concerns a novel assay device for detecting oxalate in a test sample. The assay device of the subject invention comprises enzymes and dye components immobilized on a carrier matrix. The enzyme components used with the subject device are capable of reacting with oxalate as a substrate to produce a first reaction product. The first reaction product can react with the dye components on the carrier matrix to produce a detectable second reaction product or response that is coπelated to the amount of oxalate in the sample being tested. In a preferred embodiment, the enzyme components of the subject invention comprise oxalate oxidase and a peroxidase. The oxalate oxidase can be prepared from plant tissues. The oxalate oxidase impregnated or immobilized on the carrier matrix is contacted with a sample to be tested and reacts with oxalate in the test sample and oxygen to produce hydrogen peroxide and carbon dioxide. The hydrogen peroxide produced further reacts with a peroxidase of the assay system to produce nascent oxygen, the first reaction product. The nascent oxygen thereby produced then reacts with a dye component on the carrier matrix to produce a second reaction product or response that can be detected on die matrix. Preferably, the detectable reaction product is a particular color or shade of color that is formed on the carrier matrix. The dye composition of the subject invention comprises a dye that can be oxidized by the first reaction product to a detectable oxidized form. Preferably, the dye is ortho-tolidine. In another embodiment of the present invention, the dye composition comprises dimemylaminobenzoic acid (DMAB) and 3-methyl-2-benzothiazolinone (MBTH). In an alternate embodiment, N,N-dimethylaniline (DMA) can be substituted for DMAB. These dye components can react with nascent oxygen, which is produced upon the reaction of the enzyme components with oxalate, to form an indamine dye which is blue in color. Other dye compositions are known in the art and are contemplated for use in the subject invention. The carrier matrix of the assay device can be composed of any substance capable of being impregnated witii the enzyme and dye components of the subject invention, as long as the matrix is substantially inert with respect to the analyte being assayed for. Preferably, the carrier matrix is porous and/or absorbent relative to the sample to be tested. The expression "carrier matrix" refers to either bibulous or nonbibulous matrices which are insoluble in water, and maintain their structural integrity when exposed to water or to other physiological fluids. Suitable bibulous matrices which can be used include paper, cellulose, wood, synthetic resin fleeces, woven and nonwoven fabrics and the like. Paper is a prefeπed carrier matrix. Nonbibulous matrices includes glass fiber, polymer films, preformed or microporous membranes and plastic materials, such as polypropylene and the like. In one embodiment of the assay device, purified chromatography paper can be impregnated with the enzyme and dye components of the present invention. A strip of paper, plastic, or wood with a piece of absorbent material (e.g., cotton, synthetic fibers, or slab of polymer) attached at the tip can be employed in a similar manner. A piece of cloth can be impregnated with a polymer (e.g., the polymers used to immobilize the dye system) containing the enzyme and dye components. The polymer acts as a laminate adding rigidity to the material. The cloth may be woven of natural fibers (e.g., cotton) or synthetic fibers (e.g., polyethyleneteraphthalate). A polymer matrix, such as porous polyurethane or spunbond polyolefin, which has been impregnated with the enzyme and dye compositions of the present invention can also be employed. Any improvements in conventional solid support materials used with the carrier matrix are also encompassed within the scope of the invention. Incorporation of die enzyme, dye and other reagent components on the carrier matrix can be accomplished by any method such as dipping, spreading or spraying. A preferred method is impregnation of the carrier matrix material by dipping in a reagent solution and drying to remove solvent. Drying can be accomplished by any means which will not deleteriously affect the reagents incorporated, and typically is by means of an air drying oven. Wetting agents (e.g., a dilute surfactant) can be employed to increase the efficiency of the carrier matrix. Reagent alcohol can also be added to the dye system solution to accelerate the drying process once the caπier matrix is coated with the enzyme and dye components. Reagents and solutions known in the art can be employed to stabilize and immobilize the enzyme and dye components of me subject invention to a carrier matrix. For example, salts and buffers may be used to preserve the activity of the enzyme and dye components. Ammonium sulfate, phosphate