US3926735A

US3926735A - Alkaline phosphatase assay

Info

Publication number: US3926735A
Application number: US467319A
Authority: US
Inventors: Alexander A Monte; Ching Chiang
Original assignee: Mallinckrodt Inc
Current assignee: Mallinckrodt Inc
Priority date: 1971-10-20
Filing date: 1974-05-06
Publication date: 1975-12-16
Anticipated expiration: 1992-12-16

Abstract

Alkaline phosphatase is assayed in biological specimens by using granular solid, water-soluble substantially anhydrous, storagestable reagent formulations containing a nitrogen bearing polyoxyalkylene nonionic surfactant obtained by the sequential reaction of ethylenediamine with propylene oxide and ethylene oxide in the presence of a catalyst. The surfactant has an advantageous effect upon granulation, stabilization and dissolution of the formulations, and is effective for solubilizing proteins such as enzymes in the formulations or the specimen.

Description

i United States Patent Monte et a1.

[ Dec. 16, 1975 ALKALINE PHOSPHATASE ASSAY Inventors: Alexander A. Monte; Ching Chiang,

both of Glendora, Calif.

Assignee: Mallinckrodt, Inc., St. Louis, Mo.

Filed: May 6, 1974 Appl. No.: 467,319

Related U.S. Application Data Division of Ser. No. 200,552, Nov. 19, 1971, Pat. No.

2,816,262, which is a continuation-in-part of Ser. No. 190,883, Oct. 20, 1971, abandoned.

U.S. Cl. 195/l03.5 R; 23/230 B; 195/99; 252/408 Int. Cl. COIN 31/14 Field of Search 195/63, 68, 103.5 R, 100, 195/99; 23/230 B; 252/408 References Cited UNlTED STATES PATENTS 9/1961 Babson 195/103.5 R

3,002,893 10/1961 Babson l95/103.5 R 3,413,198 11/1968 Deutsch 195/63 X 3,425,912 2/1969 Deutsch et a1. 195/103.5 R 3,627,688 12/1971 McCarty et a1. 195/63 X Primary ExaminerDavid M. Naff Attorney, Agent, or Firm-Mathew D. Madsen [5 7 ABSTRACT 12 Claims, 1 Drawing Figure ALKALINE PI-IOSPHATASE ASSAY CROSS-REFERENCE TO RELATED APPLICATION This application is a division of our copending, coassigned US. patent application Ser. No. 200,552, filed Nov. 19, 1971, now US. Pat. No. 2,816,262, which is a continuation-in-part of our US. patent application Ser. No. 190,883, filed Oct. 20, 1971, now abandoned.

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 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.

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 develop ment 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 prior art dry reagent formulations are disclosed in 2 Deutsch US. Pat. No. 3,4l3,l98 and Stern et al. US. Pat. No. 3,546,131.

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 com prises the steps of mixing a reagent capable of partici pating in a test reaction to effect a measurable change in a test system, a nitrogen-containing polyexyalkylene 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.

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 poly oxypropylene 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 multitest 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 conven tional 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 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. 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 speci mens 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 US. Pat. No. 2,979,528.

The properties and physical state of nonionic surfac tants 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 watersoluble 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 formulations. 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 properties are obtained using solid or semi-solid 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 product. 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. Solid-state 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 par ticular 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 corner of the grid.

In 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 charac teristic, 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.

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 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. 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 exam ple, 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 /2 inch to 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 performance 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. In 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 pre-determined 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 transaminase, alkaline phosphatase, glucose, inorganic phosphorus, lactate dehydrogenase-L, serum glutamic pyruvic transaminase, 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 l which more fully illustrate the invention.

TABLE 1 Exemplary Clinical Test Reagent Formulations Dry Ingredients Formu- Type of Formu (Reagents. Etc.)

