|Publication number||US3928137 A|
|Publication date||Dec 23, 1975|
|Filing date||May 6, 1974|
|Priority date||Oct 20, 1971|
|Publication number||US 3928137 A, US 3928137A, US-A-3928137, US3928137 A, US3928137A|
|Inventors||Chiang Ching, Monte Alexander A|
|Original Assignee||Mallinckrodt Inc|
|Export Citation||BiBTeX, EndNote, RefMan|
|Patent Citations (5), Referenced by (14), Classifications (8)|
|External Links: USPTO, USPTO Assignment, Espacenet|
United States Patent [1 1 Monte et al. Dec. 23, 1975 [5 1 REAGENT FORMULATION FOR URIC ACID 3.677.903 1/1912 Bittner 195/1015 R 3,858,854 1/1975 Win at al. 195/63 x ASSAY  Inventors: Alexander A. Monte; Ching Chiang,
both of Glendora, Calif.
 Assignee: Mallineknwt, Inc, St. Louis, Mo.
[22} Filed: May 6, 1974  Appl. No.: 467,431
Related US. Application Data  Division of Ser. No. 200,552, Nov. 19, 1971, Pat. No 3,816,262, which is a continuationin-part of Ser. No. 190,883, Oct. 20, 1971, abandoned.
Primary ExaminerDavid M. Naff Attorney, Agent, or Firm-Mathew D. Madsen  ABSTRACT An enzyme reagent formulation for use in assaying a biological specimen for uric acid is prepared as a granular, water-soluble, anhydrous, storage-stable mixture containing uricase, potassium chloride, mannitol, gum acacia, bovine serum albumin, glycine, sodium carbonate and a nitrogen containing polyoxyalkylene nonionic surfactant obtained by the sequential reaction of ethylenediamine with propylene oxide and ethylene oxide in the presence of a catalyst. The surfactant contains polyoxypropylene chains having an average molecular weight of between about 750 and about 6750, and polyoxyethylene chains constituting between about 10 and about 80 weight percent of the surfactant. The surfactant has an advantageous efl'ect upon granulation, dissolution and storage stability of the reagent formulation.
5 Claims, 1 Drawing Figure REAGENT FORMULATION FOR URIC ACID ASSAY CROSS-REFERENCE TO RELATED APPLICATION cation Ser. No. 190,883, filed Oct. 20, l97 l now abanl0 doned.
BACKGROUND OF THE INVENTION This invention relates to the field of clinical diagnostic testing and more particularly to novel reagents and methods for making biological assays on body fluids.
A large variety of test reagents and methods are available for use in determining the character of various body fluids to assist in the diagnosis of certain pathological conditions. Tests for determination of certain types of biological activity or the presence and quantity of certain biologically active components provide information indicating the presence or absence of disease or other physiological disorder. In accordance with such tests, the biological specimen to be analyzed, for example, a sample of a body fluid, is typically mixed with a liquid reagent formulation which contains a reagent capable of effecting a reaction which causes a measurable change in the specimen/reagent system. Very often the reaction which takes place in the test is an enzymatic reaction. Certain tests are designed, in fact, to determine the presence of a particular enzyme and in such cases the reagent formulation may contain a substrate upon which the enzyme to be determined is known to act. In other cases, the determination may be for a material which is known to be a reactive substrate in an enzymatically catalyzed reaction. In either case, the reagent formulation very commonly contains an enzyme, a coenzyrne or both. Because the catalytic activity of most enzymes is specific to a particular reaction, test reagents can be formulated which are effective to determine specific biological components or activities even in a complex body fluid containing a large number of other components which might interfere with efforts to obtain a purely chemical analysis. Moreover, many of the components which are to be determined have highly complex chemical structures which would render direct chemical analysis difficult even in the absence of any contaminants.
Unfortunately, enzymes and coenzymes are generally rather delicate materials which may be readily denatured by heating and which also tend to degenerate upon storage. Many of the substrate materials used in biological assay reagent formulations are similarly unstable. Liquid reagents containing such components are therefore not generally susceptible to storage and must be freshly prepared shortly prior to use in clinical diagnostic testing. Because of the relative expense of enzymes and coenzymes and the skill required to prepare a reagent formulation containing these materials which can be utilized to obtain accurate clinical diagnostic test results, the instability of the liquid formulations has motivated a substantial amount of research to develop reagents in a relatively storage-stable form. Much of this effort has been directed to the development of solid, dry, water-soluble formulations which can be dissolved in water at the time of testing to provide a fresh liquid reagent useful in the test. Typical 2 prior art dry reagent formulations are disclosed in Deutsch U.S. Pat. No. 3,413,198 and Stern et al. US. Pat. No. 3,546,13l.