buffer, buffered salines, citrate, and succinic acid, among others, can be used to maintain pH at an optimum level (e.g., pH 3.5 for oxalate oxidase). Sugar solutions can also be used to maintain the activity of the enzyme and dye components over time. A trehalose solution can also be used to enhance the stability of oxalate oxidase immobilized on a carrier matrix and stored at room temperature. For example, a solution of about 15% trehalose can be used to stabilize die activity of oxalate oxidase in the present assay device. Polymer solutions, such as alginate, chitosan, acrylic acid, and carboxymethylcellulose, can also be used to maintain the activity of dye components over time. Other components such as thickeners, color stabilizers, surfactants or the like can also be added. The addition of a polymer such as polyvinylpyrrolidone can be used to increase stability and the uniformity of color formation on the test device. The addition of certain minerals can increase the activity (rate at which an enzyme catalyzes a reaction) of specific enzymes. For example, copper ions enhance the activity of oxalate oxidase isolated from leaves of grain sorghum. One can also add one or more natural products to the assay device to increase Uie activity of specific enzymes (e.g., riboflavin enhances the activity of oxalate oxidase isolated from barley seedlings) Chelatmg agents, such as EDTA, can also be mcorporated m the assay device to remove certain minerals which can decrease the activity of specific enzymes (e g , iron ions inhibit the activity of oxalate oxidase isolated from barley seedlmgs). The chelation of calcium helps prevent the precipitation of calcium oxalate salts The use of other agents to enhance enzymatic activity is known in the art The amount of oxalate present in a test sample can be semi-quantitatively determined by visually comparing the degree of the color development on the matrix with a standardized color chart that has been correlated to known concentrations of oxalate Accordingly, it is preferable that the dipstick be uniformly wetted wiui the test sample so that a density of the color can be judged over a wide range of concentrations and to provide for stable color tone after the color development In this respect, the assay device of the present invention provides for excellent sensitivity, color stability and a wide color density range In addition to visual compaπson, vaπous instrumental mettiods can also be employed to detect the intensity or amount of the reaction product present on the earner matπx The color range of the present assay device when oxalate oxidase is employed as an enzyme component can be expanded by adding certain dyes to the earner matπx For example, the incorporation of FDC Yellow No 5 dye coloring to die earner matrix along with a dye component such as ortho-tohduie of the present assay device provides for a detectable color that changes from yellow to green to blue to purple in proportion to me concentration of oxalic acid in a sample The different colors or shades thereof make it easier to distinguish between vaπous concentrations of oxalate, particularly at lower concentrations of oxalate in a test sample The yellow dye does not interfere with the color development of the assay device Other dyes known in the art can be used witii the present invention In one embodiment, the assay device of the present invention can detect oxalate in a sample at concentrations as low as about 1 0 to about 0.5 milligrams (mg) per deciliter (dl) Other enzyme compositions which react with oxalate are contemplated for use with the subject invention In addition, any enzyme with peroxidase activity can be used in conjunction with the oxalate oxidase and assay device of the subject invention For example, soy bean peroxidase (Enzymol International, Inc.) may be used in lieu of horse radish peroxidase in conjunction with oxalate oxidase m the assay device. By modifying the assay methods of the subject invention (/ e , the components of the assay device which react with die substrate), other factors related to oxalate levels can also be measured The subject invention also concerns a metiiod for detecting or measuring die amount of oxalate in a sample. In one embodiment, a dipstick assay device of the present invention is contacted witii a sample for an effective penod of time. Typically, the device is contacted with the sample for between about 1 second to about 5 minutes Any oxalate that may be present m the sample can react with the enzyme and dye components to produce a detectable reaction product Optionally, the device can be washed with a suitable wash buffer, such as .025M sodium succinate buffer at pH 4 1, before and/or after contacting the assay device with uie sample to remove paniculate materials Preferably, the detectable reaction product is a visible color or shade of color deposited on the