# US 4,400,464 A · Method and agent for the stabilization of alkaline phosphatase

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- Number: US 4,400,464 A
- Document ID: US4400464A
- Status: Granted
- Kind: A
- Application number: 06/276,399
- Filed: 1981-06-22
- Granted: 1983-08-23
- Earliest claimed: 1980-06-24
- Assignees: Merck Patent GmbH
- Inventors: Rolf Vormbrock; Roland Helger
- Web page: https://patentgrep.com/patent/US4400464A

## CPC

- C12N 9/96: Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
- C12N 9/16: acting on ester bonds (3.1)
- C12Q 1/42: involving phosphatase
- Y10S 435/81: Packaged device or kit
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- Y10S: TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS

## Abstract

Alkaline phosphatase is stabilized in buffer solutions. This is accomplished by adding a water soluble complex of zinc ions and a chelating agent to the buffer.

## Claims

### Claim 1

In an aminoalcohol-containing buffer solution reagent for use in the determination of the alkaline phosphatase present in a test sample brought into contact with the buffer solution, the improvement wherein the aminoalcohol-containing buffer solution further comprises the products of the addition thereto of an amount of a water-soluble zinc chelate complex effective to stabilize the alkaline phosphatase when the buffer solution is contacted with the test sample.

### Claim 2 (depends on 1)

A buffer solution of claim 1 wherein the buffer solution comprises 2-amino-2-methylpropan-1-ol.

### Claim 3 (depends on 1, 2)

A buffer solution of claim 1 or 2 wherein the added zinc complex is zinc ethylenediaminetetraacetate, zinc trans-1,2-diaminocyclohexanetetraacetate, zinc diethylenetriaminepentaacetate or zinc ethylene glycol bis-(.beta.-amino-ethyl ether) N,N'-tetraacetate.

### Claim 4 (depends on 1, 2)

A buffer solution of claim 1 or 2 wherein the added zinc complex is zinc ethylenediaminetetraacetate.

### Claim 5 (depends on 1)

A buffer solution of claim 1 wherein the added zinc complex is formed by adding a zinc compound and a chelating agent to the buffer solution.

### Claim 6 (depends on 1)

A buffer solution of claim 1 wherein the zinc complex per se is added to the buffer solution.

### Claim 7 (depends on 1)

In a method for determining the alkaline phosphatase content of a test sample by contacting the sample with an aminoalcohol-containing buffer solution, the improvement wherein the buffer solution is that of claim 1.

### Claim 8 (depends on 1)

A buffer solution of claim 1 wherein the zinc complex comprises zinc chelated by a chelating agent which is a compound of the formula Ac.sub.2 N--Z--NAc.sub.2 wherein Z is alkylene or cycloalkylene of 2-8 C atoms and wherein any non-terminal methylene group up to 3 such methylene groups can optionally be replaced by O or N, such replacement N's optionally being substituted by acetate.

### Claim 9

A method for stabilizing the alkaline phosphatase in a buffered solution of the same containing an aminoalcohol, comprising adding to the buffered solution a stabilization effective amount of a preformed water soluble zinc chelate complex or of zinc ions and the chelating agent which make up the complex.