DETECTION OF HYDROLYZING ENZYMES

Abstract: A composition of a polysaccharide with a detectable material appended thereto and a 1,3-dione having a methylene group between the two carbonyls of the 1,3-dione, which methylene group has at least one ionizable hydrogen, provides accurate results in assays of hydrolyzing enzymes such as amylase. The composition can be incorporated in the reagent zone of a dry test element having a reagent zone and a registration zone.

Description: This invention relates to an improved composition and element for the detection and analysis of hydrolyzing enzymes in liquids, particularly the clinical analysis of amylase in biological liquids. Various techniques are used for the detection and analysis of hydrolyzing enzymes such as amylase. One useful technique is a wet chemistry assay technique involving a starch having a detectable material such as a colored or dye material attached thereto. Such an assay is described in U.S. Patent 3,694,318. In that assay, a dyed starch (a starch having a preformed dye or dye former chemically attached) is dissolved or suspended in a liquid medium. The sample to be tested is then added to the liquid medium, whereupon the amylase present in the test sample hydrolyses the starch, causing release of the dye. The non-hydrolyzed starch is then removed separated and the amount of amylase present as a function of the amount of dye released is determined. This dyed starch assay has been incorporated into dry test elements, as disclosed in U.S. Patent 4,144,306. The element of that patent comprises: i) a reagent layer containing a non-diffusible starch having attached thereto a detectable material such as a dye and ii) a registration layer adapted to receive the detectable material. The sample of liquid to be tested is applied to the reagent layer, whereupon amylase in the sample hydrolyses the starch, causing it to break down to low molecular weight polysaccharide units with the dye attached thereto. By virtue of their molecular weight, which is lower than that of the non-diffusible starch, the dye-containing low molecular weight polysaccharide units diffuse to the registration layer, where the amount of color formation and thus, the mount of amylase in the sample can be determined. Dry test elements for the assay of hydrolyzing enzymes such as amylase, like the element described above, have been very successful because of their ease of use and because of fast and reliable results. However, when the test elements have been kept for a period of a week or more at ambient conditions, they have a tendency to overpredict the amylase concentration as compared to freshly prepared elements or elements that have been stored under refrigeration. The present invention addresses the problem of overprediction of amylase concentration in elements that have been stored at ambient conditions. The problem of overprediction of amylase concentration in starch-based assays is alleviated by using a composition comprising a starch having a detectable material appended thereto and a 1,3-dione having a methylene group between two carbonyls, which methylene group has at least one ionizable hydrogen. A preferred class of 1,3-diones useful in the invention has the structure: where H' is ionizable hydrogen, R is H, alkyl, aryl, or -CO-R³, R¹ and R² each represent independently H, alkyl, aryl, -OR⁴, a heterocyclic ring, or together represent the atoms necessary to complete a 5- or 6-membered carbocyclic ring, R³ is H, alkyl, or aryl, and R⁴ is alkyl or aryl. The composition of the invention is advantageously incorporated in a dry test element. A particularly preferred test element comprises a reagent zone and a registration Zone. This element comprises a non-diffusible starch and a 1,3-dione as described above. Although the dione and starch can be in separate zones or layers in the element, it is preferred that they be combined in the reagent zone or layer. The reagent zone or layer can be considered a spreading zone or layer as well. The non-diffusible starch is interactive in the presence of the amylase being tested to provide a diffusible material comprising the detectable material that was appended to the non-diffusible starch. The registration zone of the element is adapted to receive this diffusible material. FIG. 1 shows an element according to the invention comprising a reagent layer and a registration layer on a support. FIG. 2 shows an element according to the invention comprising a reagent layer, a buffer layer, and a registration layer on a support. FIGS. 3 and 4 are graphs showing the reduced tendency of the elements of the invention to overpredict amylase concentration after storage at ambient conditions. FIG. 3 represents the tendency of elements outside the scope of the invention to overpredict amylase concentration and