earner matrix of the assay device The amount of oxalate present m die sample can be determmed by compaπng the mtensity or shade of color deposited on the assay device to a precahbrated oxalate concentration color chart In another embodiment, oxalate is detected in a liquid or aqueous sample using a spot test assay An enzyme composition and a dye composition according to die present mvention and in liquid phase are contacted with a sample to be tested for a sufficient amount of fame Alternatively, me sample is contacted with enzyme and/or dye composition as die order of combining the reagents and sample is not critical to the subject method The sample and enzyme and dye compositions are combmed on a porous or non- porous surface For example, the surface can be a glass plate, bench toweling with a cellophane backmg, or filter paper Optionally, the surface can be washed with a suitable buffer after contact with the sample Typically, the sample is reacted for between a few seconds to about 5 minutes Oxalate that may be present m the sample can react with the enzyme and dye components to produce a detectable reaction product Preferably, the detectable reaction product is deposited on the surface and can be detected coloπmetπcally The spot test assay of the present invention can be readily adapted for use witii commercially available assay systems, such as the Kodak EKTACHEM DT system Preferably, the test sample to be assayed in the spot test assay is devoid of salt or other inhibitory substances, is soluble in water or will release oxalate to an applied aqueous solution, and the sample composition or color will not mask visual or other coloπmetric means of detection of the detectable reaction product The subject mvention can be used to assay for oxalate in a sample, including biological samples Biological samples or specimens that can be assayed using the methods and devices of the invention include for example, blood, plasma, urine, stool, semen, bile, saliva, tears, ammotic fluid, synovial fluid, bone, tissue, or other biological components from an individual or subject animal In addition, contents from the stomach or gastrointestinal tract of an animal that have been erther mechanically removed or naturally expelled (e g , vomited or regurgitated) can also be assayed usmg the methods and devices of the subject invention The methods and materials of the subject invention can be used to assay for oxalate in a variety of applications For example, the methods can be used to detect oxalate m consumable products, such as, for example, beer, wine, or the water used to produce beer, wine and other beverages, to determine whether levels of oxalate have been sufficiently reduced or removed after treatment of wood or leatirer witii oxalic acid, to detect the presence of high or deleterious levels of oxalate in plant matter and animal food The present invention can also be used to assay for oxalate m Bayer liquors in the aluminum industry and pulping liquors in die paper industry In addition, the methods of the subject invention can be used to assay for oxalate oxidase in a variety of medical applications including, for example, in the diagnosis of urohthiasis, primary hyperoxaluπa, vulvar vestibuhtis, and chronic renal failure The present invention also concerns materials and methods for providing oxalate oxidase in a therapeutic protocol In one embodiment, oxalate oxidase is encapsulated in a microcapsule, for example, as described in U S Patent No 5,286,495 The encapsulated oxalate oxidase can be administered to a person in need of such treatment. Preferably, the oxalate oxidase encapsulated is oxalate oxidase produced according to die mediods of the present invention. The subject invention further concerns a novel oxalate oxidase composition and methods for producing it. Advantageously, oxalate oxidase prepared according to the present invention has greater specific activity and enzyme yield from fewer purification steps than other mediods of obtaining oxalate oxidase compositions. In addition, die oxalate oxidase of the subject invention is more stable and does not require the addition of exogenous co-factors for activity. The stability of the oxalate oxidase composition to temperature, low pH, ediyl and isopropyl alcohols, acetone, lyophilization, anionic detergents, and microencapsulation makes this preparation highly desirable for numerous medical and industrial uses in both the soluble and immobilized state. A further aspect of the present invention concerns methods for preparing oxalate oxidase compositions useful in die present assay device. The oxalate oxidase can be isolated from plant tissue. In a preferred embodiment, oxalate oxidase is prepared from germinated seeds tiiat are frozen and then thawed during the extraction of die enzyme from the plant tissue. More preferably, die seedlings are quick frozen by immersion in