A dry reagent formulation satisfactory for use in preparing liquid reagents for routine clinical diagnostic tests should satisfy a number of criteria. It must be readily soluble in a solvent compatible with the biological specimen, usually water. It should be capable of solubilizing proteinaceous material in the specimen. Moreover, it should be readily susceptible to packaging in convenient sized packages and be adapted for rapid dissolution in the solvent to provide a liquid reagent of proper strength for a given test or series of tests.
SUMMARY OF THE INVENTION It is an object of the present invention to provide improved dry, water-soluble, reagent formulations for use in conducting clinical diagnostic tests. It is a further object of the present invention to provide such formulations which can be readily granulated and shipped or stored in granular form. It is a particular object of the invention to provide such reagent formulations in freeflowing, granular form at consistent bulk densities so that they may be delivered to a volumetric packaging or tableting operation in consistent weight amounts. Additional objects of the invention include the provision of dry reagent formulations having a high capacity for solubilizing protein; the provision of such formulations having a high degree of storage stability; the provision of methods for preparing the dry reagent formulations of the invention; and the provision of methods for conducting clinical diagnostic tests utilizing such reagent formulations. Other objects and features will be in part apparent and in part pointed out hereinafter.
In one of its aspects, therefore, the present invention is directed to a reagent formulation for use in conducting a clinical diagnostic test on a biological specimen. The reagent formulation comprises a solid, water-soluble, substantially anhydrous, storage-stable mixture containing a reagent capable of participating in a test reaction to effect a measurable change in a test system, and a solid nitrogen-containing polyoxyalkylene nonionic surfactant. The surfactant has a structure corresponding to that obtained when ethylene diamine is reacted sequentially with propylene oxide and ethylene oxide in the presence of a catalyst and the polyoxypropylene chains of the surfactant have an average molecular weight of between about 750 and about 6750.
The invention is further directed to a method of conducting a clinical diagnostic test on a biological specimen using the aforementioned reagent formulation. The method comprises dissolving the reagent formulation in water to produce a liquid reagent; mixing the liquid reagent with a specimen to form a specimen/reagent test system; and measuring a change in the system resulting from the reaction between the reagent and the specimen.
The invention is also directed to a method of preparing the novel reagent formulation. The method comprises the steps of mixing a reagent capable of participating in a test reaction to effect a measurable change in a test system, a nitrogen-containing polyoxyalkylene nonionic surfactant of the above-noted character, and a solvent for the surfactant; anad removing the solvent to form a substantially anhydrous, water-soluble, freeflowing, granular solid.
DESCRIPTION OF THE DRAWING The drawing is a grid illustrating the molecular structure of various commercially available nonionic surfactants useful in the practice of the invention. The coordinates of each point on the grid correspond to the chain size of the polyoxyethylene hydrophile and polyoxypropylene hydrophobe moieties of a particular surfactant. Boundary lines set out on the grid separate the areas encompassing surfactants which assume different physical states.
DESCRIPTION OF THE PREFERRED EMBODIMENTS To facilitate preparation of liquid reagents from solid formulations in the clinical laboratory, it is highly desirable to package the solid formulations in proper unitary amounts. Thus, for example, the solid formulations may be encapsulated or tabletted with the proper quantity of reagent in each capsule or tablet for conducting a single test. Alternatively, a multi-test package can be provided from which the proper amount of liquid reagent is prepared for conducting a specified number of tests.
Where a solid reagent formulation is packaged in unitary amounts, accuracy of metering the solid material into each capsule, tablet or multi-test package is important. The metering equipment which is used for delivering solid materials in packaging and tableting operations, however, almost universally operates on a volumetric basis. Unless the solid material is free-flowing and has a consistent bulk density, therefore, it cannot be delivered in consistent weight amounts to each package, capsule or tableting station using conventional equipment.