FIG. 4 represents the reduced tendency of an element of the invention to overpredict amylase concentration. FIG. 5 is a chart representing the reduced tendency of an element of the invention to overpredict amylase concentration. Starches are well-known in the art and can be obtained from a variety of sources, such as starch from potato, corn, tapioca, wheat, rice, sweet potato, or other sources. Both water-soluble and water-insoluble starches may be used. When used in the dry test elements of the invention, the starch should have a molecular weight sufficiently high to render it non-diffusible in that element. The detectable material of the invention can be any number of well-known detectable materials, such as radioactive materials, and colored materials, such as chromogenic or fluorescent dyes. Useful detectable materials are described in U.S. Patent 4,144,306. Preferred materials are colored dyes, such as Drimarine Red Z2B sold by the Sandoz Corp. of Hanover, NJ and various halogenated cyanuric dyes described in the above-referenced U.S. Patent 4,144,306. The techniques for attaching the above-described detectable materials to starch are well-known in the art. Such techniques are disclosed in U.S. Patents 3,579,322 and 3,694,318. The composition of the invention also comprises a 1,3-dione as described above, having a methylene group between two carbonyls, which methylene group has at least one ionizable hydrogen. These 1,3-diones include beta-diketones, beta-ketoesters, beta-ketoaldehydes, and beta-dialdehydes. Examples of such 1,3-diones include: dimedone (5,5-dimethyl-1,3-cyclohexanedione) 1-benzoylacetone 1-phenyl-2,4-pentanedione 1-(2-furyl)-1,3-butanedione A preferred class of 1,3-diones useful in the invention has the structure: where H' is ionizable hydrogen, R is H, alkyl, aryl, or -CO-R³, R¹ and R² each represent independently H, alkyl, aryl, -OR⁴, a heterocyclic ring, or together represent the atoms necessary to complete a 5- or 6-membered carbocyclic ring, R³ is H, alkyl, or aryl, and R⁴ is alkyl or aryl. The hydrogen represented by H' is generally ionizable when the 1,3-dione has a pK a less than or equal to about 9. As is well known in the art, certain conditions, such as a high level of steric hindrance about H' or an easily hydratable carbonyl adjacent to the H'-bearing carbon atom, can alter the ionizability of H'. R, R¹, R², R³, and R⁴ can be any alkyl or aryl, substituted or unsubstituted, so long as the hydrogen represented by H' is ionizable. The alkyl group preferably has from 1 to 6 carbon atoms, such as methyl, ethyl, propyl, butyl, isopropyl. Examples of useful aryl groups include phenyl, tolyl, and methoxyphenyl. R¹ and R² can each be any substituted or unsubstituted heterocyclic group so long as the hydrogen represented by H' is ionizable. Preferred heterocyclic groups are those having 4 to 7 ring atoms, such as furyl, pyridyl, thienyl and pyrolyl. R¹ and R² can also together represent the atoms required to complete a carbocyclic ring having 5 to 6 carbons in the nucleus. Exemplary carbocyclic rings include cyclopentane, cyclohexane, cyclopentene and cyclohexene. All the above-described groups represented by R, R¹, R², R³, and R⁴ may be unsubstituted or substituted with groups, such as alkyl (e.g., methyl, ethyl, butyl, hexyl); aryl, (e.g., phenyl, tolyl, methoxyphenyl, hexadecyloxyphenyl, naphthyl); cyano; and carbalkoxy (e.g. carbomethoxy, carboethoxy, carbobutoxy), so long as the hydrogen represented by H' is ionizable. An especially preferred class of 1,3-diones useful in the invention are carbocyclic 1,3-diones represented by the structure: wherein R₅ and R₈ each represent a group selected from the class consisting of hydrogen; alkyl (e.g., methyl, ethyl, butyl); aryl (e.g., phenyl, tolyl, naphthyl); cyano; and carbalkoxy (e.g., carbomethoxy, carboethyoxy); and R₆ and R₇ are each selected from the class consisting of hydrogen, alkyl (e.g., methyl, ethyl, butyl, amyl); and aryl (e.g., phenyl, methoxyphenyl ethoxyphenylhexadecyloxyphenyl, tolyl, naphthyl). Examples of carbocyclic 1,3-diones useful in the present invention include: (1) 5,5-dimethylcyclohexane-1,3-dione (2) 5-phenylcyclohexane-1,3-dione (3) 5-(p-methoxyphenyl)cyclohexane-1,3-dione (4) 4-cyano-5-phenylcyclohexane-1,3-dione (5) 5-(o-decyloxyphenyl)cyclohexane-1,3-dione (6) 5-(o-hexadecyloxyphenyl)cyclohexane-1,3-dione (7) cyclohexane-1,3-dione (8) 4,6-dicarbethoxy-5-methylcyclohexane-1,3-dione (9) 4,6-dimethyl-5-phenylcyclohexane-1,3-dione (10) 4,6-dicyano-5,5-dimethylcyclohexane-1,3-dione (11) 4-phenyl-5,5-diethylcyclohexane-1,3-dione (12) 5-methyl-5-phenylcyclohexane-1,3-dione (13) cyclopentene-1,3-dione (14) 4,4-dimethylcyclopentane-1,3-dione. Further description of carbocyclic 1,3-diones, including their preparation, is found in U.S. Patent 3,447,926. In the composition