liquid nitrogen and dien thawed during homogenization of the tissue in the initial stage of purification. Typically, the seeds used are barley seeds (Hordeum vulgare). Other grain seeds are contemplated within the scope of die present invention. Preferably, the seeds are germinated by immersion in water and under conditions which exclude light during germination (etiolated) and favor root growth. The ability to freeze die germinated seedlings without significant loss of enzyme activity allows large quantities of source material to be accumulated and stored until it is ready to be used. In a prefeπed embodiment, oxalate oxidase of die present invention is isolated by homogenizing frozen plant tissue and filtering the liquid extract obtained after homogenization to remove particulate matter. Optionally, PMSF and/or EDTA can be included during the purification of die enzyme. The liquid extract is then centrifuged and the supernatant removed from the pelleted material. The supernatant is then heat-treated. Preferably, the supernatant is heated to between about 70° and 80°C for between about 5 to 12 minutes. More preferably, die supernatant is heated to about 75° for about 10 minutes. After heat- treatment, die supernatant is chilled close to freezing. Preferably, the supernatant is chilled to between about 3° to 4° C. After chilling, die supernatant is centrifuged again to pellet precipitated material. The heat-treated supernatant is separated from die pelleted material and brought to about 30% ammonium sulfate saturation. The precipitate in the 30% saturated solution is removed, preferably by centrifugation to pellet die precipitate. The supernatant is then subsequently brought to about 60% ammonium sulfate saturation. The precipitate in the 60% saturated solution is obtained, again preferably by centrifugation. The supernatant is discarded and the precipitated material, which contains the oxalate oxidase, is resuspended in solution. The method of die subject invention provides an enzyme yield of at least about 0.5 enzyme units per gram of starting plant tissue. Preferably, the enzyme yield is in the range of about 0.5 to 0.9, or greater, enzyme units per gram of plant tissue. In a preferred embodiment, the oxalate oxidase prepared by the metiiod of me present invention has a specific activity of at least about 8.5 enzyme units per milligram of protein. Preferably, the oxalate oxidase has a specific activity of at least about 10 enzyme units per milligram of protein. More preferably, die oxalate oxidase composition of me subject invention has a specific activity of at least about 20 enzyme units per milligram of protein. Optionally, the oxalate oxidase can be further purified by isoelectric focusing and collecting die fractions between about pH 2.5 and 3.0. Materials and Methods Materials. Fresh Wysor barley, lot #6321 -38B, from Wetsel Seed Co., treated with VITA VAX 200 (fungicide) and RELDAN (insecticide) and Venus barley, lot #BSCNC87-197 from Food Science and Human Nutrition, University of Florida, were used in the production of oxalate oxidase. Seed was distributed in 1 kg lots in sealed plastic bags and stored at 4-6°C. Oxalate oxidase, horse radish peroxidase (Type VI) bovine serum albumin, enzyme grade ammonium sulfate, phenylmethylsulfonylfluoride (PMSF), N, N-dimethylanaline, and 3-methyl-2-benzothiazolinone hydrazone were obtained from Sigma Biochemicals (St. Louis, MO). PHARMALYTE caπier ampholines were from Pharmacia LKB Biotechnology (Uppsala, Sweden). Seedling germination, harvesting, and storage. Seeds were germinated in 1 kg batches in a perforated, plastic pipet basket by immersion in tap water for 24 hours at 23° to 26 °C, followed by incubation beneath a shower of tap water, with shaking twice a day, for seven to nine days. Spreading imbibed seed over a cheesecloth-lined plastic rack beneath the shower and enclosing the system in a black plastic polyethylene curtain to exclude light enhanced the germination rate, favored root growth, and facilitated harvesting the appropriate tissue. After incubation, whole seedlings were blotted to remove excess water and die root rich portions harvested directly into liquid nitrogen. The frozen tissue was stored in covered plastic specimen cups (120 to 130 g) at -70 °C. Oxalate oxidase activity assays. Oxalate oxidase activity was determined using a modification of the spectrophotometiic assay method of Laker et al. ( 1980) for urinary oxalate. Fresh dye reagent containing 250 μl oxalic acid (200 mM), 2.5 ml sodium succinate, pH 4.0 (0.25 M), 250 μl 3-methyl-2- benzothiazolinone hydrazone (2.5 g/1 in 0.1 M HC1 ), 1.25 ml N,N-dimethylaniline (2.5 g/1 in 0.1 M HC1), and 350 units horse radish peroxidase was brought to 25 ml with glass distilled water in an amber bottle. A sample aliquot (10 to 200 μl) in a plastic microcuvette (1 cm light path) was diluted to 200 μl