To provide a solid formulation in free-flowing form of consistent bulk density, it is preferably granulated prior to packaging. Granulation converts a powdered material into a material constituted by small agglomerates of relatively uniformsize. Properly prepared, the granular material is free-flowing, has a consistent bulk density and is readily handled by the metering devices used in packaging operations. To granulate a powdered material, the powder is typically mixed with a binder dissolved in a volatile solvent, wet screened, dried by driving off the solvent, and dry screened following the drying step. In addition to the binder, a lubricating substance is normally incorporated in the granulation mass to further enhance the flow characteristics of the granules, especially under the compressive stress of tableting operations.
As noted, solid formulations useful as reagents for conducting clinical diagnostic tests on biological specimens should have certain additional properties. Because they are dissolved in water to produce a liquid reagent, all components, including the binder, should be readily water-soluble. Because many of the tests involve enzymatic reactions and/or proteinaceous substrates, the formulation should possess detergent properties for solubilizing protein.
It has now been discovered that the above objectives can be met and that effective clinical reagent formulations for the determination of certain biological properties of body fluids can be produced in free-flowing, granular form through the use of particular nitrogenbearing polyoxyalkylene nonionic surfactants. Test formulations granulated with the aid of these surfactants are well adapted to precision packaging and tableting operations. Because of their free-flowing character and consistent bulk density, they can be delivered to either a packaging or tableting operation in consistent weight amounts by volumetric metering. As a consequence, clinical test reagents formulated at a central location remote from a clinical laboratory can be utilized to prepare liquid test reagents for clinical use without the need for weighing, analyzing, or other procedures by the clinical chemist or technician.
The nitrogen-containing surfactants which are useful in the formulations of the invention possess the unique multiple capability of serving as binders, lubricants and solubilizers for protein. Moreover, they are themselves water-soluble, thus promoting the dissolution of the reagent formulations in water to provide clinical liquid reagents. These surfactants are sold under the trade designation Tetronic" by Wyandotte Chemical Corporation. They are normally prepared by sequential reaction of first propylene oxide and then ethylene oxide with ethylene diamine in the presence of an alkaline or acid catalyst. Normally these surfactants are prepared at elevated temperatures using alkaline catalysts such as sodium hydroxide, potassium hydroxide, sodium alkoxide, quarternary ammonium bases and the like. Other methods are available for the preparation of these surfactants. The preparation of surfactants such as those utilized in the formulations of the invention is more fully described in U.S. Pat. No. 2,979,528.
The properties and physical state of nonionic surfactants having structures corresponding to those derived from ethylene diamine, propylene oxide and ethylene oxide vary with the lengths of the polyoxypropylene and polyoxyethylene chains. As the drawing shows, the physical state of these surfactants is largely dependent upon the proportionate weight of the surfactant constituted by the polyoxyethylene chains, but is also influenced by the average molecular weight of the polyoxypropylene moieties. The polyoxypropylene chains are hydrophobic while the polyoxyethylene chains are hydrophilic. Thus, the surfactants having polyoxypropylene units of low average molecular weight are more water-soluble than those having polyoxypropylene units of a higher average molecular weight. The numbers set out on the face of the grid correspond to particular members of the Tetronic series. Each number is located at a point on the grid whose coordinates correspond to the polyoxyethylene and polyoxypropylene chain sizes of the particular product which is commercially designated by said number.
Essentially any surfactant whose structure is defined by the coordinants of a point lying in the grid of the drawing may be utilized in the formulations of the invention. It is preferred, however, that the surfactant be solid or at least semi-solid. A greater proportion of the solid surfactants can be satisfactorily incorporated in a reagent formulation and thus a greater binding and lubricating capacity is obtained without adversely affecting other properties of the formulation. Desirably, on the order of 2.5 to 5% by weight of the preferred solid surfactants are incorporated in the reagent form ulations. When the liquid formulations are used, it is not always possible to incorporate more than 2 or 3% by weight of the surfactant without imparting a somewhat waxy character to the formulation. The use of 2 to 3% by weight of a liquid Tetronic surfactant produces a useful product, but the binding and lubricating capabilities of the surfactant are not always fully exploited at such a level. Granules having the most desirable propcities are obtained using solid or semisolid surfactants.
