JP2000041698A - Composition for measuring creatine kinase activity and measurement of creatine kinase activity - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a composition for measuring creatine kinase, capable of avoiding bilirubin interference in a specimen and to provide a method for measuring creatine kinase activity. SOLUTION: In this composition for measuring creatine kinase comprising a first reagent containing no creatine phosphate and a second reagent containing creatine phosphate, the first reagent characteristically has pH <=6.1 at 30 deg.C. In this method for creatine kinase activity in a specimen in which a blank value is obtained by reacting the first reagent containing no creatine phosphate with the specimen and measuring the change of absorbance, then the reaction mixture is reacted with the second reagent containing creatine phosphate to measure the change of absorbance and the black value is subtracted from the change of absorbance, the first reagent has pH <=6.1 at 30 deg.C.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a composition for measuring the activity of creatine kinase (hereinafter abbreviated as CK), which can avoid interference with bilirubin, and a method for measuring the activity of CK.

[0002]

2. Description of the Related Art CK is the most abundant enzyme in skeletal muscle in vivo, but it is also found abundantly in brain and heart muscle, and also in smooth muscle, nervous system and normal human blood. That's the enzyme you are doing. In addition, CK transfers phosphoric acid from creatine phosphate (hereinafter abbreviated as CP) to adenosine diphosphate (hereinafter abbreviated as ADP), and adenosine triphosphate (hereinafter abbreviated as ATP) and creatine. It is an enzyme that catalyzes the reaction that produces, and plays an important role in energy metabolism. CK activity in serum is known to increase in muscular diseases such as progressive muscular dystrophy, acute myocardial infarction, myocarditis and hypothyroidism, and decrease in hyperthyroidism. It is an important index for diagnosis.

[0003] Conventionally, as a method of measuring CK activity, creatine, ATP, creatine phosphate, A
Many methods for measuring any of DP have been reported.
The equilibrium of the CK reaction is inclined toward the reverse reaction. In the reverse reaction, an activity 4 to 6 times higher than that in the forward reaction is obtained, and the sensitivity is good and the reproducibility is excellent. In addition, at the optimum pH for the correct reaction, CK
Thiol compound (hereinafter abbreviated as SH compound), which is an activator, is unstable. For these reasons, a method of measuring ATP generated continuously using a reverse reaction has been widely used. In this case, hexokinase (hereinafter abbreviated as HK) or glucokinase (hereinafter abbreviated as GlcK) and glucose-6-phosphate dehydrogenase (hereinafter abbreviated as G6PDH) are components of the CK coupling enzyme. The reaction of Formula 1, Formula 2, or Formula 3 is advanced by using a reagent to convert nicotinamide adenine dinucleotide (phosphate) (hereinafter abbreviated as NAD (P)) to reduced NAD (P) H. The method of measuring the increase in absorbance at a wavelength of 340 nm when performing
Abbreviated as CC. ) And is generally used. Table 1 shows the reagent formulations of the JSCC recommendation method (Clinical Chemistry, Vol. 19, No. 2, September 1996, pp. 184-208).

[0004]

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[0005]

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[0006]

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[0007]

[Table 1]

[0008] On the other hand, human serum has a large absorption in the visible region, such as adenylate kinase (hereinafter abbreviated as AK) or other substances that directly participate in a reaction system, or an error such as bilirubin. Substances whose absorption affects the measurement may be present at high concentrations. Methods for avoiding AK interference include adenosine monophosphate (hereinafter abbreviated as AMP) that competitively inhibits AK,
A method of adding diadenosine pentaphosphate (hereinafter abbreviated as Ap5A) or a method in which a sample is first reacted with a first reagent containing no CP to measure a blank value, and then a second reagent containing CP is added to the reaction mixture. C in the sample by the double kinetic method of subtracting the blank value from the value measured by adding the reagent
A method for measuring K activity has been established. These methods are JS
It is also recommended by the CC. However, there is currently no active workaround for bilirubin interference,
In particular, an error occurred when the CK activity in a sample containing a high concentration of bilirubin was measured by the double kinetic method.