with 25 mM sodium succinate buffer. At zero time, 2.0 ml of the dye reagent was added to the cuvette with mixing, incubated at 23 °C in the dark, and then read at 595 nm at 2 or 5 minute intervals for 8 or 20 minutes. Samples were read against a buffer blank, i.e., a microcuvette containing the same dye reagent but without the oxidase sample added in. One Enzyme Unit of activity is defined as an increase in absorbance at 595 nm of one per minute at 23 °C. Alternatively, a semi-quantitative assay was utilized for die presumptive detection of oxidase activity 100 μl of the above dye reagent was placed in one well of a plastic microtitre plate 10 μl of sample was added with mixing and mcubated at room temperature for 5 to 60 minutes Development of a purple color was observed visually and the relative mtensity scored (0 to 4) agamst that of a well contammg dye reagent devoid of sample. Protein assay The concentration of tπchloroacetic acid-insoluble protein was estimated spectrophotometncally by die bicinchoninic acid mediod (BCA) of Smith et al (1985) using bovine serum albumin (BSA) as a standard Color was developed by incubation at 60°C for 30 minutes, followed by cooling m ice water prior to readmg. Enzvme preparation 275 g of frozen barley tissue from etiolated seedlmgs and 3 ml of phenylmethylsulfonyl fluoride (PMSF) (10 mg/ml in isopropanol) were added to 600 ml of 0 1 mM ethylenediaminetetraacetic acid (EDTA) in a teflon-lined, commercial blender The mixture was ground at high speed for 15 seconds, twice, to a consistency approximating oatmeal, and immediately filtered through 4 layers of cheeseclodi into a cold cylinder The remaining liquid was expressed from the tissue by manually squeezing die bolus in the cheesecloth. This yielded about 800 ml of extract at pH 5 The extract was immediately centrifuged at 20,000 x g for 20 minutes at 0°C and the clear, amber low speed supernatant (LSS) decanted into a two liter round-bottom flask The flask was mounted on a rotating evaporator (no vacuum) witii the flask immersed in a water bath at 84 °C The temperature of the LSS was raised to about 75 °C in 10 minutes, men immediately chilled m an ice bath, centπfuged as described above, and die heat treated supernatant (HTS) retained The HTS, at pH 5, was brought to 30% saturation by adding freshly powdered ammonium sulfate ((NH 4 ) 2 S0 4 , 175 7 g/1) with constant stirring Stimng was continued for 30 minutes at 0°C and the mixture was then centπfuged as above for 10 minutes The clear, amber supernatant fraction was decanted into a cold beaker and brought to 60% saturation by the addition of more ammonium sulfate (195 g/1) as descnbed above. The white mateπal that collected at the solution surface was removed, and die mixture again centπfuged as descnbed above The supernatant fraction was discarded, the pellets drained, and the bottle walls opposite the pellets wiped free of debris The pellets were suspended m 70 ml of 0 1 mM EDTA, 100 μg/ml PMSF and dialyzed against four liters of distilled water for 36 to 48 hours at 4°C, unless noted otiierwise This preparation is referred to herem as the 30/60 ASP preparation The dialyzed 30/60ASP preparations were centrifuged at 10,000 x g for 10 minutes to clarify, if necessary, and lyophilized at -60 °C Isoelectnc focusing. Samples were introduced mto a 110 ml LKB preparative column with a 1% PHARMALYTE pH 2 5-5 earner amphohne gradient m 5 to 60% sucrose (Sugiura et al , 1979) Proteins were focused for 22 hours at 5°C with a initial current of 7 mA at a constant potential of 1500 v (anode at the bottom) accordmg to die method of Vesterberg and Svensson (1966) After stabilization of the current at 3 1 mA (22 hours), the resolved components were collected by hydrostatic displacement from the bottom of die column in 1 ml fractions at 1 ml/mm and continuously scanned at 280 nm Each fraction was dialyzed at 5°C against 0 1M NaCl, 5 mM sodium succinate, pH 4 1, 50 μg/ml PMSF for 20 hours to remove the ampholines and agamst 5 mM sodium succmate, pH 4 1 , 50 μg/ml PMSF for 24 hours, successively, to remove the salt prior to assay Lvophilization Dialyzed preparations (centπfuged at 10,000 x g for 10 minutes at 0°C to clanfy, if necessary), were decanted into a 600 ml Virtis flask, frozen as a shell on the flask wall by immersion in a dry ice/acetone bath, and lyophilized overnight at -60°C. Dry samples were transfeπed to screw cap bottles, tightly capped and stored at -20 C C Following are examples which illustrate materials, mediods and procedures, including the best mode, for practicmg the invention These examples are illustrative and should not be construed as limiting Example 1 -Seedling Germination for Isolation of Oxalate Oxidase Tnals witii 200 seeds each (Wysor barley) were undertaken to optimize germination conditions After imbibition for 21 hours in tap water, seeds were spread on trays