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Details for Patent: 5,496,716

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Details for Patent: 5,496,716

Title: Stabilized creative kinase MB composition
Abstract:A stabilized liquid enzyme composition is prepared containing at least one enzyme substrate or analog thereof and/or at least one enzyme product or analog thereof combined with the enzyme in a liquid medium containing. The liquid medium may be human, animal or artificial serum. The composition may also contain an activator chosen from a sulfhydryl compound and a divalent metal ion. In a preferred embodiment, the enzyme is isolated creatine kinase MB(CKMB), the substrate may be adenosine triphosphate, its oxidized or analog form, creatine or its analog, and the product may be adenosine diphosphate, its oxidized or analog form or phosphocreatine or its analog. The activator is preferably N-acetyl-cysteine or .beta.-mercaptoethanol. In a preferred embodiment, CKMB is stabilized with a combination of adenosine triphosphate and creatine. A preferred serum is heat-inactivated alkaline-shocked human serum. The composition displays less than 10% loss of CKMB isoenzyme concentration after 27 days storage at room temperature as measured by an immunoassay. The composition is useful as a calibrator or control reference in an immunoassay.
Inventor(s): Brandt; Douglas R. (Mundelein, IL)
Assignee: Abbott Laboratories (Abbott Park, IL)
Filing Date:May 10, 1994
Application Number:08/240,794
Claims:1. A liquid enzyme composition suitable for use as a calibrator or control reference reagent in an immunoassay for creatine kinase MB (CKMB) isoenzyme comprising

1) a predetermined amount of isolated CKMB isoenzyme which is free of CKMM and CKBB isoenzymes and which is not bound to a carrier material;

2) a CKMB stabilizer selected from the group consisting of:

a) a substrate for CKMB isoenzyme selected from the group consisting of:

i) adenosine triphosphate;

ii) oxidized adenosine triphosphate;

iii) adenosine trisposphate analogs;

iv) oxidized adenosine triphosphate analogs;

v) creatine; and

vi) creatine analogs;

b) a pair of products resulting from the action of CKMB isoenzyme on a substrate with one member selected from each group wherein

i) one group consists of adenosine diphosphate, oxidized adenosine diphosphate, adenosine diphosphate analogs and oxidized adenosine diphosphate analogs; and

ii) the other group consists of phosphocreatine, and phosphocreatine analogs;

c) a combination of a CKMB isoenzyme substrate selected from group a hereinabove and of a CKMB isoenzyme product selected from Group b hereinabove;

d) any of the stabilizers a through c and a CKMB isoenzyme activator selected from the group consisting of a sulfhydryl compound, a divalent metal ion and a combination thereof and

e) combinations thereof; and

3) a protein solution comprising human serum, animal serum or artificial serum said composition displaying less than a 10% loss of CKMB isoenzyme concentration after 27 days storage at room temperature, as measured by immunoassay.

2. A liquid enzyme composition suitable for use as a calibrator or control reference reagent in an immunoassay for creatine kinase MB (CKMB) isoenzyme formed by combining

1) a predetermined amount of isolated CKMB isoenzyme which is free of CKMM and CKBB isoenzymes and is not bound to a carrier material;

2) at least one CKMB substrate selected from the group consisting of oxidized adenosine triphosphate, adenosine triphosphate analogs, oxidized adenosine triphosphate analogs and creatine analogs; and

3) a protein solution comprising human serum, animal serum or artificial serum said composition displaying less than a 10% loss of CKMB isoenzyme concentration after 27 days storage at room temperature as measured by immunoassay.

3. A liquid enzyme composition suitable for use as a calibrator or control reference reagent in an immunoassay for creatine kinase MB (CKMB) isoenzyme comprising

1) a predetermined amount of isolated CKMB isoenzyme which is free of CKMM and CKBB isoenzymes and is not bound to a carrier material;

2) a CKMB stabilizer selected from the group consisting of

a) adenosine triphosphate;

b) adenosine triphosphate and creatine;

c) adenosine triphosphate and phosphocreatine;

d) adenosine triphosphate and adenosine diphosphate;

e) adenosine diphosphate and creatine;

f) adenosine diphosphate and phosphocreatine;

g) creatine and phosphocreatine;

h) any of stabilizers a through g and an enzyme activator selected from the group consisting of:

i) a sulfhydryl compound;

ii) a divalent metal ion; and

iii) a combination thereof;

i) combinations thereof; and

3) a protein solution comprising human serum, animal serum or artificial serum; said composition displaying less than a 10% loss of CKMB isoenzyme concentration after 27 days storage at room temperature as measured by immunoassay.

4. The liquid enzyme composition of claim 3 which comprises a CKMB activator selected from the group consisting of N-acetyl-cysteine and .beta.-mercaptoethanol.

5. The liquid enzyme composition of claim 3 which comprises magnesium as a CKMB isoenzyme activator.

6. The liquid enzyme composition of claim 5 which comprises a CKMB activator selected from the group consisting of N-acetyl-cysteine and .beta.-mercaptoethanol.

7. The liquid enzyme composition of claim 1 or 3 wherein the protein solution is heat-inactivated alkaline-shocked human serum.

8. The liquid enzyme composition of claim 3 wherein the CKMB stabilizer comprises adenosine triphosphate and creatine.

9. The liquid enzyme composition of claim 8 wherein the adenosine triphosphate concentration is between about 0.5 and 50 mM and the creatine concentration is between 5 and 160 mM when the composition is initially formed.

10. The liquid enzyme composition of claim 8 wherein the CKMB stabilizer further comprises a CKMB isoenzyme activator selected from the group consisting of N-acetyl-cysteine and .beta.-mercaptoethanol.
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