Since the dry reagent formulations of the invention are dissolved in water for use in conducting clinical diagnostic tests, it is also desirable that the surfactant component promote the dissolution of the granular productv Thus, it is preferred that the surfactant be as hydrophilic as possible, i.e., that the molecular weight of the polyoxypropylene hydrophobe moiety of the surfactant be relatively low. Thus, the preferred surfactants for use in the formulations of the invention are those which are both solid or semi-solid in physical state and relatively hydrophilic. Solidstate surfactants with polyoxypropylene chains having an average molecular weight of less than about 4000 are especially preferred, with the most suitable surfactants being those whose polyoxypropylene chains have an average molecular weight of between about 2750 and about 3750 and whose weight percentage of polyoxyethylene units is between about 70% and about 80%. Two particular surfactants whose weight and structure characteristics fall within the latter limits are those sold under the trade designations Tetronic 707 and Tetronic 908. Tetronic 707" has a polyoxypropylene hydrophobe molecular weight on the order of 2750 and a weight percentage of polyoxyethylene units of about 70 while Tetronic 908 has a polyoxypropylene molecular weight of about 3750 and a weight percentage of polyoxyethylene units of about 80%. Good results are also obtained with surfactants whose polyoxypropylene chains have an average molecular weight of between 750 and 4000 with a weight percentage of between about 35% and about 65% polyoxyethylene units. Other surfactants within the grid of the drawing are reasonably satisfactory but less effective than those represented by the right lower comer of the grid.
ln addition to their advantageous effect upon granulation and dissolution of dry clinical test reagent formulations, surfactants of the above-noted character have been found to be effective for solubilizing protein. As indicated above, this is a highly advantageous characteristic, since enzymes and other proteinaceous matter derived from either the reagent formulation or the specimen commonly participate in the test reactions. By solubili zing protein, the surfactants function to facilitate the progress of the test reaction and thus enhance the effectiveness of the reagent formulation. It may, therefore, be seen that incorporation of these surfactants in clinical test formulations uniquely provides multiple advantages in the preparation, packaging, dissolution and functional operation of clinical reagent formulations.
It has further been discovered that the dry clinical reagent formulations of the invention are quite stable and generally possess good shelf life characteristics. Although we cannot precisely account for the particular ingredient or combination of ingredients which imparts the high degree of storage stability, it appears that such stability may be a somewhat general characteristic of dry clinical reagent formulations which include the particular nitrogen-containing nonionic surfactants used in our formulations. If so, the ability to impart storage-stability represents a further aspect of the unique multiple function of this type of surfactant in such formulations.
To prepare the reagent formulations of the invention, the surfactant is mixed with a volatile solvent and at least one reagent capable of participating in a test rcaction to effect a measurable change in a reagent/specimen test system. The surfactant should be soluble up to the amount present in the solvent which is utilized. Solvents which may be used include methylene chloride, chloroform. methanol. benzene. water, methanol/water, and chloroform/methylene chloride. After thorough mixing and appropriate size classification, the solvent is removed to yield a granular product.
In a preferred embodiment of the invention, the ingredients of the formulation, in dry particulate form, are thoroughly blended in a mechanical mixer. With the mixer running, a granulating solution containing the solvent and the surfactant, preferably that sold under the trade designation Tetronic 707 or Tetronic 908, is added. Additional solvent is used as needed to produce granular agglomerates of the desired size and wetness.
The resulting wet granulation is screened through a coarse screen, for example 10 mesh, then spread in thin layers in trays and dried at reduced pressure, for example, 25 inches Hg absolute or less. Depending on the heat sensitivity of the formulation, drying is normally carried out at room temperature or at modest elevated temperature (up to about 37C.). Generally, the depth of the wet granules in the trays should not exceed about V2 inch to 4 inch.
After completion of the drying cycle, the dried granulation is rescreened through a finer screen, for example, 20 to 30 mesh, blended thoroughly and packaged in containers essentially impervious to moisture. Since the components of the reagent formulation are frequently moisture sensitive, the formulation should not be exposed to a relative humidity of more than about 5% after removal from the dryer.
The reagent formulations of the invention are adapted to be packaged in small unitary packages. For example, sufficient reagent formulation for a single assay may be tabletted or packaged in a capsule. The reagent formulations are also adapted to packaging in such containers as foil strip packets, utilizing automatic packaging machinery. Utilizing this packaging approach, sufficient reagent formulation to carry out a suitable predetermined number of tests, such as 10, 25, or 50 tests, may be accurately packaged in a single foil packet. The user then simply dissolves the contents of the multiple test packet in a predetermined volume of water and uses a suitable aliquot of the resulting liquid reagent in the preformance of each of a series of assays for the desired biological substance or property.