of the invention, the starch having the detectable material attached thereto and the 1,3-dione can be combined in any ratio which provides the invention's advantageous reduction in overprediction of amylase concentration. Since even a small amount of 1,3-dione will produce some effect, compositions having such small amounts are within the scope of the invention. The specific desirable concentrations depend on the particular starch and 1,3-dione utilized and can be easily determined by testing the composition with samples having known amylase concentrations. Preferred concentrations of 1,3-dione range from 1 to 40 parts by weight of dione per 100 parts by weight of starch. A particularly preferred 1,3-dione of the present invention is dimedone (i.e., 5,5-dimethyl-1,3-cyclohexanedione). A preferred concentration of dimedone ranges from 2 to 20 parts by weight of dimedone to 100 parts by weight of starch. In a preferred embodiment of the invention, the composition also comprises a binder, an inert particulate material, and a buffer in addition to the above-described starch and 1,3-dione. The binder can be any of the reagent carriers disclosed in the above-referenced U.S. Patent 4,144,306, but a particularly preferred class of binders are isotropically porous polymers such as blushed polymers. Blushed polymers can be formed on a substrate by dissolving a polymer in a mixture of two liquids, one of which is a lower boiling, good solvent for the polymer and the other of which has a higher boiling point and is a non-solvent or a poor solvent for the polymer. Such a polymer solution is then coated on the substrate, and dried under controlled conditions. The lower-boiling solvent evaporates more readily and the coating can become enriched in the liquid which is a poor solvent or non-solvent. As evaporation proceeds, under proper conditions, the polymer forms as an isotropically porous layer. Many different polymers can be used, singly or in combination, for preparing isotropically porous blushed polymer layers for use in this invention, for example, polycarbonates, polyamides, polyurethanes and cellulose esters such as cellulose acetate. Various microporous filters are, or are partly, blushed polymeric compositions, for example, various membrane filters of the Millipore Corporation, and they have been described in patents such as U.S. Patent No. 2,783,894 and U.S. Patent 2,772,322. The porosity of the blushed polymer should be such that the non-diffusible polysaccharide of the invention will not diffuse through it, but the diffusible material will. The inert particulate material of the invention can be any material such as pigments, diatomaceous earth, or microcrystalline colloidal materials, such as microcrystalline cellulose. Particularly preferred inert particle materials are light reflective pigments, such as barium sulfate, titanium dioxide, zinc oxide, and lead oxide. Particle size should be chosen to be compatible with the porosity of the blushed polymer. The amount of pigment to be included in the composition varies greatly and is generally between 5 and 2000 percent based on the weight of the binder. It is preferable in the dyed starch assay of amylase to utilize a buffer for maintaining the pH in the appropriate range. Preferred buffers are those that maintain the pH in the range of 6 to 8. Such buffers include triethanolamine and sodium dihydrogen phosphate. Other suitable buffers are well-known in the art. A partial listing of specific representative buffer compositions is given by Good in Biochemistry, 5, p. 467 (1966). The test element of the invention comprises one or more reagent zones and registration zones. In one embodiment of the invention, the element also comprises one or more buffer zones between the reagent zone(s) and the registration zone(s). The elements may be self-supporting, but are preferably on a support. Referring to the drawings, FIG. 1 represents a test element of the invention having a support 2, a registration layer 6, and a reagent layer 10. FIG. 2 represents a test element of the invention having a support 14, a registration layer 18, a buffer layer 22, and a reagent layer 26. Reagent zones of the invention comprise the above-described starch and 1,3-dione and are preferably permeable and porous. Permeability, incuding permeability arising from porosity, can be achieved through the presence of various carriers, matrices, or binders, such as fibrous materials or porous, non-fibrous materials described in the above-referenced U.S. Patent 4,144,306. A preferred permeable binder of the invention is the above-described class of blushed polymers. Also useful as a porous carrier is a polymer binder with an inert particulate material, such as microcrystalline cellulose, dispersed therein. Pigment particles may be incorporated in the reagent