[0009]

DISCLOSURE OF THE INVENTION The present invention has been made in view of the above-mentioned circumstances, and a composition for measuring the activity of CK, which can avoid interference by bilirubin in a specimen,
It is an object of the present invention to provide a method for measuring the activity of K.

[0010]

Means for Solving the Problems The present inventors have made intensive studies to solve such problems, and as a result, have determined the pH at 30 ° C. of the first reagent used in the CK activity measurement by the double kinetic method. It has been found that by setting the ratio to 6.1 or less, interference by bilirubin can be avoided, and the present invention has been achieved. That is, the present invention relates to a composition for measuring the activity of CK comprising a first reagent containing no CP and a second reagent containing CP, wherein p at 30 ° C.
A blank is obtained by reacting a sample with a composition for measuring the activity of CK, wherein H is not more than 6.1, and a first reagent not containing CP, and measuring a change in absorbance. A method of measuring the change in absorbance by adding a second reagent containing CP to the reaction mixture, reacting the mixture, and subtracting a blank value from the change in absorbance to measure the activity of CK in the sample. PH of reagent at 30 ° C
Is set to 6.1 or less.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.

The composition for measuring the activity of CK of the present invention comprises CP
And a second reagent containing CP. When the CK in the sample acts on the CP in the second reagent, the first reagent receives a substance to which the phosphate of the CP is transferred, a coupling enzyme and a substrate of the CK measurement system, and a reducing equivalent derived from the substrate. It is preferable to use a substance containing a substance or other substances necessary for these reactions. For example, ADP, glucose, H
K or GlcK, NAD or NADP, G6PDH, magnesium salts, SH compounds as CK activators, ED
Examples include those containing a chelating agent such as TA or DTPA as a component. The second reagent only needs to contain at least CP.

The concentration of the SH compound in the composition for measuring the activity of CK may be not less than the concentration at which CK activating ability is generated. For example, N-acetylcysteine (hereinafter abbreviated as NAC) is used as the SH compound. When used, the concentration in the reaction solution is 0.1 to 200 mM, preferably 20 to 100 m
What is necessary is just to add so that it may become M. Further, a plurality of SH compounds may be used in appropriate combination.

EDTA or DTPA in the composition for measuring the activity of CK is 0.005 to 20 mM, preferably 0.1 to 20 mM.
What is necessary is just to add so that it may be set to about 5 mM.

The source of HK used in the present invention is not limited. For example, HK derived from Bakers Yeast can be used. The source of GlcK, which has a higher substrate specificity for glucose than HK, is not limited. For example, various sources such as those derived from microorganisms such as Aerobacter aerogenes and those derived from animals are used. can do.

HK and GlcK may be added so that the concentration at the time of measuring CK activity is within the concentration range according to a known measuring method.
What is necessary is just to add so that it may become 5-20 U / ml, Preferably it becomes 1-6 U / ml.

The source of G6PDH is not limited, similarly to HK and GlcK, but G6PDH acts not only on NADP but also on NAD as a coenzyme.
H (eg, Leuconostoc mesenteroides, Pseudomonas fluorescens, enzymes derived from thermophilic bacteria, etc.) are preferred. G6PDH may be added so that the concentration at the time of measuring CK activity falls within the concentration range according to a known measuring method.
What is necessary is just to add so that it may become 5-40 U / ml, Preferably it becomes 0.5-10 U / ml.

NAD or NADP used in the present invention
May be added so that the concentration at the time of measuring the CK activity falls within the concentration range according to a known measuring method. For example, the concentration in the reaction solution at the time of measuring the CK activity is 0.1 to 10 mM, preferably 1 to 10.
What is necessary is just to add so that it may be set to about 5 mM.

The ADP used in the present invention, when present in a high concentration in the reaction mixture, inhibits CK activity. For example, the concentration of the ADP in the reaction mixture at the time of measuring the CK activity is 0.1 to 10%.
mM, preferably 1 to 5 mM.

When CP used in the present invention is present in a reaction solution at a high concentration, inhibition of CK activity is recognized. For example, when the CK activity is measured, the concentration in the reaction solution is 5 to 70 mM.
Preferably, it is added so as to have a concentration of 20 to 50 mM.