In some instances, depending on the nature of the components and their compatibility, all of the reagents necessary in a single assay or determination may be included in a single formulation. ln other instances, incompatabilities and/or other considerations may make it desirable to segregate certain reagents in which case two or more reagent formulations are prepared in accordance with the invention.
To conduct a clinical diagnostic test using the formulations of the invention, the liquid reagent produced by dissolving the dry formulation in a predetermined amount of water is mixed with the biological specimen in a predetermined volumetric or weight ratio. With the aid of appropriate instrumentation as required, the resulting specimen/reagent system is observed for the presence, absence, nature and extent of a physical, chemical or biological change. Such change as does occur is measured to provide the desired information for use in the clinical diagnosis.
Exemplary reagent formulations prepared in accordance with the invention and useful for the determination of hemoglobin, blood urea nitrogen, total protein, serum glutamic oxaloacetic transaminasc, alkaline phosphatase, glucose. inorganic phosphorus. lactate dehydrogenase-L, serum glutamic pyruvic transami nase, uric acid (colorimetric) and uric acid (u.v.) are set forth in Table l. The preferred compositions of these reagent formulations and methods for preparing them are described in the examples following Table 1 which more fully illustrate the invention.
Table l Exemplary Clinical Test Reagent Formulations Granulating Solution Poly- No. of Dry Ingredients ethylene Theo- Tests Formu- Type of Formu (Reagents, Etc.) TETRONIC glycol CH Cl rctical (Thoulation lation Name/Formula Wt. (g.) 707 (g.) 6000 (g) (ml.) I) Yield (g.) sands) A Reagent Formu- NaHCO 300) 30 (a) 200 1000 50 lation for Hemo- K,-,Fe(CN),; 50) (h) 300 globin Assay KCN 30) Mannitol 590) T Enzyme Formu- Uricasc Formulation for Uric lation (equiva- Acid Assay lent to 0.05
units) Glycine H315) 4.50 (a) 25 7.5 Sodium carbonate. anhydrous 39.75) (h) U Copper Reagent Tris-(hydroxy- Formulation for methyl)-amino- Uric Acid Assay methane H150) 7.05 (a) [5 234 7.5
Sodium Bicar bonate l 12.50) (b) 5 Copper Sulfate, anhydrous L95) V Neocuproine Formu- Neocuproine HCl 5.25) 4.75 (a) 30 I60 7.5
lation for Uric Renex 35 150 (b) )0 Acid Assay W Blank Formu- Uricase Placebo 19.50) 4.50 (a) 177 7.5
lation for Uric Glycine l 13.25 Acid Assay Sodium carbonate,
1 l (:1) indicates amount of CH.,CI used as carrier for Tetronic 707 (b) indicates amount of additional CH,Clused to optimize granulation EXAMPLE 1 Hemoglobin Reagent Formulation and Assay Composition of the reagent formulation useful for hemoglobin assay is set forth as formulation A in Table I.
To prepare this formulation, sodium bicarbonate (300 g), milled potassium ferricyanide (50 g.) and potassium cyanide g.) were initially added to 21 Hobart bowl and mixed with a stainless steel spatula. Mannitol (590 g.) was then added and the resulting blend was agitated for five minutes in the mixer. While agitation was continued, a solution of Tetronic 707 (30 g.) in methylene chloride (200 ml.) was added. An additional amount of methylene chloride (300 ml.) was then added to produce the proper granulation.
The wet granulation was screened through a No. 10 mesh stainless steel screen and the wet screened material was transferred to 8 inches X 12 inches Pyrex drying trays, at a depth of between about /2 inch and about 54 inches in each tray. The granulation was then dried in a vacuum oven for 15 hours at a temperature of C. and a pressure of 25 inches Hg.
The dried granulation was removed from the vacuum oven in an environment where the relative humidity was not more than 5%. The dried granulation was then screened through a No. 20 mesh stainless steel screen using an Erweka oscillator. The screened, dried granuof the dissolved reagent, erythocytes in the blood are hemolyzed releasing hemoglobin which is oxidized to methemoglobin. Methemoglobin is converted to cyanmethemoglobin whose formation alters the optical density of the reagent/specimen system. The optical density of the reagent/specimen system is measured at 540 nm. using a suitable spectrophotometer and compared against a reagent blank set at transmission. The hemoglobin level is then determined by reference to a standard curve.
To prepare a liquid reagent sufficient for 50 tests, formulation A (1.00 g.) is dissolved in distilled water and the resulting solution is diluted to 250 ml. and mixed thoroughly. The reagent solution thus produced is stable for three months at room temperature if protected from light.