zone for light reflecting purposes. Registration zones according to the present invention are described in the above-referenced U.S. Patent 4,144,306. The registration zone is permeable to the detectable material-containing diffusible material and is preferably radiation-transmissive to allow detection of the detectable material. The registration zone is adapted to receive the diffusible material from the reagent zone. When the diffusible material comprises a detectable material that is a dye, the registration zone may contain a mordant for that dye. Such mordants are well-known in the photographic arts. Particularly useful mordants are polymeric mordants having repeating units of the formula: wherein A represents an organo group, such as an alkylene group, forming a portion of the polymer backbone, Q represents a chemical bond or an organo group linking M⁺ to A, M⁺ represents a quaternary ammonium or phosphonium group, and X⁻ represents an anion. Such mordants are described in U.S. Patent 4,069,017. The elements of the present invention may include other zones well-known in the clinical analysis art. These other zones include spreading zones, radiation blocking zones including reflecting zones, filter zones, subbing layers. The composition of these zones and their location in the element are known in the art and are described in U.S. Patents 3,992,158 and the above-referenced U.S. Patent 4,144,306. For example, spreading zones can be isotropically porous zones, achieving such porosity through the use of inert particle materials and/or blush polymers, and can be positioned adjacent to the reagent zone as the outermost layer of the element (if a multilayer element is used). Reflecting zones can include pigments such as titanium dioxide, barium sulfate, and/or blushed polymers, which are generally reflective in nature. Reflecting zones may be positioned between the reagent zone and the registration zone or the spreading zone. Filter zones, which may be the same as or different from the reflecting zone, may also include particulate materials and/or blushed polymers and may be positioned between the reagent zone and the registration zone or the spreading zone. Subbing layers are well-known in the clinical analysis and photographic arts and may be positioned anywhere in the element. Any of the zones of the present invention may also include well-known addenda, such as buffers, surfactants, or coating aids, as described in the above-referenced U.S. Patent 4,144,306. Multilayer elements of the invention can be prepared by various laminating or coating techniques well-known in the art, such as hand-coating, blade coating; bead coating, or dip coating. The elements may be self-supporting or carried on a support. Useful support materials include a variety of polymeric materials, such as cellulose acetate, poly(ethyleneterephthalate), polycarbonates, and polyvinyl compounds such as polystyrenes. The support should be chosen so that the presence of the detectable material may be detected through the support. For example, if the detectable material is a colored dye, the support is preferably transparent to light. Coating and laminating techniques, along with support materials, are further described in the above-referenced U.S. Patent 4,144,306. For coatable reagent zones, a coating solution or dispersion including a binder and the starch can be prepared, coated as discussed herein and dried to form a dimensionally stable zone. The thickness of any reagent zone and its degree of permeability are widely variable and depend on actual usage. Dry thicknesses of from 10 to 100 microns are convenient, although more widely varying thicknesses can be used. For example, if comparatively large amounts of starch are required, it may be desirable to use slightly thicker reagent zones. Fibrous reagent zones can be formed by impregnation of a fibrous matrix, in accordance with well-known techniques. Registration zones and other zones can be prepared using methods and thicknesses as used when preparing coatable reagent zones, but with constituents appropriate for the particular zone. The elements of the invention are used by applying a sample of the liquid to be tested to the element. Generally, the element will be formed so that the liquid first contacts one or more spreading zones, if present, or the reagent zone(s). After application of the liquid, the element may be exposed to any conditioning, such as heating or humidification, that is desirable to quicken or otherwise facilitate any test result. After an appropriate time to allow for diffusion from the reagent zone to the registration zone of any detectable material-containing diffusible material released from the starch, the amount of detectable material in the registration zone is determined. Such a determination may be made by passing