Alkaline phosphatase assay

Claim 2

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;

Claim 3

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

Claim 4

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

Claim 5

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

Claim 6

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

Claim 7

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

Claim 8

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

Claim 9

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;

Claim 10

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;

Claim 11

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.

Claim 12

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.

Claim 13

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.

Claim 14

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

Claim 15

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

Claim 16

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

Claim 17

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

Claim 18

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

Claim 19

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

Claim 20

A REDUCER QUENCH REAGENT FORMULATION CONTAINING: A. L-ASCORBIC ACID; B. SULFAMIC ACID; AND C. SAID NITROGEN-CONTAINING POLYOXYALKYLENE NONIONIC SURFACTANT; AND

Claim 21

A REDUCER QUENCH REAGENT FORMULATION CONTAINING: A. L-ASCORBIC ACID; B. SULFAMIC ACID; AND C. SAID NITROGEN-CONTAINING POLYOXYALKYLENE NONIONIC SURFACTANT; AND

Claim 22

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

Claim 23

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

Claim 24

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.

Claim 25

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.

Claim 26

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.

Claim 27

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.

Claim 28

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.

Claim 29

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.

Claim 30

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.

Claim 31

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.

Claim 32

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.

Claim 33

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.

Claim 36

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.

Claim 37

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.