The glucose used in the present invention may be added so that the concentration at the time of measuring the CK activity falls within the concentration range according to a known measuring method. It may be added so as to be 200 mM, preferably 5-50 mM.

As the magnesium salt used in the present invention, for example, all magnesium salts usually used in the art such as magnesium acetate, magnesium sulfate and magnesium hydrochloride can be used. However, since CK is known to undergo non-competitive inhibition by anions, it is preferable to use magnesium acetate, magnesium hydrochloride, or the like that dissociates anions having a small degree of inhibition. These magnesium salts may be added so that the concentration at the time of measuring CK activity falls within the concentration range according to a known measuring method. For example, the concentration in the reaction solution at the time of measuring CK activity is 2 to 50 mM, preferably 5 mM.
What is necessary is just to add so that it may be set to -25 mM.

In the composition for measuring the activity of CK of the present invention, in addition to the above-mentioned substances, preservatives such as sodium azide, stabilizers, surfactants, buffers and the like used in the art may be appropriately used. it can.

Since the body fluid sample contains AK which gives a positive error to CK activity, the CK to which the present invention is applied is used.
It is preferable to add AMP, Ap5A, etc., which have an activity of inhibiting AK activity, to the composition for activity measurement.

The measurement of CK activity using the composition for measuring CK activity of the present invention may be performed according to the following known operation method (double kinetic method). First, a sample is reacted with a first reagent containing no CP, and the change in absorbance is measured to obtain a blank value. Next, a second reagent containing CP is added to the mixed reaction product to cause a reaction. Is measured, and a CK activity value in the sample is obtained by subtracting a blank value from the absorbance change amount.

The method for measuring the activity of CK of the present invention uses a first reagent containing no CP and a second reagent containing CP. The method for measuring the activity of CK is to first react a sample with a CP-free first reagent, measure the change in absorbance to obtain a blank value, and then add a second reagent containing CP to the reaction mixture. This is a double kinetic method in which the amount of change in absorbance is measured by reacting, and the CK activity value in the sample is obtained by subtracting a blank value from the amount of change in absorbance. The first reagent does not contain CP, but when CK in the sample acts on CP in the second reagent, a substance to which phosphate of CP is transferred, a coupling enzyme and a substrate of the CK measurement system, It is preferable to use a receptor containing a reducing equivalent receptor derived from a substrate, and other components necessary for these reactions. For example, ADP, glucose, HK or GlcK, NAD
Alternatively, those containing, as components, NADP, G6PDH, magnesium salts, a chelating agent such as an SH compound, EDTA or DTPA as a CK activator. The second reagent only needs to contain at least CP.

The method of the present invention for measuring the activity of CK capable of avoiding interference with bilirubin may be carried out by adjusting the pH of the above-mentioned first reagent at 30 ° C. to 6.1 or less, preferably from 5.0 to 5.0.
6.1, more preferably in the range of 5.8 to 6.1.

[0028]

Next, the present invention will be described in detail with reference to examples.

Example 1 [Preparation of first reagent] Imidazole buffer 125 mM, magnesium acetate 12.5 mM, ADP 2.5 mM, NA
DP 2.5 mM, NAC 25 mM, Ap5A0.013
mM, AMP 6.25 mM, glucose 25 mM, sodium azide 15 mM, EDTA 2 mM, and then adjusted to pH 5.0 with acetic acid at 30 ° C., and then GlcK (manufactured by Unitika) 4 U / ml, G6
PDH (Boehringer Mannheim) 1.9 U / ml
Was added.

[Preparation of Second Reagent] A 150 mM solution of CP was used as a second reagent.

[Preparation of Sample] Bilirubin F of interference check A from International Reagents was added to standard serum at a concentration of 40 mg / dl, and this was added to the same standard serum.
This was diluted with serum to which an equal amount of distilled water had been added to prepare a 10-step dilution series.