the element through an area in which an apparatus suitable for reflection, transmission, or fluorescence spectrophotometry is provided. Such apparatus serves to direct a beam of energy, such as light, through the support and the registration zone or the reagent zone. The light is then reflected, such as from the reagent zone or a reflection zone in the element, back to a detecting means or passes through the element to a detector, in the case of transmission detection. Use of reflection spectrophotometry can be advantageous in some situations as it can effectively avoid interference from residues, such as blood cells, which may be left on or in the zones of the element. Conventional techniques of fluorescence spectrophotometry can also be employed if the detectable species is a fluorescent material. Detection would be accomplished using energy that excites the fluorescent species and a detector that senses its fluorescent emission. Furthermore, when blood serum is tested or means are provided for eliminating unwanted whole blood residues, transmission techniques can be used to detect and quantify the indicating reaction products by directing a flow of radiant energy, for example, U.V., visible, or I.R. radiation at one surface of the element and measuring the output of that energy from the opposing surface of the element. Generally, electromagnetic radiation in the range of from 200 to 900 nm has been found useful for such measurements, although any radiation to which the element is permeable and which is capable of quantifying the product produced in the element can be used. Various calibration techniques can be used to provide a control for the analysis. As one example, a sample of a standard test liquid can be applied adjacent to the area where the drop of sample is placed in order to permit the use of differential measurements in the analysis. The following examples further illustrate the present invention: Example 1 An element for the assay of amylase according to the invention is prepared having the configuration shown in FIG. 2 on a poly(ethylene terephthalate) support. Additionally, the element has a subbing layer of polyvinylpyrrolidone coated at 0.540 g/m² between the reagent layer and the registration layer. The reagent layer is coated with the following coverages; The buffer layer is coated with the following coverages: The registration layer is coated with the following coverages: For comparison, an element is prepared identically to the above-described element, except that no dimedone is present in the registration layer. Samples of both element formulations (with and without dimedone) are aged for various periods of time at various conditions (1 week at 22°C (72°F)/80% relative humidity (RH), 3 weeks at 22°C (72°F)/80% RH, 3 weeks at -18°C (0°F)/50% RH, and 6 weeks at 22°C (72°F)/80% RH). The aged elements and fresh elements are tested using serum based control solutions having known amylase concentrations. The results of this comparative test are shown in FIGS. 3 and 4. FIG. 3 represents the results for the element outside the scope of the invention without dimedone and FIG. 4 represents the results for the element of the invention with dimedone. The graphs of FIGS. 3 and 4 show the rate of dye released in the registration layer as a function of the actual concentration of amylase in the test liquid for: fresh elements, elements aged 1 week at 22°C/80% RH, elements aged 3 weeks at 22°C/80% RH, elements aged 3 weeks at -18°C/50% RH, and elements aged 6 weeks at 22°C/80% RH. As shown in FIG. 3, the elements outside the scope of the invention containing no dimedone show a significantly greater tendency to overpredict the concentration of amylase after aging at 22°C/80% RH than do the elements of the invention in FIG. 4. The improved performance of the elements of the invention is also shown in FIG. 5. FIG. 5 is a bar chart showing the % bias (the percent by which the element over- or under-predicts the actual concentration of amylase) for amylase concentrations of 104, 184, and 735 U/L. FIG. 5 shows that elements of the invention (those containing dimedone) show a significantly reduced tendency to overpredict the concentration of amylase in the sample. EXAMPLES 2-5 Elements according to the invention are prepared as in Example 1 using the components shown in Table 1 in place of dimedone: The elements along with a control containing no dione in the reagent layer are kept for 16 days at 22°C/80% RH and tested with solutions of amylase at concentrations of 44 and 746 U/L. The % bias (as defined In Example 1) for each of the elements is shown in Table 2. The results shown in Table 2 indicate a much lower tendency to overpredict the concentration of amylase with elements of the invention than with the control element outside the scope of the invention.