To conduct the hemoglobin assay test, a reagent/- specimen test system is prepared by adding 20 microliters of well mixed blood (collected with an anticoagulant) to 5 ml. of the above solution of formulation A in a clean test tube. The contents of the tube are mixed thoroughly and allowed to stand at room temperature for at least five minutes. The optical density is then measured as described above to determine the hemoglobin level.
EXAMPLE 2 Colorimetric Formulations and Assay for Uric Acid For the colorimetric uric acid test, three separate formulations are provided. These three formulations are set forth in Table l as formulations T, U and V. Predetermined amounts of these formulations are dissolved in separate portions of water to provide liquid reagents for use in making the uric acid assay.
Preparatory to blending the constituents of enzyme reagent formulation T, the modified uricase component thereof was produced. To produce the modified uricase, a borate buffer was initially prepared by dissolving boric acid (50 g.) in distilled water (3.5 l.) and titrating the resulting solution to a pH of 9 with 10% solution of sodium hydroxide. The titrated solution was then diluted to a total volume of 4 l. (0.2 M in borate) and chilled in refrigerator prior to use. Uricase (about 40 mg.) was transferred to a 250 ml. beaker by streams of the borate buffer delivered from a wash bottle. The uricase used was uricase solution in 50% glycerol obtained from Boehringer Mannheim Corporation (Cat. No. 15074 EVAC) and having a specific activity of about 4.5 U/mg. at 25C. and about 10.5 U/mg. at 37C. After the uricase was transferred, additional borate buffer was added to bring the total volume of uricase solution in the beaker to approximately 100 ml. The diluted uricase solution was then dialyzed against approximately 2 l. of 0.2 M borate buffer for 4 hours, contaminated buffer being replaced with fresh buffer at the end of the first 2 hours of dialysis. While dialysis was in progress potassium chloride (6 g.), mannitol 4 g.) and gum acacia (4 g.) were dissolved in 0.2 M borate buffer (100 ml.). The resulting solution was clarilied by centrifugation at 10,000 rpm and 10C. for 16 minutes using a No. 872 angle rotor in an [EC B-20 refrigerated centrifuge. Bovine serum albumin (0.4 g.) and approximately 67,200 units of catalase were added to the clarified solution to produce a solution referred to hereinafter as the inert solution.
After dialysis of the uricase solution was complete, the dialyzed uricase solution was transferred to a 500 ml. beaker and combined with a 200 ml. portion of the inert solution. Distilled water (200 ml.) was then added and the resulting solution was thoroughly mixed. This solution was then transferred into twoseparate freezedrying vessels and shell frozen in a dry ice/alcohol bath at a temperature of 60C. or below. The frozen solution was lyophilized at -60C. to -70C. and an absolute pressure of millimicrons mercury for 2024 hours. The resultant lyophilized powder was collected under an atmosphere having a relative humidity of less than 5% and stored in a dessicator at 4C. Approximately 19.5 g. of modified uricase was obtained.
To prepare uricase reagent T, modified uricase (equivalent to 0.05 units/test). glycine l 13.25 g.) and anhydrous sodium carbonate (39.75g.) were blended in a Hobart bowl and thoroughly agitated to promote intimate mixing. With the mixer running, a solution of Tetronic 707 (4.50 g.) in methylene chloride (25 ml.) was introduced. Additional methylene chloride (approximately ml.) was subsequently added to produce the desired degree of granulation and wetnessv The wet granulation was then screened and dried and the resulting dry granulation rescreened and packaged in the manner described in Example 1 for hemoglobin reagent formulation A.
In the preparation of copper reagent formulation U, tris-(hydroxymethyl)-aminomethane (112.50 g.), sodium bicarbonate (112.5 g.) and anhydrous cupric sulfate (1.95 g.) were blended in a Hobart bowl and agitated to promote intimate mixing. With the mixer running, a solution of Tetronic 707 (7.05 g.) in methylene chloride (15 ml.) was added. Additional methylene chloride (approximately 5 ml.) was subsequently introduced to produce the desired degree of granulation and wetness. The wet granulation was then screened and dried and the resulting dry granulation rescreened and packaged in the manner described in Example 1 for hemoglobin reagent formulation A.
