US3816262A - Reagent formulations for assaying biological specimens amd methods of preparing and using same - Google Patents

Reagent formulations for assaying biological specimens amd methods of preparing and using same Download PDF

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US3816262A
US3816262A US00200552A US20055271A US3816262A US 3816262 A US3816262 A US 3816262A US 00200552 A US00200552 A US 00200552A US 20055271 A US20055271 A US 20055271A US 3816262 A US3816262 A US 3816262A
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reagent
formulation
surfactant
solution
formulations
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A Monte
C Chiang
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Mallinckrodt Chemical Works
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Mallinckrodt Chemical Works
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Priority to BE790338D priority Critical patent/BE790338A/xx
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Priority to US00200552A priority patent/US3816262A/en
Priority to CA153,136A priority patent/CA992857A/en
Priority to GB4558172A priority patent/GB1363335A/en
Priority to AU47444/72A priority patent/AU445936B2/en
Priority to DE2250886A priority patent/DE2250886A1/de
Priority to FR7237158A priority patent/FR2157599A5/fr
Priority to JP47105179A priority patent/JPS4850795A/ja
Priority to IT7252503Q priority patent/IT972384B/it
Priority to NL7214285A priority patent/NL7214285A/xx
Priority to US467319A priority patent/US3926735A/en
Priority to US05/467,550 priority patent/US3950133A/en
Priority to US05/467,423 priority patent/US3953295A/en
Priority to US467551A priority patent/US3876502A/en
Priority to US05/467,320 priority patent/US3953294A/en
Priority to US467431A priority patent/US3928137A/en
Priority to US05/468,464 priority patent/US3997470A/en
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/008—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions for determining co-enzymes or co-factors, e.g. NAD, ATP
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/72—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood
    • G01N33/721—Haemoglobin