Claims: A composition for detecting amylase comprising a starch having a detectable material appended thereto and a 1,3-dione having a methylene group between the two carbonyls of said 1,3-dione, which methylene group comprises at least one ionizable hydrogen. The composition of claim 1 wherein said 1,3-dione is a beta-diketone. The composition of claims 1 or 2 wherein said 1,3-dione has the structure: where H' is ionizable hydrogen, R is H, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, or -CO-R³, R¹ and R² each represent independently H, alkyl, aryl, -OR⁴, a heterocyclic ring, or together represent the atoms necessary to complete a 5- or 6-membered carbocyclic ring, R³ is H, alkyl, or aryl, and R⁴ is alkyl or aryl. The composition of any of claims 1-3 wherein said 1,3-dione is a carbocyclic 1,3-dione. The composition of any of claims 1-4 wherein said 1,3-dione is dimedone. The composition of any of claims 1-5 wherein said detectable material is a dye. A test element for detecting amylase comprising a support having thereon the composition of any of claims 1-6. A test element for analysis of amylase in a liquid, comprising a support having thereon: a reagent zone including a non-diffusible starch having a detectable material appended thereto, said non-diffusible starch being interactive in the presence of amylase to provide a diffusible material comprising said detectable material, and a registration zone adapted to receive said diffusible material, characterized in that said reagent zone also comprises a 1,3-dione having a methylene group between the two carbonyls of said 1,3-dione, which methylene group comprises at least one ionizable hydrogen The test element of claim 8 wherein said 1,3-dione has the structure: where H' is ionizable hydrogen, R is H, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, or -CO-R³, R¹ and R² each represent independently H, alkyl, aryl, -OR⁴, a heterocyclic ring, or together represent the atoms necessary to complete a 5- or 6-membered carbocyclic ring, R³ is H, alkyl, or aryl, and R⁴ is alkyl or aryl. The test element of claims 8 or 9 wherein said 1,3-dione is dimedone. The test element of any of claims 8-10 wherein said diffusible material is a starch having a molecular weight low enough to render it diffusible, and said detectable material is a dye, and wherein the registration zone comprises a mordant for said dye, said test element further comprising a buffer zone between said reagent zone and said registration zone. A method for detecting amylase in a liquid, comprising the steps of: a) contacting the liquid with the reagent zone of the test element of any of claims 7-11, so as to cause diffusion of said diffusible material to the registration zone, and b) detecting the amount of said amylase in the sample as a function of the amount of said detectable material in the registration zone.

Inventors: EIKENBERRY, JON, HUMBERT, DOLORES L., SCHLEGEL, BROOKE, P., WARREN, KAREN L.
Applicants: EASTMAN KODAK COMPANY (A NEW JERSEY CORPORATION)
Application year: 1987
Main classification: G01N33
IPC codes: C12Q1, G01N33