lined wrth three layers of Whatman #3 filter paper, wetted with tap water and mcubated in constant fluorescent light or m darkness at 23 °C for seven days Alternatively, imbibed seeds were placed m a perforated plastic basket under a shower of tap water (23 °C) for seven days Maximum germination occurred with seed m die tray mcubated in the dark Those seeds exhibited a preponderance of root emergence Upon prolonged germination, no more shoots emerged and no further increase in germination rate was noted on the trays However, continued incubation of the seeds under the tap water shower increased botii die number of seed germinated and the amount of tissue generated per seedling Since the objective was to obtain large amounts of tissue, die shower mode of germination was modified to accommodate larger amounts of grain (See the Materials and Methods section) Yields typically ranged from about 1 kg to about 3 6 kg of frozen tissue (roots, gram, and shoots) per 1 kg of dry seed A preparation from green sprouts, pruned from the seedlings and frozen separately yielded no activity, tiius, efforts were made to exclude light during germination Example 2 — Preparation of Barlev Oxalate Oxidase Compositions Oxalate oxidase was punfied as descnbed in die Materials and Mediods section Particulate material in die initial tissue homogenate precluded a quantitative assay of oxalate oxidase activity However, using the semi-quantitative microtitre plate assay it was determined that less than half of die oxalate oxidase activity detected m the homogenate was recovered in the low speed supernatant (LSS) Contamination with insoluble starch in the root-rich preparation was minimized by selectively harvesting root-rich tissue to the exclusion of the ungermmated barley grains The addition of PMSF (dissolved in isopropanol) to the initial tissue homogenate dramatically altered the behavior and die appearance of die barley extract and successive fractions deπved from it The cloudy opalescence in supernatant fractions was eliminated and die pellets obtained upon centrifugation were discrete and compact. In one preparation, an eight-fold enrichment m oxalate oxidase activity with a ten-fold concentration of protein was obtamed from tissue homogenates prepared in the presence of PMSF. More than 90% of die enzymatic activity detected m me low speed supernatant was recovered in the ammonium sulfate precipitated protems (See Table 1). Table 1. Enrichment of oxalate oxidase isolated from barley seedlmgs. Fraction Vol 1 Total Activity Specific Yield (ml) (units) 2 Activity 3 (units/g (units/mg) tissue) LSS (Low Speed Supernatant) 720 276 1.08 1.00 HT (Heat T reated) 720 171 0.62 0.62 HTS (Heat Treated Supernatant) 705 168 0.68 0.61 30/60ASP (Ammonium sulfate ppt 72 257 8.50 0.93 between 30 and 60% saturation) 1 Fraction dialyzed prior to assay 2 Unit of activity is the change in absorption at 595 nm per minute in the spectrophotometπc assay defined in the Mateπals and Methods section 3 Protein determined with BCA as described in the Mateπals and Methods section Further purification of the 30/60ASP oxalate oxidase preparation was achieved by preparative isoelectric focusing of dus material between about pH 2.5 and 5.0 A 30/60ASP fraction (43 ml), prepared in the presence of PMSF and dialyzed against four liters of 1% glycine for 45 hours was immediately loaded widi a sucrose/PHARMALYTE gradient into a cooled focusing apparatus. The sample was focused for 22 hours and 1 ml fractions collected and prepared for assay as descnbed in the Material and Methods section. As seen in Figure 1 , essentially all of die recovered activity (55% of mat applied) was focused as a single peak. A band of tan precipitate was visible in the lower portion of the column. The oxalate oxidase obtained after isoelectric focusing was concentrated 14-fold over the starting material, but exhibited only a slight increase in specific activity (12 units/mg). The pH of die active fractions was estimated to he between about pH 2.75 and 2.9, assuming that the pH gradient was linear from pH 2.5 to 5 0 in 1 10 ml. The low levels of activity detected in the more alkaline fractions of the upper portion of the gradient conesponded to cross contamination with residual precipitated material observed as the fractions were collected from the column Example 3 — Preparation of Dipstick Device A dipstick assay device accordmg to the subject invention was prepared by saturating a 0.25 in by 3 m strip of high punty chromatography paper witii a solution of die enzyme and dye components The solution compnsed 0.1 g carboxymemylcellulose, 150 units oxalate oxidase (the dialyzed 30/60 ASP fraction prepared as descnbed in die Mateπals and Mediods section), 120 PZ units horseradish peroxidase, and 4 5 mg ortho-tohdme, in 10 mL of 0 025 M sodium succmate buffer (pH 4 0) The dipsticks were contacted widi die