Definitions

  • the reagent formulation comprises a mixture containing a reagent capable of participating in a test reaction to effect a measurable change in a test system; and a nitrogen bearing 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.
  • the polyoxypropylene chains of said surfactant have an average molecular weight of between about 750 and about 6,750.
  • 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.
  • 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.
  • 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 ma gent capable of effecting a reaction which causes a measurable change in the specimen/reagent system.
  • 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 coenzyme 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.
  • 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.
  • 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.
  • 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, watersoluble, 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 6,750.
  • 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 the 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; and removing the solvent to form a substantially anhydrous, water-soluble, freeflowing, granular solid.
  • 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.
  • the solid formulations may be encapsulated or tabletted with the proper quantity of reagent in each capsule or tablet for conducting a single test.
  • a multi-test package can be provided from'which the proper amount of liquid reagent is prepared for conducting a specified number of tests.
  • Granulation converts a powdered material into a material constituted by small agglomerates of relatively uniform size. 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.
  • 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.
  • 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.
  • 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.
  • test formulations granulated with the acid 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.
  • 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 re action 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, quaternary 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.
  • 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.
  • 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.
  • 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 grip 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.
  • 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 percent by weight of the preferred solid surfactants are incorporated in the reagent formulations. When the liquid formulations are used, it is not always possible to incorporate more than 2 or 3 percent by weight of the surfactant without imparting a somewhat waxy character to the formulation.
  • Tetronic surfactant 2 to ,3 percent 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 properties are obtained using solid or semi-solid surfactants.
  • the surfactant component promote the dissolution of the granular product.
  • the surfactant be as hydrophilic as possible, i.e., that the molecular weight of the polyoxypropylene hydrophobe moiety of the surfactant be relatively low.
  • 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.
  • Solid-state surfactants with polyoxypropylene chains having an average molecular weight of less than about 4,000 are especially preferred, with the most suitable surfactants being those whose polyoxypropylene chains have an average molecular weight of between about 2,750 and about 3,750 and whose weight percentage of polyoxyethylene units is between about 70 percent and about 80 percent.
  • Two particular surfactants whoe weight and structure characteristics fall within the latter limits are those sold under the trade designations Tetronic 707 and I tronic 9018.
  • Tetronic 707 has a polyoxypropylene hydrophobe molecular weight on the order of 2,750 and a weight percentage of polyoxyethylene units of about 70 percent while Tetronic 908 has a polyoxypropylene molecular weight of about 3,750 and a weight percentage of polyoxyethylene units of about 80 percent.
  • Good results are also obtained with surfactants whose polyoxypropylene chains have an average molecular weight of between 750 and 4,000 with a weight percentage of between about percent and about 65 percent polyoxyethylene units.
  • Other surfactants within the grid of the drawing are reasonably satisfactory but less effective than those represented by the right lower corner of the grid.
  • 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 solubilizing 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.
  • the surfactant is mixed with a volatile solvent and at least one reagent capable of participating in a test reaction 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.
  • Sol vents 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 sol vent is removed to yield a granular product.
  • the ingredients of the formulation, in dry particulate form are thoroughly blended in a mechanical mixer.
  • a granulating solution containing stability may be a somewhat general characteristic of dry clinical reagent formulations which include the particular nitrogen-containing nonionicsurfactants used in our formulations. If so, the ability to impart storagestability represents a further aspect of the uniquemultithe solvent and the surfactant, preferably that sold under the trade designation Tetronic V" 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 A inch to inch.
  • 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 percent 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 performanceof each of aseries of assays for the desired biological substance or property.
  • 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 orweight 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.
  • Table I Exemplary Clinical Test Reagent Formulations Granulating Solution Poly- No. of Dry Ingredients ethylene Theo Tests Formu- Type Of Fonnu- TETRONIC glycol CH CI retical (Thoulation lation Name/Formula Wt.
  • Acid Assay l l indicates amount of CH C], used as carrier for Tetronic 707 (b) indicates amount of additional CH,Cl, used to optimize granulation Blank Formu- Uricase Placebo l9.50 4.50 (a) 177 7.5 lation for Uric Glycine l 13.25 (b) l0 Acid Assay Sodium carbonate,
  • the wet granulation was screened through a No. 10 mesh stainless steel screen and the wet screened material was transferred to 8 inch X 12 inch Pyrex drying trays, at a depth of between about A inch and about /1 inches in each tray. The granulation was then dried in a vacuum oven for 15 hours at a temperature of 35C. 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 percent.
  • the dried granulation was then screened through a No. 20 mesh stainless steel screen using an Erweka oscillator.
  • the screened, dried granulation was transferred to a P.K. blender and mixed for 5 minutes, then packaged in tightly closed 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/specimem system is measured at 540 nm. using a suitable spectrophotometer and compared against a reagent blank set at percent transmission. The hemoglobin level is then determined by reference to a standard curve.
  • 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 3 months at room temperature if protected from light.
  • 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 5 minutes. The optical density is then measured as described above to determine the hemoglobin level.
  • EXAMPLE 2 Blood Urea Nitrogen Formulations and Assay Because certain components of the total number of reagents required for a blood urea nitrogen (BUN) test tend to interact with one another in the dry state, three separate formulations are provided to segregate the interacting components. The three formulations prepared are set forth in Table l as formulations B, C and D. Predetermined amounts of each of these formulations are dissolved in separate portions of water to provide liquid reagents for use in making the BUN assay.
  • BUN blood urea nitrogen
  • alkaline chlorine reagent formulation B sodium dichloroisocyanurate, sold under the trade designation ACL-60 by Monsanto Co. (120 g.), anhydrous lithium hydroxide (240 g. and mannitol powder (180 g.) were blended in a Hobart bowl and agitated to promote intimate mixing. With the mixer running, a solution of Tetronic 707 (15 g.) in methylene chloride (200 ml.) was introduced. Additional methylene chloride (100 ml.) was subsequently added to produce the desired degree of granulation and wetness. The wet granulation was screened and dried, and the resultant dry granulation rescreened and packaged, in the manner described in Example 1 for hemoglobin reagent formulation A.
  • the lyophilized urease/ethylenediaminetetraacetic acid (EDTA) component was prepared.
  • disodium EDTA (24 g.) and tetrasodium EDTA (15.12 g.) were dissolved in distilled water (about 900 ml.) with heating as required.
  • a carbohydrate polymer having a number average molecular weight of about 1,850, a dextrose equivalent of ll3, a pH of 4.5-5.5, and a decomposition point of 440F., sold under the trade designation of Amisol by Corn Products Company 1 l g.) was added and the solution was cooled to about 4C.
  • Lyophilized urease powder having a specific activity of approximately 200 lU/mg., such as that sold by Worthington Biochemical Corporation, Code No. URPC, 100,000 International Units) was dissolved in this solution.
  • the solution was filtered through glass wool or cotton to remove any turbidity.