To prepare neocuproine reagent formulation V, neocuproine hydrochloride (5.25 g.), Renex-35" 150 g.) and Tetronic 707 (4.75 g.) were blended in 21 Hobart bowl and thoroughly agitated to promote intimate mixing. Methylene chloride (approximately 40 ml.) was then introduced to produce the desired degree of granulation and wetness. The wet granulation was then screened and dried and the resulting dry granulation rescreened and packaged in the manner described in Example 1 for hemoglobin reagent formulation A.
Dissolved in separate portions of water, formulations T, U and V provide liquid reagents useful in assaying a biological specimen for uric acid. Uric acid in the specimen reduces cupric ion of formulation U to cuprous ion which in turn reacts with neocuproine of formulation V in buffered solution to form a color complex. The resulting optical density of the test system is compared with the optical density of a blank prepared in the same manner as the test system but further including uricase from formulation T which destroys uric acid. The differences in absorbances between the test system and the blank is proportional to the serum uric acid content.
To prepare the liquid enzymatic reagent, formulation T (1.18 g.) is dissolved in distilled water (150 ml.). A uricase blank solution (formulation W) is prepared by dissolving (1.18 g.) in distilled water (150 ml.). The copper-bearing liquid reagent is prepared by dissolving formulation U (1.55 g.) in distilled water (25 ml.). A neocuproine-bearing liquid reagent is prepared by dissolving formulation V (1.06 g.) in distilled water (25 ml). The liquid reagent solutions of formulations U and V are stable indefinitely at room temperature while the solution of formulation T should be prepared fresh daily. The resulting solutions are sufiicient for conducting 50 tests.
In the conduct of the test, a 3 ml. portion of the solution of formulation T is added to one test tube and 3 ml. portion of formulation W is added to a second test tube. 0.1 ml. of serum is then added to both tubes to provide a specimen/reagent test system in the tube containing distilled water and a blank test system in the tube containing the solution of formulation T. The contents of both test tubes are then incubated for 15 minutes at 37C., following which 1 ml. of a combined color reagent mixture, prepared by mixing equal volumes of the solutions of formulations U and V, is added to both the specimen/reagent test system and the blank test system. Both of the test systems are allowed to stand at room temperature for 15 minutes after addition of the combined color reagent mixture and the light absorbance of each system is then measured at 455 nm on a spectrophotometer set at transmis sion on a water blank. To provide the data required for the calculation of uric acid in the serum. another optical density measurement is taken on a standard reagent blank. The standard reagent blank is prepared by adding uric acid l mg.) and lithium carbonate (60 mg.) to distilled water (about 500 ml.) and warming the mixture to 60C. to dissolve the additives. The resulting solution is cooled to room temperature and diluted to a total volume of l000 ml. with additional quantities of distilled water. 3 ml. of this reagent blank is then added to a test tube and processed in the same fashion as the blank and the specimen/reagent test system including addition of serum, incubation, addition of the abovenoted combined color reagent mixture and a -minute hold prior to measurement of optical density. The mg% uric acid in the serum specimen is then determined in accordance with the following calculation:
Test O.D. (vs. water) Blank O.D. (vs. water) Standard O.D. (vs. reagent blank) x 10mg.
Two formulations are used in the uric acid (U.V.) test. One of these formulations is formulation T of Example 10 while the other is set forth in Table l as formulation W. Predetermined amounts of these formulations are dissolved in separate portions of water to provide liquid reagents for use in making the uric acid (U.V.) assay.
in preparing formulation W, a uricase placebo is used. This is prepared in the same manner as the modifled uricase component of formulation T as described in Example If) except that the uricase is omitted.
To prepare formulation W, uricase placebo (19.50 g.), glycine 1 I325 g.) and anhydrous sodium carbonate (39.75 g.) were blended in a Hobart bowl and thoroughly agitated to promote intimate mixing. With the mixer running, a solution 'of Tetronic 707 (4.50 g.) in methylene chloride (25 ml.) was introduced. Additional methylene chloride (approximately 10 ml.) was subsequently added to provide the desired degree of granulation and wetness. The wet granulation was screened and dried and the resultant dried granulation rescreened and packaged in the manner described in Example 1 for hemoglobin reagent formulation A.
A liquid reagent solution of formulation W is used in conjunction with a liquid reagent solution of formulation T in practicing the uric acid (U.V.) test. In the presence of the uricase of formulation T, uric acid from the specimen reacts with water and oxygen to form allantoin, carbon dioxide, and hydrogen peroxide. Light absorbance at 293 nm, the absorption peak of uric acid, is measured before and after treatment of the specimen with uricase from formulation T with the difference in absorbance being proportional to the uric acid present in the system. Allantoin, the product of the uricase catalyzed reaction of water, uric acid and oxygen, does not absorb at 293 nm.