solution, moroughly coating die entire dipstick, and removed to dry The dipsticks can be stored in a dry and dark place, preferably below 30 °C, pnor to use A dipstick coated witii die enzyme and dye components as descnbed above was contacted with a sample to be tested for die presence of oxalate After contact with die sample, color was allowed to develop for a certain penod of time and die dipstick color was dien compared to a standard color chart for estimating oxalate concentration A concentration calibrated color chart can be prepared by dipping individual dipsticks in solutions containing oxalic acid concentrations of 1, 2, 4, 8 and 16 mM, respectively The colors observed on the dipsticks tested witii each known oxalate solution are aπanged on a chart mat shows a color or color mtensity for a given concentration of oxalate after a specified time of development The concentiation of oxalate in a sample can be estimated by comparing die color on the test dipstick that develops after a specific period of tune to the colors shown on the standardized color chart A dipstick device of die present mvention detected oxalate in a test sample at a concentration of about 09 mg/dl or about 100 μM based on visual observation of the color development and comparison to me standardized color chart Example 4 — Spot Test for oxalate A spot test assay for oxalate according to the subject invention was performed several different ways ( 1 ) on a twelve place, glass spot plate, 50 microhters (0 05 ml) of a fraction containmg oxalate oxidase and a dye reagent accordmg to die present uiventton were added to a well containmg 0 5 ml of test sample The glass plate was viewed over a white surface Several samples were tested in other wells, simultaneously When oxalate was present m the fraction, a purple color was observed in less than 1 minute Trace amounts of oxalate could be detected after 20-30 minutes, (2) one drop (about 0025 ml) of a fraction containmg oxalate oxidase and dye reagent accordmg to die present mvention was placed on white, porous bench towelmg (backed wim cellophane) The liquid was quickly absorbed to yield a small wet circle on die towelmg One drop of test sample was placed within die wet circle Several spots were prepared with several test samples and tested simultaneously When oxalate was present in a sample, die spot became dark purple in die center and dimmer toward the outer fringes of the circle (less man 5mm in diameter) widun 2 to 5 minutes, (3) oxalate was also detected usmg die procedure as descnbed m (2), except mat a strip of Whatman #1 filter paper was used as die surface mateπai instead of bench towelmg It should be understood tiiat die examples and embodiments described herein are for illustrative purposes only and mat vaπous modifications or changes m light thereof will be suggested to persons skilled in die art and are to be included within the spirit and purview of tins application and die scope of die appended claims References 1. Costello et al (1976) "An enzymatic metiiod for me spectrophotometnc determmation of oxalic acid" J. Lab. Cltn. Med. 87(5):903-908. 2. Chiπboga, J. (1966) "Punfication and Properties of Oxalic Acid Oxidase" Arch. Bwchem Biophys. 116:516-523. 3. Japanese patent application No. 63074500, published April 4, 1988 entitled "Test Strip for Detectmg Oxalate m Body Fluid." 4. Laker, M.F., A.F. Hofmann, B.J.D. Meeuse (1980) "Spectrophotometnc determination of urinary oxalate witii oxalate oxidase prepared from moss" Cltn Chem 26.827-830. 5. Santamana, J.R., R. Coll, E. Fuentespma (1993) "Comparative Study of Two Commercial Enzymatic Kits for Determining Oxalate Concentrations in Urine" Chn Bwchem 26:93-96 6. Smith, P.K., R.I. Krophn, G.T. Hermanson, A.K. Malha, F.H. Gartner, M.D. Provenzano, E.K. Fujimoto, N.M. Goeke, B.J. Oloon, D.C. Klenk (1985) Anal Bwchem 150:76-85 7. Smith, Lynwood H. (1991) "Diet and hyperoxaluπa m die syndrome of ldiopatinc calcium oxalate urolitinasis" Am J of Kidney Diseases XV1Ϊ370-375. 8 Sugiura, M., H Yamamura, K. Hnano, M. Sasaki, M. Moπkawa, M. Tsuboi (1979) "Puπfication and properties of oxalate oxidase from barley seedlmgs" Chem Pharm Bull 27.2003-2007 9. Thun, M.J., S. Schober (1991) "Urohthiasis in Tennessee: An Occupational Wmdow into a Regional Problem" American Journal of Public Health 81(5):587-591. 10. Vesterberg, O., H. Svensson (1966) "Isoelectπc frachonation, analysis, and characterization of ampholytes in natural pH gradients. VI Further studies of die resolving power in connection with the separation of myoglobms" y4c/α Chem Scand 20.820-834. 11. Yπberπ, J. and Posen, S. (1980) "A Semi-Automatic Enzymatic Metiiod for Estimating Urinary Oxalate." Chn Chem 26:881-884 12. U.S. Patent No. 4,455,371 issued to Richardson, K.E. and Obzansky, D.M. on June 19, 1984. 13. U.S. Patent No. 5,286,495 issued to Bahch, C. and Vaghefi, F. on February 15, 1994.