  • the clarified solution was divided and each portion placed in one of several freeze drying vessels and the portions frozen in thin layers by rotating the vessels in a dry ice/alcohol bath at 60 C. or below.
  • the frozen thin layers were lyophilized at --60"C. to -70C. at a total pressure of 5 m,u Hg. for 16 to 24 hours.
  • the resultant lyophilized powder was collected under an atmosphere having a relative humidity of less than 5 percent and stored in a desiccator at 4C. Assay of the specific urease activity of the dried powder was in the range of 2 to 3 l.U.lmg. at 37C. It was found that the modified urease product thus produced is stable at room temperature and does not require refrigeration.
  • the following exemplary procedure provides approximately 600 grams of granular enzyme formulation containing 12 I.U. urease/ mg. utilizing a lyophilized urease/EDTA formulation having a specific urease activity of 3.0 l.U.lmg.
  • granular enzyme formulation D To prepare granular enzyme formulation D, lumpfree mannitol (459 mg.) and the lyophilized ureaselEDTA formulation prepared as above and having an activity of 3.0 l.U.lmg. (120 g.) were blended in a Hobart bowl and agitated to promote intimate mixing. With the mixer running, a solution of "Tetronic 707 15 g.) and polyethylene glycol having a molecular weight of about 6,000 (6 g.) in methylene chloride ml.) was added to the blend. Additional methylene chloride (approximately 200 ml.) was subsequently introduced to produce the desired degree of granulation. The wet granulation was then screened and dried at room temperature, and the resulting dry granulation rescreened and packaged, in the manner described for hemoglobin reagent formulation A in Example 1.
  • formulations B, C and D provide liquid reagents useful in the determination of blood urea nitrogen.
  • the urease of formulation D specifically hydrolyzes urea to ammonium carbonate, which is subsequently decomposed by the addition of the alkali in formulation B to produce carbon dioxide and ammonia.
  • sodium nitroferricyanide, of chromogenic reagent formulation C ammonia reacts with the phenol derivative of formulation C and active chlorine from formulation B to form an emerald green color complex.
  • the intensity of the color is measured colorimetrically at 650 nm. and is proportional to the urea present.
  • formulation B (0.925 g.), formulation C (6.25 g.) and formulation D 1.0 g.) are each dissolved in water in separate containers.
  • the solution of formulation D is diluted to 50 ml. while the solutions of formulations B and C are each diluted to 25 ml. All of the diluted solutions are thoroughly mixed.
  • the solution of formulation D is stable for one month under refrigeration.
  • the solutions of formulations B and D are stable for 3 months if kept in amber bottles under refrigeration. The resulting solutions are sufficient for conducting 50 tests.
  • anhydrous cupric tartrate 500 g.
  • dihydrated sodium tartrate 750 g.
  • lithium hydroxide 2,000 g.
  • a surfactant comprising a polyoxyethylene ether alcohol and urea sold under the trade designation Renex 35 by Atlas Chemical Industries (500 g.) were blended and thoroughly agitated in a Hobart bowl mixer.
  • a solution of Tetronic 707 100 g.
  • methylene chloride 1,500 ml.
  • Additional methylene chloride 250 ml.
  • 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.
  • the above-prepared formulation Upon being stored at a temperature of 45C., the above-prepared formulation was found to be stable for at least 4 weeks which is equavilent to a stability period of 16 weeks at room temperature.
  • formulation E Dissolved in water, formulation E yields a liquid reagent useful in assaying a body fluid for total protein.
  • formulation E (1.925 g.) is dissolved in distilled water and the solution is diluted to 100 ml. and thoroughly mixed. This reagent solution is stable for at least 3 months at room temperature, and is sufficient to conduct 25 tests.
  • a reagent/- specimen assay system is prepared by adding 50 microliters of serum to 4.0 ml. of the solution of formulation E in a test tube. The contents of the tube are mixed thoroughly and incubated at 37C. for minutes. The 30 optical density at 540 nm. is then read using a spectrophotometer and compared with a reagent blank set at 100 percent transmission. The total protein level is then determined by reference to a standard curve.
  • the modified malate dehydrogenase and reduced nicotinamide adenine dinucleotide components are prepared.
  • Modified malate dehydrogenase was derived from yellow split peas.
  • the peas were pulverized to a fine powder using a mill or a micromill.
  • a saturated solution of potassium chloride was prepared at room temperature by stirring potassium chloride (500 g.) in approximately 1 1. of distilled water for 5 minutes and allowing the solution to stand at room temperature overnight.
  • Pulverized pea powder 10 g. was stirred into a 50 ml. portion of the saturated potassium chloride solution, which extracted the malate dehydrogenase therefrom. Extraction was carried out at room temperature for approximately 3 hours with occasional stirring. At the end of the 3 hour extraction period, the resulting suspension was centrifuged at 10,000 rpm using a No.
  • MDH malate dehydrogenase
  • GOT glutamic oxaloacetic transaminase
  • modified reduced nicotinamide adenine 7' Y dinucleotide NAD + modified reduced nicotinamide adenine 7' Y dinucleotide
  • gum arabic 15 g.
  • Amisol 10 g. and bovine serum albumin (0.5 g.) were dissolved in distilled water (450 ml.) and the resulting solution was titrated to pH 9.0 with 12N sulfuric acid. After titration, the solution was diluted to 500 milliliters with distilled water and clarified by centrifugation at 10,000 rpm with a No. 872 angle rotor in an IEC B-20 refrigerated centrifuge at 10C. for 16 minutes.
  • NADH powder reduced nicotinamide adenine dinucleotide disodium salt
  • the resulting mixture stirred for at least 5 minutes to insure good mixing.
  • This mixture was transferred into freeze drying vessels and frozen in thin layers by rotating the vessels in a dry ice/alcohol bath at 60C. or below.
  • the frozen thin layers were lyophilized for 20 to 24 hours at 60C. to 70C. and an absolute pressure of 5 mp Hg.
  • the resultant lyophilized powder was collected under an atmosphere having a relative humidity of less than 5 percent and stored in a dessicator at 4C. Approximately 50 g. of dry lyophilized powder was obtained.
  • L-aspartic acid (1.250 g. the above modified MDH (equivalent to 1.4 l.U. or 6.0 milligrams MDH), the above modified NADH (equivalent to 0.5 milligrams NADH), tris- (hydroxymethyl)-aminoethane (1,875 g.), and milled succinic acid (187.5 g.) were blended in a Hobart bowl mixer and agitated to promote intimate mixing. With the mixer running, a solution of Tetronic 707 (25 g.) in methylene chloride (800 ml.) was added to the blend.
  • Tetronic 707 25 g.
  • methylene chloride 800 ml.
  • formulations F and G provide liquid reagents useful in assaying for serum glutamic oxaloacetic transaminase.
  • SGOT present in the specimen catalyzes the transamination of L- aspartic acid and aketoglutaric acid producing oxaloacetate and glutamate.
  • the oxaloacetate and NADH in the presence of MDH are converted to malate and NAD.
  • the extent of reaction is indicative of the SGOT present and is measured by observing a decrease in optical density at 340 nm. at 37C.
  • the liquid reagent solution of formulation F is prepared by dissolving 7.2 g. of the formulation in distilled water, diluting to 125 ml. and mixing well.
  • the solution of formulation G is prepared in similar fashion utilizing a 1.0 g. of the formulation and diluting to 25 ml.
  • the formulation F solution' should be prepared fresh daily while the formulation G solution is stable for a week under refrigeration.
  • An SGOT unit is defined as that amount of enzyme which catalyzes the oxidation of 0.001 micromole of NADH to NAD per minute, at 37C. Other conditions for determining SGOT are known. The Karmen procedure known to the art carries out the above reactions at 25C. instead of 37C.
  • EXAMPLE 5 Alkaline Phosphatase Formulations and Assay Three separate formulations are provided for the alkaline phosphatase test. These formulations are set forth in Table l as formulations H, I and J. Predetermined amounts of these formulations dissolved in separate portions of water provide liquid reagents for use in making the alkaline phosphatase assay.
  • chromogenic reagent formulation I Prior to the preparation of chromogenic reagent formulation I, the sodium molybdate component of the reagent blend was dried. Sodium molybdate was transferred into tared 8 inch X 12 inch Pyrex trays at a depth of between about Va and 4 inch. The trays were then placed in a vacuum oven and the sodium molybdate dried at 55C. and a total pressure of 25 inches Hg until a weight loss of not less than 13 percent was observed.
  • chromogenic reagent formulation I To prepare chromogenic reagent formulation I, the dried sodium molybdate (475 g.) and a dry sodium lau ryl sulfate product sold under the trade designation Duponol ME by E. I. DuPont de Nemours and Company (737.5 g.) were blended in a Hobart bowl and agitated to promote intimate mixing. With the mixer running, a solution of Tetronic 707 (37.5 g.) in methylene chloride (300 ml.) was introduced. Additional methylene chloride 150 ml.) was subsequently added to produce the desired degree of granulation and wetness. The wet granulation was screened and dried and the resultant dry granulation rescreened and packaged in the manner described in Example 1 for hemoglobin reagent formulation A.
  • buffer substrate reagent formulation J Preparation of buffer substrate reagent formulation J was initiated by blending B-glycerophosphoric acid, disodium salt (500 g.) tris-(hydroxymethyl)-aminomethane (1.5 kg), milled succinic acid (12.5 g.) and Duponol ME (500 g.) in a Hobart bowl and agitating the blend to promote intimate mixing. With the mixer running, a solution of Tetronic 707 (87.5 g.) in methylene chloride (400 ml.) was introduced. Additional methylene chloride ml.) was subsequently added to produce the desired degree of granulation and wetness. The wet granulation was screened and dried and the resultant dry granulation rescreened and packed in the manner described in Example 1 for hemoglobin reagent fonnulation A.
  • formulations I H, I and .I provide liquid reagents useful in the determination of the alkaline phosphatase content of biological specimens such as blood serum.
  • Alkaline phosphatase in the specimen promotes the release of phosphate from the B-glycerophosphate constituent of substrate reagent formulation J.
  • the phosphate thus released reacts with sodium molybdate of chromogenic reagent fonnulation I in the presence of sulfamic acid and ascorbic acid of reducer quench formulation H.
  • the ascorbic acid reduces the phosphomolybdic acid thus formed to phosphomolybdenum blue. The color of the latter is indicative of the concentration of alkaline phosphatase present in the reagent specimen system.
  • the dry reagent formulations are each dissolved in water to provide liquid reagents.
  • Formulation I 1.25 g.) is dissolved in 25 ml. of distilled water to provide a chromogenic liquid reagent;
  • formulation H (5.00 g.) is dissolved in 25 ml. of water to provide a reducer quench liquid reagent;
  • formulation I (2.60 g.) is dissolved in ml. of water to provide a substrate buffer liquid reagent.
  • the solution of formulation I is stable for a week at room temperature.
  • the solution of formulation J is stable for 2 weeks when refrigerated.
  • the liquid reagent constituted by the solution of formulation H should be prepared fresh daily and protected from light. The resulting solutions are sufficient for conducting 50 tests.
  • Conduct of an alkaline phosphatase determination is initiated by adding 3 ml. portions of the solution of substrate formulation J to each of two test tubes and incubating each tube for 2 minutes at 37C. One of the two test tubes is then used for the test reaction while the other tube is used for a blank.
  • 0.1 ml. (100 lambda) of serum is added to the tube used for the test reaction and mixed thoroughly with the liquid reagent therein to provide a specimen/reagent test system.
  • the test system is then incubated for exactly 15 minutes at 37C., following which 0.5 ml. portions of the liquid reagent solution of formulation I and 0.5 ml. portions of the liquid reagent solution of formulation H are added to both the test system and the tube carrying the test blank. Both the test system and the blank are then incubated at 37C. for another 2 0 minutes, and the optical density of each determined at 700 nm against a water blank set at 100 percent transmission.
  • the inorganic phosphorus content of each test solution is determined from its optical density compared to the water blank. Since both the test system and test blank originally contain the same amount or proportion of B-glycerophosphate, the inorganic phosphorus released by action of alkaline phosphatase is determined by substracting the total inorganic phosphorus in the test system from the total amount of inorganic phosphorus in the blank. 7
  • the mg percent inorganic phosphorus released is multiplied by 4.
  • the procedure of Bodansky known to the art, there is a one-to-one correspondence between the mg percent phosphorus released and the alkaline phosphatase units. Since the test ofthe invention utilizes only a 15 minute incubation time instead of the 60 minute incubation time of Bodansky, the factor of 4 is applied to obtain corresponding results.
  • the standard curve used to determine absolute inorganic phosphorus is obtained by spectrophotometric measurements of the optical densities of potassium dihydrogen phosphate solutions containing 0, 2.5, 5.0, 7.5 and 10 mg percent phosphorus at 700 nm, against a reagent blank set at 100 percent transmission.
  • a standard potassium dihydrogen phosphate solution for use in establishing the standard curve is prepared by weighing out KH PO (438.1 mg.) in a 100 ml. volumetric flask, diluting to volume with high quality distilled water, and storing the diluted solution at 4C. This stock solution contains 100 mg. phosphorus per 100 ml. (100 mg percent) and appropriately diluted aliquots of this stock solution are used in establishing the standard curve.
  • phosphorus standard curves obtained from spectrophotometric measurements of varying concentrations of potassium dihydrogen phosphate are linear only up to about mg percent. Where high phosphorus sera (above 7.5 mg percent) are analyzed for alkaline phosphatase content, therefore, a 50 lambda serum sample is used and the final result multiplied by 2.
  • An alkaline phosphatase unit is defined as the amount of enzyme in 100 ml. of serum which releases 1 mg. phosphorus per hour at 37C.
  • EXAMPLE 6 Glucose Reagent Formulations and Assay For the glucose test. Three separate formulations are provided. These are set forth in Table l as formulations K, L and M. Predetermined amounts of these formulations are dissolved in separate portions of water to provide liquid reagents for use in making the glucose assay.
  • chromogenic reagent formulation K To prepare chromogenic reagent formulation K, mannitol (423.6 g.) and o-dianisidine dihydrochloride (3,3-dimethoxybenzidine dihydrochloride) (7.5 g.) were blended in a Hobart bowl and agitated to promote intimate-mixing. With the mixer running, a solution of Tetronic 707 (14.4 g.) and polyethylene glycol- 6,000 (4.5 g.) in methylene chloride (150 ml.) was introduced. Additional methylene chloride ml.) was subsequently added to produce the desired degree of granulation and wetness. The wet granulation was screened and dried at room temperature, and the resultant dry granulation rescreened and packaged in the manner described in Example 1 for hemoglobin reagent formulation A.
  • Buffer reagent formulation L was prepared by blending monobasic sodium phosphate (311.4 g.), dibasic sodium phosphate (190.8 g.), mannitol (52.8 g.) and spray dried gum arabic (30.0 g.) in a Hobart bowl and agitating the resulting blend to promote intimate mixing. With the mixer running, a solution of Tetronic 707 (15.0 g.) in distilled water (40 ml.) was introduced. Additional distilled water (10 ml.) was subsequently added to produce the desired degree of granulation and wetness. The wet granulation was screened and dried and the resultant dry granulation rescreened and packaged in the manner described in Example 1 for hemoglobin reagent formulation A.
  • modified glucose oxidase gum arabic (60 g.) and mannitol (40 g.) were dissolved in water (about 1,600 ml.) and the resulting colloidal solution was titrated to pH 7.0 with 2 percent sodium hydroxide. After titration, the solution was diluted to 2 liters with distilled water and clarified by centrifugation at 10,000 rpm with a No. 872 angle rotor in an lEC B-20 refrigerated centrifuge. The clear supernatant inert solution was collected and stored at 4C. Bovine serum albumin (2g) was dissolved in 200 ml. of the above-prepared inert solution with the aid of a magnetic stirrer.
  • This solution was transferred into freeze drying vessels, each vessel being filled with -200 ml. of the solution.
  • the solution was frozen in thin layers by rotating the vessels in a dry ice/alcohol bath at 60C. or below.
  • the frozen layers were lyophilized for 16 to 20 hours at -60C. to 70C. and an absolute pressure of Smp. Hg.
  • the resultant lyophilized powder was collected under an atmosphere having a relative humidity of less than 5 percent and stored in a dessicator at 4C.
  • Approximately 14 g. of dry lyophilized glucose oxidase powder with a specific activity range of 1416 lU/mg. at 37C. was obtained.
  • glucose oxidase subformulation was initially produced.
  • the above modified glucose oxidase (suffcient to provide 45 units of unmodified material/test) and mannitol (sufficient to provide 14 mg. total of modified GOD plus mannitol 'per test) were blended in a Hobart bowl and thoroughly agitated to promote intimate mixing.
  • a solution of Tetronic 707 (15.0 g.) and polyethylene glycol- 6,000 (3.0 g.) in distilled water (40 ml.) was introduced. Additional distilled water ml.) was subsequently added to produce the desired degree of granulation and wetness.
  • the wet granulation was screened and dried at room temperature, and the resultant dry granulation rescreened in the'manner described in Example 1 for hemoglobin reagent formulation A.
  • the dry, rescreened granulated glucose oxidase subformulation was transferred to a tared 1,500 cc amber bottle and stored in a dry roompending subsequent intermixture with the other constituents of formulation M, as described below.
  • Each gram of this granulated glucose oxidase subformulation contains sufficient GOD for the conduct of 71 glucose tests.
  • the yield of this formulation in terms of potential test units may therefore be calculated as follows:
  • a peroxidase trituration subformulation for formulation M was prepared by blending,with mortar and pestle, an amount of horseradish peroxidase (POD) and an amount of mannitol sufficient for the number of tests calculated above and determinedby the following respective calculations:
  • POD horseradish peroxidase
  • the dispersion 50 g. of the GOD subformulation After preparation of the GOD/ POD dispersion 50 g. of the GOD subformulation, the dispersion, and an ad ditional 50 g. of the GOD subformulation, were sequentially screened through a 40 mesh stainless steel screen onto aclean receiving surface. All of the screened material was then transferred to aPK blender, along with the remainder of the GOD subformulation, and mixed for more than 5 minutes. The resultant blended granulation was transferred into 8 inch X 12 inch Pyrex trays at a depth of V2 inch to /1 inch and dried for about 15 hours at a room temperature and a total pressure of inches Hg.
  • liquid reagent solutions usefulin the determination of true glucose in a biological specimen such as blood serum.
  • a biological specimen such as blood serum.
  • glucose in the specimen is oxidized to gluconic acid with hydrogen peroxide formed as a by product.
  • the byproduct, hydrogen peroxide oxidizes the o-dianisidine constituent of chromogenic reagent formulation K, in the presence of horseradish peroxidase, producing a colored product which causes an increase in optical density at 445 nm. The extent of the increase indicates the concentration of glucose in the test system.
  • a chromogenic liquid reagent is prepared by dissolving formulation K (0.75 g.) in 50 ml. water.
  • a liquid reagent having both enzyme activity and buffering capacity is prepared by dissolving formulation M (0.75 g.) and formulation L (1.0 g.) in another 50 ml. portion of distilled water.
  • the chromogenic liquid reagent should be prepared fresh daily.
  • the liquid reagent containing formulations L and M is stable for at least 1 week if refrigerated. The resulting solutions are sufficient for conducting 50 tests.
  • Glucose standard curves are linear up to 300 mg percent using the formulations and assay procedure of this example. Sera with high glucose levels 300 mg percent) should be diluted with saline (0.9% NaCl) before analysis and the calculation corrected with the dilution factor.
  • EXAMPLE 7 Inorganic Phosphorus Formulations and Assay Two separate formulations are provided for the inorganic phosphorus test. The two fonnulations which are utilized are set forth in 'Table 1 as formulations N and O. Predetermined amounts of these formulations are dissolved in separate portions of water to provide liquid reagents for use in making the inorganic phosphorus assay.
  • chromogenic reagent formulation the sodium molybdate constituent of the formulation was dried in the manner described in Example 5. Tris- (hydroxymethyl)-aminomethane (1.50 kg), Duponol ME” (470 g.) and dried sodium molybdate (950 g.) were then blended in a Hobart bowl and thoroughly agitated to promote intimate mixing. With the mixer running, a solution of Tetronic 707 (80.0 g.) in methylene chloride (475 ml.) was added. Additional methylene chloride ml.) was then introduced to produce the desired degree of granulation and wetness. The wet granulation was screened and dried and the resultant dry granulation rescreened and packaged in the manner described in Example 1 for hemoglobin reagent formulation A.
  • reagent formulations N and 0 Dissolved in separate portions of water, reagent formulations N and 0 yield liquid reagents useful in assaying a body fluid for inorganic phosphorus.
  • Sodium molybdate from the solution of chromogenic formulation 0 reacts i h... ,t 0 s. niphqsphqrusfr m the sa m to produce phosphomolybdic acid.
  • Phosphomolybdic acid is in turn reduced by the ascorbic acid component of the solution of reducer quench formulation Nto produce a phosphomolybdenum blue, the color of which is indicative of the inorganic phosphorus content of the specimen.
  • the liquid reagent solution of formulation N is prepared by dissolving 1.5 g. of the formulation in 150 ml. of water.
  • the solution of formulation is prepared by dissolving 4.9 g. of that formulation in 50 ml. of water.
  • the chromogenic liquid reagent solution of formulation N is stable for 1 week at room temperature.
  • the liquid reagent solution of formulation 0 should be prepared fresh daily and protected from light. The resulting solutions are sufficient for conducting 50 tests.
  • the phosphorus standard curves are linear up to mg percent.
  • nicotinamide adenine dinucleotide modified as described below (equivalent to 225 mg. nicotinamide adenine dinucleotide) and mannitol (sufficient to provide 25.0 mg./test) were blended in a Hobart bowl and agitated to promote intimate mixing.
  • Additional methylene chloride (approximately 250 ml.) was subsequently introduced to produce the desired degree of granulation and wetness.
  • the wet granulation was then screened and dried at room temperature, and the resultant dry' granulation rescreened and packaged in the manner described in Example I for hemoglobin reagent formulation A.
  • NAD free acid 10 g. was dissolved in distilled water (approximately 150 ml.) and the resulting solution was titrated slowly with 10 percent sodium hydroxide solution to pH 6.4 to 6.5 (approximately 6.5 ml. of sodium hydroxide solution was required). Lactose (20.5 g.) was then added together with a sufficient amount of water to make a total volume of approximately 400 ml. The solution was stirred until all solids were completely dissolved. The resulting solution was transferred into freeze drying vessels and shell frozen by rotating the vessels in a dry ice/alcohol bath at -C. or below.
  • the frozen 'product was lyophilized for 20 to 24 hours at 60C. to C. and an absolute pressure of 5 my Hg.
  • the resultant lyophilized powder was collected under an atmosphere having a relative humidity of less than 5 percent and stored in a dessicator at 4C. Approximately 30.8 g. of dry modified NAD powder was obtained.
  • substrate formulation Q lithium lactate (2.25 kg), tris-(hydroxymethyl)-aminomethane (225 g.), sodium bicarbonate (450 g.) and Renex 35" 175 g.) were blended in a Hobart bowl and agitated to promote intimate mixing. With the mixer running, a solution of Tetronic 707" g.) and polyethylene glycol- 6,000 (25 g.) in methylene chloride (500 ml.) was added to the blend. Additional methylene chloride (approximately 1,l75 ml.) was subsequently introduced to produce the desired degree of granulation and wetness. The wet granulation was then screened and dried at room temperature, and the resultant dry granulation rescreened and packaged in the manner described in Example 1 for hemoglobin reagent formulation A.
  • formulations P and Q provide a liquid reagent useful in assaying a biological specimen for lactate dehydrogenase-L.
  • Catalyzed by LDH-L from the specimen lactate from substrate formulation Q and NAD from coenzyme formulation P are converted to pyruvate and NADH.
  • the extent of this reaction which corresponds to the LDH enzymatic activity of the specimen, is measured by observing an increase in optical density at 340
  • formulation P (1.25 g.) and formulation Q (3.20 g.) are added to the distilled water ml.) in a suitable container and the container shaken gently until the granules are completely dissolved.
  • the liquid reagent solution thus prepared should be used within 8 hours.
  • the LDH units present in the specimen are determined by the following calculation: (A A X 4,900/L LDH units per ml. serum where A, equals the absorbance read at a 1 minute, A equals the absorbance read at 3 minutes and L equals the light path in centimeters.
  • the spectrophotometer which is used includes a temperature control means for the cuvette compartment in which the sample cell is held during measurement, and the temperature of the sample is controlled at 37C.
  • the absorption cell should not be removed from the heating unit for more than seconds when reading the sample.
  • the LDH unit is defined as that amount of enzyme which catalyzes the conversion of 0.001 micromoles per minute NAD to NADH- under the above-described test conditions.
  • the Wacker (or Amador) procedure also known to the art, employs the same primary reaction but is carried out at 25C. instead of 37C.
  • one Wacker (or Amador) unit is equivalent to 1.15 LDH units.
  • the LDH units are multiplied by 0.87.
  • the modified lactate dehydrogenase and reduced nicotinamide adenine dinucleotide components are prepared.
  • the modified NADH component was prepared in accordance with the method described in Example 4.
  • Modified lactate dehydrogenase was prepared as described below.
  • the resulting enzyme mixture was stirred for 5 minutes to insure good mixing and then divided and each portion placed in one of several freeze-drying vessels. Each portion was then frozen in thin layers by rotating the vessel containing it in a dry ice/alcohol bath at 60C. or below. The frozen thin layers were lyophilized at 60 C. to 70C. at a total pressure of 5 mp. Hg for a period of 18-20 hours. The lyophilized powder obtained was collected under an atmosphere having a relative humidity of less than 5 percent stored in a dessicator at 4C. Approximately 26 g. of dry powder was obtained. This powder was assayed for the enzymatic activities of both glutamic pyruvic transaminase and LDH. The GPT specific activity was found to be less than 0.04 percent in relation to the LDH specific activity and the modified LDH lyophilized powder was therefore suitable for use in preparing formulation R.
  • modified NADH having an equivalent NADH content of 0.4 mg
  • modified LDH having an LDH equivalent of two units
  • DL-alanine 1.20 kg.
  • dibasic sodium phosphate 585 g.
  • milled monobasic sodium phosphate 750 g.
  • Tetronic 707 50.4 g.
  • methylene dichloride 200 ml.
  • Additional methylene chloride approximately ml.
  • the wet granulation was then screened and dried at room temperature and the resultant dry granulation rescreened and packaged in the manner described in Example 1 for hemoglobin reagent formulation A.
  • Substrate reagent formulation S has essentially the same composition and was prepared in essentially the same manner as was formulation G of Example 4.
  • formulations R and S provide liquid reagents useful in assaying for 'serum glutamic pyruvic transaminase (SGPT).
  • SGPT present in the specimen catalyzes the transamination of L-alanine and cz-ketoglutaric acid producing a pyruvate and glutamate.
  • Pyruvate and NADH, in the presence of LDH, are converted to lactate and NAD. The extent of reaction is indicative of the SGPT present and is measured by observing a change in optical density at 340 nm at 37C.
  • formulation R (6.55 g.) is dissolved in distilled water (125 ml.).
  • a substrate liquid reagent is prepared by dissolving formulation S (1.00 g.) in distilled water (25 ml.). The resulting solutions are sufficient for 50 tests.
  • the substrate liquid reagent is stable for 1 week when refrigerated.
  • the coenzyme reagent formulation should be prepared fresh daily.
  • an aliquot of the solution of formulation R (2.5 ml.) is mixed with a serum specimen ul) to form a reagent/specimen test system which is then preincubated at 37C. for 7-10 minutes in a heating unit.
  • an aliquot of the solution of reagent formulation S (0.5 ml.) is added to the test system and mixed in less than 10 seconds at room temperature.
  • the containers holding the system is returned to the heating unit where it is held at 37C. Exactly 2 minutes after the solution of formulation S is added, the container is removed from the heating unit and the absorbance of the test system measured using a spectrophotometer which has previously been set at zero absorbance using distilled water as a blank.
  • the container is returned to the heating unit and held at 37C. Exactly 5 minutes after the solution of formulation S is added (i.e., 3 minutes after the first reading), the container holding the test system is again removed from the heating unit and the absorbance again measured on the spectrophotometer.
  • the SGPT content of the system is then determined in accordance with the following calculations: A, A X 1,6)65/L SGPT units per ml. serum where A equals absorbance read at 2 minutes, A equals absorbance read at 5 minutes and L equals the light path of the absorption cell or the ID. of the container in centimeters.
  • An SGPT unit is defined as that amount of enzyme which catalyzes the oxidation of 0.001 micromoles per minute of NADH to NAD at 37C. under the above test conditions.
  • the Karmen procedure known to the art carries out the above reactions at 25C. instead of 37C.
  • one Karrnen unit is equal to 1.09 SGPT units, and SGPT units may therefore be converted to Karmen units by multiplying the SGPT units by 0.917.
  • the cuvette compartment of I the spectrophotometer used includes means for temperature control and the temperature of the specimen is controlled at 37C.
  • the absorption cell should not be removed from the heating unit for more than seconds when making an optical density determination on the test system.
  • EXAMPLE l0 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.
  • the modified uricase component thereof was produced.
  • a borate buffer was initially prepared by dissolving boric acid (50 g.) in distilled water (3.5 l.) and.
  • alase were added to the clarified solution to produce a solution referred to hereinafter as the inert solution.
  • 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 two separate 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 5 millimicrons mercury for 20-24 hours. The resultant lyophilized powder was collected under an atmosphere having a relative humidity of less than 5 percent and stored in a dessicator at 4C. Approximately 19.5 g. of modified uricase was obtained.
  • 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 10 ml.) was subsequently added 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.
  • Uricase (0.2 M in borate) 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 percent glycerol obtained from Boehringer Mannheim Corporation (Cat. No. 15,074 EVAC) and having a specific activity of about 4.5 U/mg. at 25C. and about 10.5 U/mg. at 37C.
  • 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.
  • neocuproine hydrochloride 5.25 g.
  • Renex-35 g. Renex-35 g.
  • Tetronic 707 4.75 g.
  • Methylene chloride approximately 40 ml.
  • 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.
  • 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.
  • formulation T 1.18 g.
  • 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 forrnulation 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 sufficient for conducting 50 tests.
  • 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 100 percent transmission on a water blank.
  • a spectrophotometer set at 100 percent transmission on a water blank.
  • Another optical density measurement is taken on a standard reagent blank.
  • the standard reagent blank is prepared by adding uric acid (100 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 1,000 ml. with additional quantities of distilled water. 3 ml.
  • 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 above-noted combined color reagent mixture and a 15 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:
  • EXAMPLE 1 l U.V. Formulations and Assay for Uric Acid 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.
  • a uricase placebo is used. This is prepared in the same manner as the moditied uricase component of formulation T as described in Example 10 except that the uricase is omitted.
  • uricase placebo (19.50 g.), glycine l 13.25 g.) and anhydrous sodium carbonate (39.75 g.) were blended in a Hobart bowl and thoroughly agitated to promote intimate mixing.
  • 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.
  • 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 10 above.
  • formulation W (1.18 g.) is dissolved in distilled water 150 ml.
  • 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.
  • a blank system is prepared by mixing the solution of formulation W (3.0 ml.) with a specimen of serum 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 (100pt1). 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 1 centimeter light path. The instrument is zeroed at 0.800 0D. with the blank at 293 nm. The absorbance of the unknown is then read and the mg percent uric acid in the specimen determined in accordance with the following calculation:
  • a coenzyme reagent formulation for use in assaying a biological specimen for lactate dehydrogenase comprising a solid, water-soluble, substantially anhydrous, storage-stable mixture containing:
  • a nitrogen-containing polyoxyalkylene nonionic surfactant obtained by the sequential reaction of ethylenediamine first with propylene oxide and then with'ethylene oxide in the presence of a catalyst, the polyoxypropylene chains of said surfactant having an average molecular weight of between 31 about 750 and about 6,750 and the polyoxyethylene chains constituting between about to about 80 weight percent of said surfactant.
  • a substrate reagent formulation for use in assaying a biological specimen for lactate dehydrogenase comprising a solid, water-soluble, substantially anhydrous, storage-stable mixture containing:
  • a surfactant comprising polyoxyethylene ether alcohol and urea
  • a nitrogen-containing polyoxylalkylene nonionic surfactant obtained by the sequential reaction of ethylenediamine first with propylene oxide and then with ethylene oxide in the presence of a catalyst, the polyoxypropylene chains of said surfactant having an average molecular weight of between about 750 and about 6,750 and the polyoxyethylene chains constituting between about 10 to about 80 weight percent of said surfactant.
  • a nitrogen-containing polyoxyalkylene non-ionic surfactant obtained by the sequential reaction of ethylenediamine first with propylene oxide and then with ethylene oxide in the presence of a catalyst, the polyoxypropylene chains of said surfactant having an average molecular weight of between about 750 and about 6,750 and the polyoxyethylene chains constituting between about 10 to about 80 weight percent of said surfactant;
  • a surfactant comprising polyoxyethylene ether alcohol and urea
  • a surfactant comprising polyoxyethylene ether alcohol and urea
  • a nitrogen-containing polyoxyalkylene nonionic surfactant obtained by the sequential reaction of ethylenediamine first with propylene oxide and then with ethylene oxide in the presence of a catalyst, the polyoxypropylene chains of said surfactant having an average molecular weight of between about 750 and about 6,750; and the polyoxyethylene chains constituting between about l0 to about weight percent of said surfactant; and
  • a nitrogen-containing polyoxyalkylene nonionic surfactant obtained by the sequential reaction of ethylenediamine first with propylene oxide and then with ethylene oxide in the presence of a catalyst, the polyoxypropylene chains of said surfactant having an average molecular weight of between about 750 and about 6,750; and the polyoxyethylene chains constituting between about 10 to about 80 weight percent of said surfactant; and

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US00200552A 1971-10-20 1971-11-19 Reagent formulations for assaying biological specimens amd methods of preparing and using same Expired - Lifetime US3816262A (en)

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BE790338D BE790338A (fr) 1971-10-20 Compositions reactives de recherche sur des echantillons biologiques, et leurs procedes de preparation et d'utilisation
US00200552A US3816262A (en) 1971-10-20 1971-11-19 Reagent formulations for assaying biological specimens amd methods of preparing and using same
CA153,136A CA992857A (en) 1971-10-20 1972-10-03 Reagent formulations for assaying biological specimens and methods of preparing and using same
GB4558172A GB1363335A (en) 1971-10-20 1972-10-03 Reagent formulations for assaying biological specimens
AU47444/72A AU445936B2 (en) 1971-10-20 1972-10-05 Reagent formulations for assaying biological specimens and methods of preparing and using same
DE2250886A DE2250886A1 (de) 1971-10-20 1972-10-17 Reagensformulierungen zur untersuchung biologischer proben, verfahren zu ihrer herstellung und deren verwendung
FR7237158A FR2157599A5 (enrdf_load_stackoverflow) 1971-10-20 1972-10-19
NL7214285A NL7214285A (enrdf_load_stackoverflow) 1971-10-20 1972-10-20
JP47105179A JPS4850795A (enrdf_load_stackoverflow) 1971-10-20 1972-10-20
IT7252503Q IT972384B (it) 1971-10-20 1972-10-20 Processo per preparare formulazioni di reagenti per titolare campioni biologici relative formulazioni e loro uso
US05/467,550 US3950133A (en) 1971-10-20 1974-05-06 Reagent formulations for assaying biological specimens and methods of preparing and using same
US05/467,423 US3953295A (en) 1971-10-20 1974-05-06 Reagent formulations for glucose assay
US467551A US3876502A (en) 1971-10-20 1974-05-06 Reagent formulations for assaying urea nitrogen in biological specimens and methods of preparing and using same
US467319A US3926735A (en) 1971-10-20 1974-05-06 Alkaline phosphatase assay
US05/467,320 US3953294A (en) 1971-10-20 1974-05-06 Transaminase assay
US467431A US3928137A (en) 1971-10-20 1974-05-06 Reagent formulation for uric acid assay
US05/468,464 US3997470A (en) 1971-10-20 1974-05-09 Surfactant containing reagent formulations for assaying biological specimens and methods of preparing same

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US19088371A 1971-10-20 1971-10-20
US00200552A US3816262A (en) 1971-10-20 1971-11-19 Reagent formulations for assaying biological specimens amd methods of preparing and using same

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US05/467,320 Division US3953294A (en) 1971-10-20 1974-05-06 Transaminase assay
US05/467,423 Division US3953295A (en) 1971-10-20 1974-05-06 Reagent formulations for glucose assay
US05/467,550 Division US3950133A (en) 1971-10-20 1974-05-06 Reagent formulations for assaying biological specimens and methods of preparing and using same
US05/468,464 Division US3997470A (en) 1971-10-20 1974-05-09 Surfactant containing reagent formulations for assaying biological specimens and methods of preparing same

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US (1) US3816262A (enrdf_load_stackoverflow)
JP (1) JPS4850795A (enrdf_load_stackoverflow)
AU (1) AU445936B2 (enrdf_load_stackoverflow)
BE (1) BE790338A (enrdf_load_stackoverflow)
CA (1) CA992857A (enrdf_load_stackoverflow)
DE (1) DE2250886A1 (enrdf_load_stackoverflow)
FR (1) FR2157599A5 (enrdf_load_stackoverflow)
GB (1) GB1363335A (enrdf_load_stackoverflow)
IT (1) IT972384B (enrdf_load_stackoverflow)
NL (1) NL7214285A (enrdf_load_stackoverflow)

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3915643A (en) * 1973-07-12 1975-10-28 Pierce Chemical Co Determination of salicylate
US3935071A (en) * 1972-03-24 1976-01-27 Boehringer Mannheim G.M.B.H. Process for the conversion of glucose into gluconic acid
US4014651A (en) * 1973-05-14 1977-03-29 Mallinckrodt, Inc. Method for determining thyroid function and reagent composition therefor
US4017365A (en) * 1973-10-09 1977-04-12 Chugai Seiyaku Kabushiki Kaisha Method for determining enzyme activity
US4210557A (en) * 1978-12-15 1980-07-01 Beckman Instruments, Inc. Non-high density lipoprotein precipitant
US4341527A (en) * 1978-01-25 1982-07-27 Rolf Zander Process and reagent for determination of the hemoglobin content of blood
US4452903A (en) * 1981-02-17 1984-06-05 Lee Jin P Assay method and reagent kit means for lipid-containing body fluid
US4547460A (en) * 1984-04-16 1985-10-15 Eastman Kodak Company Multizone analytical element and method for analyte determination
EP0212642A1 (de) * 1985-08-28 1987-03-04 Roche Diagnostics GmbH Verfahren zur Herstellung von präzisen Dosiseinheiten eines Reagenzbestandteiles
EP2420147A1 (en) * 2010-08-17 2012-02-22 Vitae Natural Nutrition, S.L. Nutritional supplement compositon
CN104677895A (zh) * 2015-01-26 2015-06-03 山东省果树研究所 一种测定板栗淀粉含量的方法

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6038666B2 (ja) * 1974-02-07 1985-09-02 和光純薬工業株式会社 クレアチニン測定用試薬
GB1522127A (en) * 1976-07-23 1978-08-23 Mallinckrodt Inc Limulus amebocyte lysate reagent compositions
JPS5433094A (en) * 1977-08-18 1979-03-10 Terumo Corp Test piece for detecting bilirubin
DE3024835A1 (de) * 1980-07-01 1982-01-28 Bayer Ag, 5090 Leverkusen Reagenz fuer die haemoglobinbestimmung
DE3107060A1 (de) * 1981-02-25 1982-09-09 Boehringer Mannheim Gmbh, 6800 Mannheim Kontroll- oder eichserum und verfahren zu seiner herstellung
GB2328228B (en) * 1997-08-16 2000-08-16 Sofitech Nv Shale-stabilizing additives

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3935071A (en) * 1972-03-24 1976-01-27 Boehringer Mannheim G.M.B.H. Process for the conversion of glucose into gluconic acid
US4014651A (en) * 1973-05-14 1977-03-29 Mallinckrodt, Inc. Method for determining thyroid function and reagent composition therefor
US3915643A (en) * 1973-07-12 1975-10-28 Pierce Chemical Co Determination of salicylate
US4017365A (en) * 1973-10-09 1977-04-12 Chugai Seiyaku Kabushiki Kaisha Method for determining enzyme activity
US4341527A (en) * 1978-01-25 1982-07-27 Rolf Zander Process and reagent for determination of the hemoglobin content of blood
US4210557A (en) * 1978-12-15 1980-07-01 Beckman Instruments, Inc. Non-high density lipoprotein precipitant
US4452903A (en) * 1981-02-17 1984-06-05 Lee Jin P Assay method and reagent kit means for lipid-containing body fluid
US4547460A (en) * 1984-04-16 1985-10-15 Eastman Kodak Company Multizone analytical element and method for analyte determination
EP0212642A1 (de) * 1985-08-28 1987-03-04 Roche Diagnostics GmbH Verfahren zur Herstellung von präzisen Dosiseinheiten eines Reagenzbestandteiles
EP2420147A1 (en) * 2010-08-17 2012-02-22 Vitae Natural Nutrition, S.L. Nutritional supplement compositon
CN104677895A (zh) * 2015-01-26 2015-06-03 山东省果树研究所 一种测定板栗淀粉含量的方法

Also Published As

Publication number Publication date
GB1363335A (en) 1974-08-14
AU4744472A (en) 1974-03-07
FR2157599A5 (enrdf_load_stackoverflow) 1973-06-01
JPS4850795A (enrdf_load_stackoverflow) 1973-07-17
IT972384B (it) 1974-05-20
DE2250886A1 (de) 1973-05-03
CA992857A (en) 1976-07-13
NL7214285A (enrdf_load_stackoverflow) 1973-04-25
AU445936B2 (en) 1974-03-07
BE790338A (fr) 1973-04-20

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