Granulating Solution Poly No. of ethylene Theo- Tests TETRONIC glycol CH. ,Cl retical tThou lation lation Name/Formula WT. (g) 707 (g.) 6000 (g.) (ml) (1) Yield (g.) sands) A Reagent Formu NaHCO; 300 30 (a) 200 I000 50 lation for Hemo- K;,Fe(CN). 50 (b) 300 glohin Assay KCN 30 Mannitol 590 H Reducer Formu Ascorbic Acid 3250 l50 (a) 500 5000 50 lation for Alkaline Sulfamic Acid 1600 (b) 200 Phosphatase Assay l Molybdate Formu Sodium Molyh- 475 37.5 (a) 300 l250 50 lation for Alkaline date 27(H O) Phosphatase Assay Duponol ME Dry 737.5 (b) 150 J Substrate Formu Disodium B-Glycerolation for Alkaphosphate 500 87.5 (a) 400 2600 50 line Phosphatase Tris-(hydroxymethyU- Assay aminomethane 1500 100 Succinic Acid 125 Duponol ME Dry 500 t l (:1) indicates amount of CH Cl used as carrier for Tetronic 707. (h) indicates amount of additional CH- .Cl. used 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 1.

To prepare this formulation, sodium bicarbonate (300 g.). milled potassium ferricyanide (50 g.) and potassium cyanide (30 g.) were initially added to a Hobart bowl and mixed with a stainless steel spatula. Mannitol (590 g.) was then added and the resulting blend was agitated for 5 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. 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 /2 inch and about 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 granulation was transferred to a P. K. blender and mixed for 5 minutes, then packaged in tightly closed containers. Approximately 1000 g. of a water-soluble, substantially anhydrous reagent formulation, sufficient for 50,000 tests, was obtained.

LII

sity of the reagent/specimen system is measures at 540 nm. using a suitable spectrophotometer and compared against a reagent blank set at 100% transmission. The hemoglobin level is then determined by reference to a standard curve.

To prepare a liquid reagent sufficient for 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 solutionthus 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 5 minutes. The optical density is then measured as described above to determine the hemoglobin level.

EXAMPLE 2 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.

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 /2 inch and /1 inch. The trays were 9 then placed in a vacuum oven and the sodium molybdate dried at 55C. and a total pressure of inches Hg until a weight loss of not less than 13% was observed.

To prepare chromogenic reagent formulation 1, the dried sodium molybdate (475 g.) and a dry sodium lauryl sulfate product sold under the trade designation Duponol ME by E. l. 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.

In the preparation of reducer quench formulation H, L-ascorbic acid 3.25 kg.) and sulfamic acid 1.60 kg.) were blended in a Hobart bowl and agitated to promote intimate mixing. With the mixer running, a solution of Tetronic 707 (150 g.) in methylene chloride (500 ml.) was introduced. Additional methylene chloride (200 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.

Preparation of buffer substrate reagent formulation J was initiated by blending B-glycerophospheric 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 (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 packed in the manner described in Example 1 for hemoglobin reagent formulation A.

Dissolved in separate portions of water, formulations H, I and J 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 formulation I in the presence of sulfamic acid and as corbic 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.

In preparation for a test, 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: and formulation J (2.60 g.) is dissolved in 150 m1. 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 two weeks when refrigerated. The liquid reagent constituted by 10 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 tubes is then used for the test reaction while the other tube is used for a blank.

0.1 ml. 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 20 minutes, and the optical density of each determined at 700 nm against a water blank set at 100% transmission. Using a standard curve, the inorganic phosphorous 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 phosphorous released by action of alkaline phosphataseis determined by subtracting the total inorganic phosphorous in the test system from the total amount of inorganic phosphorus in the blank.

To calculate the'alkaline phosphatase units present in the test system, the mg% inorganic phosphorus released is multiplied by 4. In the procedure of Bodansky, known to the art, there is a one-to-one correspondence between the mg% phosphorus released and the alkaline phosphatase units. Since the test of the 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% phosphorus at 700 nm, against a reagent blank set at 100% transmission. A standard potassium dihydrogen phosphate solution for use in establishing the standardcurve 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 m1. (100 mg%) and appropriately diluted aliquots of this stock solution are used in establishing the standard curve.