Claims: Claims We claim: 1 An assay device for detecting oxalate in a test sample, comprising: (a) an enzyme composition, wherem oxalate is a substrate for said enzyme composition, and wherein said enzyme composition reacts witii oxalate in said sample to produce a first reaction product, (b) a dye composition, wherem said dye composition is reactive witii said first reaction product to produce a second reaction product, wherem said second reaction product can be detected; and (c) a earner matπx, wherem said enzyme composition and said dye composition are immobilized on said earner matnx. 2 The device accordmg to claim 1 , wherem said enzyme composition compπses oxalate oxidase 3 The device accordmg to claim 1, wherem said dye composition is selected from the group consistmg of ortho-tohdme, DMAB/MBTH, and DMA/MBTH 4 The device accordmg to claim 1, wherein said earner matnx is selected from the group consistmg of plastic, paper, cloth, and polymer 5 The device accordmg to claim 2, wherein said oxalate oxidase is an oxalate oxidase having a specific activity of at least about 8.5 enzyme units per milligram of protem after ammonium sulfate ((NH 4 ) 2 S0 4 ) precipitation 6 The device according to claim 2, wherein said enzyme composition further compπses a peroxidase 7. A process for preparing an oxalate oxidase composition compπsmg the steps of preparing an extract from plant tissue, wherem said plant tissue compπses a root-πch fraction of germinated seedlings, precipitatmg protems m said extract by brmgmg said extract to at least about 30% saturation with ammonium sulfate, removmg said precipitated protems and bringing said extract to at least about 60% saturation with ammonium sulfate to precipitate said oxalate oxidase present m said extract 8. The process, accordmg to claim 7, wherein PMSF is mciuded during said process 9 The process accordmg to claim 7, wherein said germinated seedlmgs are germinated under conditions which exclude light 10 The process accordmg to claim 7, wherein said germinated seedlmgs are frozen and men thawed dunng die preparation of said extract from said plant tissue 1 1. The process accordmg to claim 7, wherem said plant tissue compπses barley plant tissue 12 The process accordmg to claim 7, wherein pπor to precipitating said protems witii ammonium sulfate, said extract is heated and tiien subsequently chilled to between about 3 ° to 4 C C 13 The process, accordmg to claim 12, wherein the step of heating said extract is at a temperature of between about 70° to 80° C for between about 5 to 12 minutes 14 An oxalate oxidase composition havmg a specific activity of at least about 8 5 enzyme units per milligram of protem after ammonium sulfate ((NH 4 ) 2 S0 4 ) precipitation 15 The oxalate oxidase according to claim 14, havmg a specific activity of at least about 10 enzyme units per milligram of protem 16 The oxalate oxidase according to claim 14, havmg a specific activity of at least about 20 enzyme units per milligram of protein 17 The oxalate oxidase accordmg to claim 14 wherein said oxalate oxidase is produced accordmg to a process compnsmg die steps of preparing an extract from plant tissue, precipitating proteins m said extract by brmgmg said extract to at least about 30% saturation with ammonium sulfate, removing said precipitated proteins and bringing said extract to at least about 60% saturation witii ammomum sulfate to precipitate said oxalate oxidase present in said extract 18 A method for detecting oxalate m a test sample, compnsmg the steps of (a) contacting die device of claim 1 witii said test sample for an effective penod of time, wherem a detectable reaction product is produced when oxalate is present in said test sample, and (b) detecting said oxalate by detectmg the production of said reaction product 19 The method according to claim 18, wherein said detectable reaction product compnses a visible dye on said support matπx, wherein said visible dye can be detected visually 20 The metiiod accordmg to claim 18, wherem said device is contacted with said test sample for between about 1 second to about 5 minutes 21 A metiiod for detectmg oxalate m a test sample, compnsmg the steps of (a) combining an enzyme composition and a dye composition with said sample, wherem oxalate is a substrate for said enzyme composition, and wherem said enzyme composition reacts witii oxalate in said sample to produce a first reaction product, wherem said dye composition is reactive with said first reaction product to produce a second reaction product, wherem said second reaction product can be detected; and (b) detecting said oxalate by detectmg the production of said second reaction product 22 The metiiod, accordmg to claim 21, wherem said enzyme composition and said dye composition are combined witii said sample on a non-porous surface 23 The metiiod, accordmg to claim 21, wherein said enzyme composition and said dye composition are combined with said sample on a porous surface 24 The method, accordmg to claim 21, wherein said enzyme composition comprises oxalate oxidase 25 The metiiod, accordmg to claim 21 , wherem said dye composition is selected from the group consistmg of ortho-tolidine, DMAB/MBTH, and DMA/MBTH 26 The method, accordmg to claim 21, wherein said enzyme composition further compnses a peroxidase

Inventors: BATICH CHRISTOPHER D, MANS RUSTY JAY, MCFETRIDGE IAN, BATICH, CHRISTOPHER, D., MANS, RUSTY, JAY, MCFETRIDGE, IAN
Applicants: UNIV FLORIDA, UNIVERSITY OF FLORIDA
Application year: 1997
Main classification: C12Q1
IPC codes: C12N9, C12Q1, G01N31