Stabilized liquid phosphate containing diagnostic compositions and method of preparing same

Claim 3

The method of claim 1 further characterized in that said method comprises mixing a buffer compound with a magnesium compound to form a mixture thereof, and introducing said mixture into a water containing liquid to form an aqueous buffer composition such that the buffer compound is present in the buffer composition to create a proper pH within the desired range for a phosphatase enzyme determination.

Claim 4

The method of claim 2 further characterized in that said magnesium compound is magnesium aspertate.

Claim 5

The method of claim 1 further characterized in that said method comprises mixing a buffer compound with a magnesium compount to form a mixture thereof, and introducing said mixture into a water containing liquid to form an aqueous buffer composition such that the buffer compound is present in the buffer composition to create an alkaline pH within the range of 9.5 to 10.7 for determination of alkaline phosphatase.

Claim 6

The method of claim 1 further characterized in that said method comprises mixing a buffer compound with a magnesium compound to form a mixture thereof, and introducing said mixture into a water containing liquid to form an aqueous buffer composition such that the buffer compound is present in the buffer composition to create an alkaline pH within the range of 10.0 to 10.5 for determination of alkaline phosphatase.

Claim 7

The method of claim 1 further characterized in that said method comprises mixing a buffer compound with a magnesium compound to form a mixture thereof, and introducing said mixture into a water containing liquid to form an aqueous buffer composition such that the buffer compound is present in the buffer composition to create an acid pH within the range of 3.5 to 4.8 for determination of acid phosphatase.

Claim 8

The method of claim 1 further characterized in that said method comprises mixing a buffer compound with a magnesium compound to form a mixture thereof, and introducing said mixture into a water containing liquid to form an aqueous buffer composition such that the buffer compound is present in the buffer composition to create an acid pH within the range of 4.0 to 4.5 for determination of acid phosphatase.

Claim 9

The method of claim 1 further characterized in that said stabilizing agent is phenol and phenolic compounds.

Claim 10

The method of claim 1 further characterized in that said diagnostic reagent compound is a labile substrate used in determination of a phosphatase enzyme.

Claim 11

The method of claim 8 further characterized in that a solubilizing agent is included in the liquid solution to prevent phase separation of the stabilizing agent.

Claim 12

The method of claim 8 further characterized in that said substrate is paranitrophenolphosphate.

Claim 13

The method of claim 1 further characterized in that said diagnostic reagent compound is a labile substrate used in phosphatase enzyme determination, said substrate is paranithrophenolphosphate, and said stabilizing agent is phenol and phenolic compounds.

Claim 14

A composition comprising a labile organic diagnostic reagent compound stabilized against hydrolysis in an aqueous solution, said compound containing a cyclic or aromatic group with a phosphate moiety thereon, said compound being stabilized in the presence of a stabilizing agent selected from the class consisting of (1) phenol and phenolic compounds, (2) imidazol and (3) nitro aliphatic groups containing from 1 to 6 carbon atoms, said stabilizing agent approximating the effect of the cyclic or aromatic group without the phosphate moiety thereon as a reaction product or portion thereof in a hydrolysis reaction, or the effect of the phosphate moiety which would be produced in a hydrolysis reaction, and which thereby inhibits a hydrolization reaction of said compound, and a water containing liquid in which the mixed reagent compound and stabilizing agent are substantially disolved to form the aqueous composition thereof.

Claim 15

A composition comprising a labile organic diagnostic reagent compound stabilized against hydrolysis in an aqueous solution, said compound containing a cyclic or aromatic group with a phosphate moiety thereon, said compound being stabilized in the presence of a stabilizing agent selected from the class consisting of (1) phenol and phenolic compounds, (2) imidazol and (3) nitro aliphatic groups containing from 1 to 6 carbon atoms, said stabilizing agent approximating the effect of the cyclic or aromatic group without the phosphate moiety thereon as a reaction product or portion thereof in a hydrolysis reaction, or the effect of the phosphate moiety which would be produced in a hydrolysis reaction, and which thereby inhibits a hydrolization reaction of said compound, and a water containing liquid in which the mixed reagent compound and stabilizing agent are substantially disolved to form the aqueous composition thereof.