As those skilled in the art will appreciate, phosphorus standard curves obtained from spectrophotometric measurements of varying concentrations of potassium dihydrogen phosphate are linear only up to about 15 mg%. Where high phosphorus sera (above 7.5 mg%) 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.

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:

1. A buffer substrate reagent formulation for usein assaying a biological specimen for alkaline phosphatase comprising a solid. water-soluble. substantially anhydrous. storage-stable mixture containing:

' a. B-glycerophosphoric acid disodium salt:

b. tris-( hydroxymethyl kaminomethane;

c. succinic acid:

d. sodium lauryl sulfate; and

e. 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 chains constituting between about 10 and about 80 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. A reducer quench reagent formulation for use in assaying a biological specimen for alkaline phosphatase comprising a solid. water-soluble substantially anhydrous. storage-stable mixture containing:

a. L-ascorbic acid:

b. sulfamic acid; and

c. 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 chains constituting between about 10 and about 80 weight percent of said surfactant.

4. A reagent formulation as set forth in claim 3 wherein said nitrogen-containing surfactant is solid and the' polyoxypropylene chains thereof have an average molecular weight of less than about 4000. 5. A chromogenic reagent formulation for use in assaying a biological specimen for alkaline phosphatase comprising a solid. water-soluble substantially anhydrous. storage-stable mixture containing:

a. sodium molybdate;

b. sodium lauryl sulfate; and i c. a nitrogen-containing polyoxyalkylene nonionic surfactant obtained by the sequential reaction of ethylene diamine 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 chains constituting between about 10 and about 80 weight percent of said surfactant.

6. A reagent formulation as set forth in claim 5 wherein said nitrogen-containing surfactant is solid and the polyoxypropylene chains thereof have an average molecular weight of less than about 4000.

7. The method of assaying a biological specimen for alkaline phosphatase using a plurality of solid. water- 12 soluble. substantially anhydrous. storage-stable reagent formulations respectively comprising:

1. a chromogenic reagent formulation containing:

a. sodium molybdate;

b. sodium lauryl sulfate; and

c. 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 chains constituting between about 10 and about weight percent of said surfactant;

2. a reducer quench reagent formulation containing:

a. L-ascorbic acid; b. sulfamic acid; and c. said nitrogen-containing polyoxyalkylene nonionic surfactant; and 3. a buffer substrate reagent formulation containing:

a. B-glycerophosphoric acid disodium salt; b. tris-( hydroxymethyl )-aminomethane: c. succinic acid; d. sodium lauryl sulfate; and e. said nitrogen-containing polyoxyalkylene nonionic surfactant;

the method comprising the steps of:

i. dissolving each of said reagent formulations in separate portions of water to produce a plurality of liquid reagents;

ii. preheating an aliquot of predetermined volume of the liquid reagent containing said buffer substrate reagent formulation for a predetermined period'of time;

iii. mixing the preheated aliquot of said liquid reagent containing said buffer substrate reagent formulation with a biological specimen to form a specimen/reagent test system;

iv. maintaining the specimen/reagent test system at a predetermined elevated temperature for a predetermined period of time;

v. adding both the liquid reagent containing said chromogenic reagent formulation and the liquid reagent containing said reduced quench reagent formulation to said system;

vi. thereafter maintaining said system at a predetermined elevated temperature for a further predetermined period of time: and

vii. measuring the optical density of said system resulting from the interaction between said reagents and said specimen.

8. The method as set forth in claim 7 wherein the optical density of said system is compared with the optical density of a blank processed under such conditions as to provide the same proportion of inorganic phosphorus as would be provided in said test system in the absence of alkaline phosphatase.

9. The method as set forth in claim 7 wherein said nitrogen-containing polyoxyalkylene surfactant is solid and the average molecular weight of the polyoxypropylene chains thereof is less than about 4000.