[Measurement of CK activity value] Using the first reagent and the second reagent, the CK activity value in the sample was measured by the following method. First, after reacting 320 μl of the first reagent with 8 μl of the sample, using a Hitachi 7070 automatic analyzer (analysis method:
Rate B, main wavelength: 340 nm, sub-wavelength: 405 nm,
Measurement points: 10 to 16, molecular absorption coefficient used: ε =
6.22), the amount of change in absorbance was measured and used as a blank value. Next, 80 μl of the second reagent was added to the mixed reaction product to cause a reaction, and the reaction was performed using a Hitachi 7070 type automatic analyzer (analysis method: rate B, main wavelength: 340 nm, auxiliary wavelength: 405).
nm, measurement point: 25-31, molecular extinction coefficient used:
ε = 6.22), and the amount of change in absorbance was measured. The CK activity value was determined by subtracting a blank value from the absorbance change amount.
Table 2 shows the results. The values in Table 2 indicate C in the serum supplemented with bilirubin (bilirubin / F concentration 4 to 40 mg / dl).
The K activity value was shown as a relative value (%) to the CK activity value in the standard serum without bilirubin.

Example 2 The CK activity value of a sample was measured in the same manner as in Example 1 except that the pH of the first reagent at 30 ° C. was changed to 5.2. Table 2 shows the results.

Example 3 The CK activity value of a sample was measured in the same manner as in Example 1 except that the pH of the first reagent at 30 ° C. was changed to 5.4. Table 2 shows the results.

Example 4 The CK activity value of a sample was measured in the same manner as in Example 1 except that the pH of the first reagent at 30 ° C. was changed to 5.6. Table 2 shows the results.

Example 5 A CK activity value in a sample was measured in the same manner as in Example 1 except that the pH of the first reagent at 30 ° C. was changed to 5.8. Table 2 shows the results.

Example 6 The CK activity value of a sample was measured in the same manner as in Example 1 except that the pH of the first reagent at 30 ° C. was changed to 6.0. Table 2 shows the results.

Example 7 The CK activity value of a sample was measured in the same manner as in Example 1 except that the pH of the first reagent at 30 ° C. was changed to 6.1. Table 2 shows the results.

Comparative Example 1 A CK activity value in a sample was measured in the same manner as in Example 1 except that the pH of the first reagent at 30 ° C. was changed to 6.2. Table 2 shows the results.

Comparative Example 2 The CK activity value of a sample was measured in the same manner as in Example 1 except that the pH of the first reagent at 30 ° C. was changed to 6.4. Table 2 shows the results.

Comparative Example 3 The CK activity value of a sample was measured in the same manner as in Example 1 except that the pH of the first reagent at 30 ° C. was changed to 6.6. Table 2 shows the results.

Comparative Example 4 The CK activity value of a sample was measured in the same manner as in Example 1 except that the pH of the first reagent at 30 ° C. was changed to 6.8. Table 2 shows the results.

[0043]

[Table 2]

In Table 2, portions having an error of ± 5% or more are marked with *.

From the results in Table 2, it was found that by setting the pH of the first reagent at 30 ° C. to 6.1 or less (pH 5.0 to 6.1), interference by bilirubin at a high concentration of up to 40 mg / dl was ± 5%. It is clear that it can be avoided below%.
Further, the pH of the first reagent at 30 ° C. is adjusted to 5.8 to 6.1.
, The interference by bilirubin is ± 3.
It is clear that it can be avoided below%.

[0046]

According to the composition for measuring the activity of CK of the present invention, the interference of bilirubin in a sample is not received, so that the CK activity value can be measured more accurately. Further, the method for measuring the activity of CK of the present invention can avoid interference by bilirubin in a sample, so that the CK activity value can be accurately measured.

Claims (2)

[Claims] 1. A composition for measuring the activity of creatine kinase comprising a first reagent not containing creatine phosphate and a second reagent containing creatine phosphate, wherein the pH of the first reagent at 30 ° C. is 6.1 or less. A composition for measuring creatine kinase activity. 2. A blank is determined by reacting a sample with a first reagent not containing creatine phosphate and measuring a change in absorbance, and then adding a second reagent containing creatine phosphate to the reaction mixture. In the method of measuring the change in absorbance by adding and reacting, and subtracting a blank value from the change in absorbance to measure the activity of creatine kinase in the sample, the pH of the first reagent at 30 ° C. is adjusted to 6.1 or less. A method for measuring creatine kinase activity.