Claim 16

The composition of claim 13 further characterized in that said composition is used in conjunction with a buffer composition comprised of a mixture of a buffer compound and a magnesium compound, said mixture being present in a water containing liquid to form an aqueous buffer composition such that the buffer compound is present in the buffer composition to create a proper pH range for a phosphatase enzyme determination.

Claim 17

The buffer composition of claim 14 further characterized in that the pH range is 9.5 to 10.7 for alkaline phosphatase determination.

Claim 18

The buffer composition of claim 14 further characterized in that the pH range is 10.0 to 10.5 for alkaline phosphatase determination.

Claim 19

The buffer composition of claim 14 further characterized in that the pH range is 3.5 to 4.8 for acid phosphatase determination.

Claim 20

The buffer composition of claim 14 further characterized in that the pH range is 4.0 to 4.5 for acid phosphatase determination.

Claim 21

The composition of claim 14 further characterized in that said substrate is paranitrophenolphosphate.

Claim 22

The buffer composition of claim 14 further characterized in that said magnesium compound is magnesium aspertate.

Claim 23

The composition of claim 13 further characterized in that said stabilizing agent is phenol and phenolic compounds.

Claim 26

The method of claim 22 further characterized in that said stabilizing agent is phenol and phenolic compounds.

Claim 27

The method of claim 23 further characterized in that a solubilizing agent is included in the aqueous solution of substrate and stabilizing agent to prevent phase separation of the stabilizing agent.

Claim 28

The method of claim 22 further characterized in that said substrate is paranithrophenolphosphate and said stabilizing agent is phenol and phenolic compounds.

Claim 29

A stabilized liquid reagent used in phosphatase enzyme determination, said system comprising a substrate composition comprised of a labile organic diagnostic reagent substrate stabilized against hydrolysis in an aqueous solution, said substrate containing a cyclic or aromatic group with a phosphate moiety thereon, said reagent substrate being stabilized in the presence of a stabilizing agent selected from the class consisting of (1) phenol and phenolic compounds, (2) imidazol and (3) nitro aliphatic groups containing from 1 to 6 carbon atoms, said stabilizing agent approximating the effect of the cyclic or aromatic group without the phosphate moiety thereon as a reaction product or portion thereof in a hydrolysis reaction, or the effect of the phosphate moiety which would be produced in a hydrolysis reaction, and which thereby inhibits a hydrolization reaction of said substrate, said substrate being specific with respect to a phosphatase enzyme such that the enzyme will be affected by the substrate or the end product in a determination reaction or both and will not be affected by the stabilizing agent, the mixed reagent substrate and stabilizing agent being substantially dissolved in a water containing liquid to form an aqueous solution thereof, said solution being stored in a container, and said reagent system comprising an aqueous buffer composition comprised of a mixture of a buffer compound and a magnesium compound in a water containing liquid to form said aqueous buffer composition, said buffer compound being present in an amount to create a proper pH range for a phosphatase enzyme determination, said buffer composition being stored in another container.

Claim 30

A stabilized liquid reagent used in phosphatase enzyme determination, said system comprising a substrate composition comprised of a labile organic diagnostic reagent substrate stabilized against hydrolysis in an aqueous solution, said substrate containing a cyclic or aromatic group with a phosphate moiety thereon, said reagent substrate being stabilized in the presence of a stabilizing agent selected from the class consisting of (1) phenol and phenolic compounds, (2) imidazol and (3) nitro aliphatic groups containing from 1 to 6 carbon atoms, said stabilizing agent approximating the effect of the cyclic or aromatic group without the phosphate moiety thereon as a reaction product or portion thereof in a hydrolysis reaction, or the effect of the phosphate moiety which would be produced in a hydrolysis reaction, and which thereby inhibits a hydrolization reaction of said substrate, said substrate being specific with respect to a phosphatase enzyme such that the enzyme will be affected by the substrate or the end product in a determination reaction or both and will not be affected by the stabilizing agent, the mixed reagent substrate and stabilizing agent being substantially dissolved in a water containing liquid to form an aqueous solution thereof, said solution being stored in a container, and said reagent system comprising an aqueous buffer composition comprised of a mixture of a buffer compound and a magnesium compound in a water containing liquid to form said aqueous buffer composition, said buffer compound being present in an amount to create a proper pH range for a phosphatase enzyme determination, said buffer composition being stored in another container.

Claim 31

The method of claim 26 further characterized in that said stabilizing agent is phenol and phenolic compounds.

Claim 32

The composition of claim 26 further characterized in that said diagnostic reagent compound is a labile substrate used in determination of a phosphatase enzyme, said substrate is paranithrophenolphosphate and said stabilizing agent is phenol and phenolic compounds.

Claim 35

The method of claim 29 further characterized in that said method comprises mixing a buffer compound with a magnesium compound to form a mixture thereof and introducing said mixture into a water containing liquid to form an aqueous buffer composition such that the buffer compound is present in the buffer composition to create a proper pH within the desired range for a phosphatase enzyme determination.

Claim 36

The method of claim 29 further characterized in that said method comprises mixing a buffer compound with a magnesium compound to form a mixture thereof and introducing said mixture into a water containing liquid to form an aqueous buffer composition such that the buffer compound is present in the buffer composition to create an alkaline pH within the range of 9.5 to 10.7 for determination of alkaline phosphatase.

Claim 37

The method of claim 29 further characterized in that said method comprises mixing a buffer compound with a magnesium compound to form a mixture thereof and introducing said mixture into a water containing liquid to form an aqueous buffer composition such that the buffer compound is present in the buffer composition to create an acid pH within the range of 3.5 to 4.8 for determination of acid phosphatase.

Claim 38

The method of claim 29 further characterized in that said stabilizing agent is phenol and phenolic compounds.

Claim 39

The method of claim 29 further characterized in that said diagnostic reagent compound is a labile substrate used in determination of a phosphatase enzyme, said labile substrate is paranithrophenolphosphate and said stabilizing agent is phenol and phenolic compounds.

Claim 40

A composition comprising a labile organic diagnostic reagent compound stabilized against hydrolysis in an aqueous solution, said compound containing a cyclic group or aromatic group with a nitro group and phosphate group attached thereto by weak oxygen bonding, said compound being stabilized in the presence of a stabilizing agent selected from the class consisting of (1) phenol and phenolic compounds, (2) imidazol and (3) nitro aliphatic groups containing from 1 to 6 carbon atoms, said stabilizing agent approximating the effect of the cyclic or aromatic group without the phosphate moiety thereon as a reaction product or portion thereof in a hydrolysis reaction, or the effect of the phosphate moiety which would be produced in a hydrolysis reaction, and which thereby inhibits a hydrolization reaction of said substrate, and a water containing liquid in which the mixed reagent compound and stabilizing agent are substantially dissolved to form the aqueous composition thereof.

Claim 41

A composition comprising a labile organic diagnostic reagent compound stabilized against hydrolysis in an aqueous solution, said compound containing a cyclic group or aromatic group with a nitro group and phosphate group attached thereto by weak oxygen bonding, said compound being stabilized in the presence of a stabilizing agent selected from the class consisting of (1) phenol and phenolic compounds, (2) imidazol and (3) nitro aliphatic groups containing from 1 to 6 carbon atoms, said stabilizing agent approximating the effect of the cyclic or aromatic group without the phosphate moiety thereon as a reaction product or portion thereof in a hydrolysis reaction, or the effect of the phosphate moiety which would be produced in a hydrolysis reaction, and which thereby inhibits a hydrolization reaction of said substrate, and a water containing liquid in which the mixed reagent compound and stabilizing agent are substantially dissolved to form the aqueous composition thereof.

Claim 42

The composition of claim 35 further characterized in that said composition is used in conjunction with a buffer composition comprised of a mixture of a buffer compound and a magnesium compound, said mixture being present in a water containing liquid to form an aqueous buffer composition such that the buffer compound is present in the buffer composition to create a proper pH range for a phosphatase enzyme determination.

Claim 43

The composition of claim 35 further characterized in that said stabilizing agent is phenol and phenolic compounds.

Claim 44

The composition of claim 35 further characterized in that said diagnostic reagent compound is a labile substrate used in determination of a phosphatase enzyme.

Claim 45

The composition of claim 35 further characterized in that said reagent compound is a labile substrate used in phosphatase enzyme determination, said substrate is paranithrophenolphosphate and said stabilizing agent is phenol and phenolic compounds.

Claim 48

The method of claim 40 further characterized in that said method comprises mixing a buffer compound with a magnesium compound to form a mixture thereof, and introducing said mixture into a water containing liquid to form an aqueous buffer composition such that the buffer compound is present in the buffer composition to create a proper pH within the desired range for a phosphatase enzyme determination, and storing said buffer composition in a separate container.

Claim 49

The method of claim 40 further characterized in that said substrate is paranitrophenolphosphate or salts thereof and said stabilizing agent is phenol or a phenolic compound.

Claim 50

The method of claim 42 further characterized in that the substrate is present in an amount from about 100 millimoles to about a saturation level, and said stabilizing agent is present in an amount from about 100 millimoles to about at least twice the amount of substrate.

Claim 51

A stabilized liquid reagent system used in phosphatase enzyme determination, said system comprising a substrate composition comprised of a labile organic diagnostic reagent substrate having a general formula R -- PO 4 and which is stabilized in an aqueous solution against hydrolysis in a hydroysis reaction and which reaction is represented by: R -- PO.sub.4 + H.sup.+ ⃡ R.sup.+ + PO.sub.4, and where R -- PO 4 represents a cyclic or aromatic group with a phosphate moiety thereon and PO 4 represents a phosphate group hydrolyzed therefrom and R + represents a reaction product based on R with the phosphate moiety hydrolized therefrom, said reaction normally proceeding to saturation in absence of any stabilization against hydrolysis, said reagent substrate being stabilized in the presence of a stabilizing agent selected from the class consisting of (1) phenol and phenolic compounds, (2) imidazol and (3) nitro aliphatic groups containing from 1 to 6 carbon atoms, said stabilizing agent approximating the effect of the cyclic or aromatic group without the phosphate moiety thereon as a reaction product or portion thereof in a hydrolysis reaction, or the effect of the phosphate moiety which would be produced in a hydrolysis reaction, and which thereby inhibits a hydrolization reaction of said substrate, said substrate being specific with respect to and used in a determination of a phosphatase enzyme such that the enzyme will be affected by the substrate or the end product in a determination reaction or both and will not be affected by the stabilizing agent, the mixed reagent substrate and stabilizing agent being substantially disolved in a water containing liquid to form an aqueous solution thereof, and said solution being stored in a container.

Claim 52

A stabilized liquid reagent system used in phosphatase enzyme determination, said system comprising a substrate composition comprised of a labile organic diagnostic reagent substrate having a general formula R -- PO 4 and which is stabilized in an aqueous solution against hydrolysis in a hydroysis reaction and which reaction is represented by: R -- PO.sub.4 + H.sup.+ ⃡ R.sup.+ + PO.sub.4, and where R -- PO 4 represents a cyclic or aromatic group with a phosphate moiety thereon and PO 4 represents a phosphate group hydrolyzed therefrom and R + represents a reaction product based on R with the phosphate moiety hydrolized therefrom, said reaction normally proceeding to saturation in absence of any stabilization against hydrolysis, said reagent substrate being stabilized in the presence of a stabilizing agent selected from the class consisting of (1) phenol and phenolic compounds, (2) imidazol and (3) nitro aliphatic groups containing from 1 to 6 carbon atoms, said stabilizing agent approximating the effect of the cyclic or aromatic group without the phosphate moiety thereon as a reaction product or portion thereof in a hydrolysis reaction, or the effect of the phosphate moiety which would be produced in a hydrolysis reaction, and which thereby inhibits a hydrolization reaction of said substrate, said substrate being specific with respect to and used in a determination of a phosphatase enzyme such that the enzyme will be affected by the substrate or the end product in a determination reaction or both and will not be affected by the stabilizing agent, the mixed reagent substrate and stabilizing agent being substantially disolved in a water containing liquid to form an aqueous solution thereof, and said solution being stored in a container.

Claim 53

The composition of claim 44 further characterized in that said composition is used in conjunction with a buffer composition comprised of a mixture of a buffer compound and a magnesium compound, said mixture being present in water containing liquid to form an aqueous buffer composition such that the buffer compound is present in the buffer composition to create a proper pH within the desired range for a phosphatase enzyme determination.

Claim 54

The composition of claim 45 further characterized in that said substrate is paranitrophenolphosphate or salts thereof and said stabilizing agent is phenol or a phenolic compound.

Claim 55

The composition of claim 46 further characterized in that the substrate is present in an amount from about 100 millimoles to about a saturation level, and said stabilizing agent is present in an amount from about 100 millimoles to about at least twice the amount of substrate.