10. The method of preparing a solid. water-soluble. free-flowing. substantially anhydrous. storage-stable chromogenic reagent formulation for use in assaying a biological specimen for' alkaline phosphatase. said method comprising the steps of:

l. prreparing a mixture containing:

a. sodium molybdate;

b. sodium lauryl sulfate;

c. 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 hav ing an average molecular weight of between about 750 and about 6750 and polyoxyethylene chains constituting between about and about 80 weight percent of said surfactant; and

d. a solvent for said surfactant; and

2. removing the solvent to form a substantially anhy drous, free-flowing, water-soluble, granular solid.

11. The method of preparing a solid, water-soluble, free-flowing, substantially anhydrous, storage-stable buffer substrate reagent formulation for use in assaying a biological specimen for alkaline phosphatase, said method comprising the steps of:

1. preparing a mixture containing:

a. fi-glycerophosphoric acid disodium salt;

bi tris-( hydroxymethyl )-aminomethane;

c. succinic acid;

d. sodium lauryl sulfate;

e. 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 hav- U reducer quench reagent formulation for use in assaying a biological specimen for alkaline phosphatase, said method comprising the steps of:

l. preparing a mixture containing:

a. L-ascorbi'c acid;

b. sulfamic acid;

c. 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 chains constituting between about 10 and about weight percent of said surfactant; and

d. a solvent for said surfactant; and

2. removing the solvent to form a substantially anhydrous, free-flowing, water-soluble, granular solid.

l l i

Claims

1. A CHROMOGENIC REAGENT FORMULATION CONTAINING: A. SODIUM MOLYBDATE; B. SODIUM LAURYL SULFATE; AND C. A NITROGEN-CONTAINING POLYOXYALKYL 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 CHAINS CONSTITUTING BETWEEN ABOUT 10 AND ABOUT 80 WEIGHT PERCENT OF SAID SURFACTANT;

1. preparing a mixture containing: a. L-ascorbic acid; b. sulfamic acid; c. 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 chains constituting between about 10 and about 80 weight percent of said surfactant; and d. a solvent for said surfactant; and

1. preparing a mixture containing: a. Beta -glycerophosphoric acid disodium salt; b. tris-(hydroxymethyl)-aminomethane; c. succinic acid; d. sodium lauryl sulfate; e. 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 chains constituting between about 10 and about 80 weight percent of said surfactant; and f. a solvent for said surfactant; and

1. prreparing a mixture containing: a. sodium molybdate; b. sodium lauryl sulfate; c. 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 chains constituting between about 10 and about 80 weight percent of said surfactant; and d. a solvent for said surfactant; and

1. a chromogenic reagent formulation containing: a. sodium molybdate; b. sodium lauryl sulfate; and c. 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 chains constituting between about 10 and about 80 weight percent of said surfactant;

1. A buffer substrate reagent formulation for use in assaying a biological specimen for alkaline phosphatase comprising a solid, water-soluble, substantially anhydrous, storage-stable mixture containing: a. Beta -glycerophosphoric acid disodium salt; b. tris-(hydroxymethyl)-aminomethane; c. succinic acid; d. sodium lauryl sulfate; and e. 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 chains constituting between about 10 and about 80 weight percent of said surfactant.

2. a reducer quench reagent formulation containing: A. L-ascorbic acid; b. sulfamic acid; and c. said nitrogen-containing polyoxyalkylene nonionic surfactant; and

3. a buffer substrate reagent formulation containing: a. Beta -glycerophosphoric acid disodium salt; b. tris-(hydroxymethyl)-aminomethane; c. succinic acid; d. sodium lauryl sulfate; and e. said nitrogen-containing polyoxyalkylene nonionic surfactant; the method comprising the steps of: i. dissolving each of said reagent formulations in separate portions of water to produce a plurality of liquid reagents; ii. preheating an aliquot of predetermined volume of the liquid reagent containing said buffer substrate reagent formulation for a predetermined period of time; iii. mixing the preheated aliquot of said liquid reagent containing said buffer substrate reagent formulation with a biological specimen to form a specimen/reagent test system; iv. maintaining the specimen/reagent test system at a predetermined elevated temperature for a predetermined period of time; v. adding both the liquid reagent containing said chromogenic reagent formulation and the liquid reagent containing said reduced quench reagent formulation to said system; vi. thereafter maintaining said system at a predetermined elevated temperature for a further predetermined period of time; and vii. measuring the optical density of said system resulting from the interaction between said reagents and said specimen.

