Liquid reagent to detect creatine kinase

Details Liquid reagent to detect creatine kinase Liquid reagent to detect creatine kinase

1999-12-20

2001-10-23

Leary, Louise N. (Department: 1623)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving transferase

C435S183000, C435S194000

Reexamination Certificate

active

06306617

ABSTRACT:

BACKGROUND OF THE INVENTION
The present invention relates to a liquid reagent for detecting creatine kinase.
Creatine kinase is used for bodily catalysis of the ATP-dependent phosphorylation of creatine into creatine phosphate (to-and-fro reaction). Creatine kinase is found in the body, for instance in the brain, in the heart muscle and the skeletal musculature. Increased creatine kinase values in the heart muscle are present at cardiac infarction, and in the skeletal musculature they indicate muscle pathologies (for instance muscular dystrophy). Renal insufficiency is accompanied for instance with a rise in creatine kinase activity in the serum.
Accordingly creatine kinase activity represents a routinely checked and significant clinical parameter.
A current detection system for creatine kinase activity operates according to the reaction below:
In this detection, creatine kinase activity is determined indirectly by means of the NAD(P)H content with absorption measurement at 340 nm.
Conventional liquid reagents used in detecting creatine kinase, for instance by means of the above reaction, contain the required substrates, co-substrates and enzymes and optionally and illustratively stabilizing or bactericidal substances etc. In many cases, the reagents required for the reactions of detection are split in such manner into two reaction batches so that no undesired enzymatic decomposition reactions take place during storage. The reagent batches are mixed to form the liquid reagent (test reagent) and the reactions of detection take place only thereafter in the test sample as a function of creatine kinase activity.
The above described detection procedure is relatively reliable and is used in many creatine kinase activity tests.
However, it does entail a problem in that creatine kinase is comparatively rapidly inactivated for instance in serum. For that reason, liquid reagents of this species contain a creatine kinase reactivator. A suitable reactivator is a component containing SH groups, typically N-acetylcysteine, (NAC hereafter), which is suitable for freeze drying.
However, NAC is rated only as a conditionally appropriate reactivator in the literature (Morin, Clinical chemistry, vol 23, #9, September 1977, pp 1569-1575). In particular it was found that the reactivator effect of NAC drops following a substantial length of storage because of formation of inhibitors, and as a result special substances to stabilize NAC are present in many detection systems containing NAC.
On this ground, the above cited publication recommends other reactivators, in particular thioglycerol to which is ascribed substantially increased reactivation performance and storage stability compared to NAC.
However, applicant has found that problems remain even when using thioglycerol in liquid reagents for detecting creatine kinase, in particular, as regards stability.
As a result, thioglycerol is predominantly used separately in the known liquid reagents. Illustratively, the German patent 31 38 602 discloses a liquid reagent of two reagent batches, one of which is essentially a thioglycerol buffer. In this batch, the thioglycerol is not meant to be a reactivator. Instead, before the analysis proper, a sample is added to the reagent batch containing thioglycerol to eliminate clouding. Thereupon the cleared sample is mixed with the second reagent batch which contains the actual detection reagents.
The patent document WO 95/30769 describes a liquid reagent of which one batch acts as the activator solution of which the main ingredient is thioglycerol. The other substances needed for detection are contained in the other reagent batches. The described liquid reagent offers improved thioglycerol stability. However, on account of the additional batch (almost always the known liquid reagent consists of a total of three batches) handling is made more difficult.
SUMMARY OF THE INVENTION
Therefore it is the objective of the invention to create a liquid reagent of the initially cited kind which contains thioglycerol as the reactivator, which is stable over a long time and allows detection with fair simplicity.
Accordingly, the invention provides a liquid reagent to detect creatine kinase activity and containing the following substances: glucose-6-phosphate dehydrogenase (hereafter G6PDG), hexokinase, creatine phosphate, glucose, ADO, NAD(P) and thioglycerol, these substances being split in such manner into two separate batches of reagents that thioglycerol and at least one further substance are contained in one batch, and G6PDH and any remaining substances are contained in the other batch, of reagents.
It was found that on account of the separation of the invention of G6DPH and thioglycerol, and where called for further conventionally used substances such as NADP, glucose, hexokinase, ADP, AMP or MG, the storage stability of both batches of reagents, and thereby that of the liquid reagent, can be substantially increased as a whole.


REFERENCES:
patent: 5716797 (1998-02-01), Danno et al.
patent: 31 38602 A1 (1998-12-01), None
patent: 95/30769 (1994-05-01), None

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