The liquid reagent solution of formulation T is prepared as described in Example l0 above. To prepare a blank liquid reagent, formulation W (1.18 g.) is dissolved in distilled water 9(ml.). As noted above, the solution of formulation T should be prepared fresh daily. The liquid reagent solution of formulation W is stable for 1 month when refrigerated. The resulting solution is sufficient for conducting 50 tests.
In conducting the test, a blank system is prepared by mixing the solution of formulation W (3.0 ml.) with a specimen of serum (100 pl) while a specimen/reagent test system is prepared by mixing the solution of formulation T (3.0 ml.) with a specimen of the same serum [00 #1). Both the blank system and the specimen/reagent test system are incubated at 37C. for 15 minutes. The incubated mixtures are then transferred to cuvettes of a spectrophotometer having a l centimeter light path. The instrument is zeroed at 0.800 O.D. with the blank at 293 nm. The absorbance of the unknown is then read and the mg% uric acid in the specimen determined in accordance with the following calculatlOnI (0.8 O.D. of unknown) X 4|.36
In view of the above, it will be seen that the several objects of the invention are achieved and other advantageous results attained.
As various changes could be made in the above methods and products without departing from the scope of the invention, it is intended that all matter contained in the above description shall be interpreted as illustrative and not in a limiting sense.
What is claimed is:
I. An enzyme reagent formulation for use in assaying a biological specimen for uric acid comprising a granular, wate r-soluble, anhydrous, storage-stable mixture containing:
b. potassium chloride;
mannitol; gum acacia; bovine serum albumin; glycine;
sodium carbonate; and
a nitrogen-containing polyoxyalkylene nonionic surfactant obtained by the sequential reaction of ethylenediamine with propylene oxide and ethylene oxide in the presence of a catalyst, said surfactant containing polyoxypropylene chains having an average molecular weight of between about 750 and 6750 and polyoxyethylene chains constituting between about 10 and about weight percent of said surfactant.
2. A reagent formulation as set forth in claim 1 wherein said nitrogen-containing surfactant is solid and the polyoxypropylene chains thereof have an average molecular weight of less than about 4000.
3. The method of preparing a granular, water-soluble, free-flowing, substantially anhydrous, storage-sta ble enzyme reagent formulation for use in assaying a biological specimen for uric acid, said method comprising the steps of:
l. preparing a mixture containing:
b. potassium chloride;
d. gum acacia;
e. bovine serum albumin;
g. sodium carbonate;
h. a nitrogen-containing polyoxyalkylene nonionic surfactant obtained by the sequential reaction of ethylenediamine with propylene oxide and ethylene oxide in the presence of a catalyst, said surfactant containing polyoxypropylene chains having an average molecular weight of between about 750 and about 6750 and polyoxyethylene 14 propylene chains thereof have an average molecular weight of less than about 4000.
S. The method as set forth in claim 4 wherein the polyoxypropylene chains of the surfactant have an average molecular Weight of about 2750 and the weight percentage of the polyoxyethylene units thereof is about UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION PATENT NO. 2 3,928,137 DATED I December 23, 1975 INVENTORt'S) I Alexander A Monte: Ching Chiang, Both of Glendora, California It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:
In col. 2, line 66. "anad" should read and In col. 6, line 48, "preformance" should read performance In columns 7 and 8, Table l, the first two lines of the text relating to Formulation W should read as follows:
- W Blank Formu- Uricase Placebo 19.50) 4.50 (a) 25 177 7.5
lation for Uric Glycine 113.25 (b) 10 In col. 11, line 6 "9(ml.)" should read (150 ml.
In col. l2 (Claim 1), line 42. "and 6750" should read and about 6750 Signed and Sealed this twentieth D3) Of ApriI1976 [SEAL] AIIESI.
RUTH C. M ASON C. MARSHALL DANN Alluring ()jfr'ter ('mmnissimwr nj'lau'nls and Trademarks
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|U.S. Classification||435/10, 435/27|
|International Classification||G01N33/72, C12Q1/62|
|Cooperative Classification||C12Q1/62, G01N33/721|
|European Classification||G01N33/72B, C12Q1/62|