3. A reducer quench reagent formulation for use in assaying a biological specimen for alkaline phosphatase comprising a solid, water-soluble substantially anhydrous, storage-stable mixture containing: a. L-ascorbic acid; b. sulfamic acid; and c. 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 chains constituting between about 10 and about 80 weight percent of said surfactant.

3. A BUFFER SUBSTRATE REAGENT FORMULATION CONTAINING: A. B-GLYCEROPHOSPHORIC ACID DISPDIUM SALT; B. TRIS-(HYDROXYMETHYL)-AMINOMETHANE; C. SUCCINIC ACID; D. SODIUM LAURYL SULFATE; AND E. SAID NITROGEN-CONTAINING POLYOXYALKYLENE NONIONIC SURFACTANT; THE METHOD COMPRISING THE STEPS OF: I. DISSOLVING EACH OF SAID REAGENT FOMRULATIONS IN SEPARATE PORTIONS OF WATER TO PRODUCE A PLURALITY OF LIQUID REAGENTS; II. PREHEATING AN ALIQUOT OF PREDTERMINED VOLUME OF THE LIQUID REAGENT CONTAINING SAID BUFFER SUBSTRATE REAGENT FORULATION FOR A PREDETERMINED PERIOD OF TIME; III. MIXING THE PREHEATED ALIQUOT OF SAID LIQUID REAGENT COMTAINING SAID BUFFER SUBSTRATE REAGENT FORMULATION WITH A BIOLOGICAL SPECIMEN TO FORM A SPECIMEN/REAGENT TEST SYSTEM; IV. MAINTAINING THE SPECIMEN/REAGENT TEST SYSTEM AT A PREDETERMINED ELEVATED TEMPERATURE FOR A PREDETERMINED PERIOD OF TIME; V. ADDING BOTH THE LIQUID REAGENT CONTAINING SAID CHROMOGENIC REAGENT FORMULATION AND THE LIQUID REAGENT CONTAINING SAID REDUCED QUENCH REAGENT FORMULATION TO SAID SYSTEM; VI. THEREAFTER MAINTAINING SAID SYSTEM AT A PREDETERMINED ELEVATED TEMPERATURE FOR A FURTHER PREDETERMINED PERIOD OF TIME; AND VII. MEASURING THE OPTICAL DENSITY OF SAID SYSTEM RESULTING FROM THE INTERACTION BETWEEN SAID REAGENTS AND SAID SPECIMEN.

4. A reagent formulation as set forth in claim 3 wherein said nitrogen-containing surfactant is solid and the polyoxypropylene chains thereof have an average molecular weight of less than about 4000.

5. A chromogenic reagent formulation for use in assaying a biological specimen for alkaline phosphatase comprising a solid, water-soluble substantially anhydrous, storage-stable mixture containing: a. sodium molybdate; b. sodium lauryl sulfate; and c. a nitrogen-containing polyoxyalkylene nonionic surfactant obtained by the sequential reaction of ethylene diamine 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 chains constituting between about 10 and about 80 weight percent of said surfactant.

7. THE METHOD OF ASSAYING A BIOLOGICAL SPECIMEN FOR ALKALINE PHOSPHATASE USING A PLURALITY OF SOLIDS WATER-SOLUBLE, SUBSTANTIALLY ANHYDROUS, STORAGE-STABLE REAGENT FORMULATIONS RESPECTIVELY COMPRISING:

10. The method of preparing a solid, water-soluble, free-flowing, substantially anhydrous, storage-stable chromogenic reagent formulation for use in assaying a biological specimen for alkaline phosphatase, said method comprising the steps of:

12. The method of prepAring a solid, water-soluble, free-flowing, substantially anhydrous, storage-stable reducer quench reagent formulation for use in assaying a biological specimen for alkaline phosphatase, said method comprising the steps of: