JP3173780B2 - Reagent for simultaneous measurement of total creatine kinase and creatine kinase-isoform activity - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial application field) The present invention relates to creatine kinase (hereinafter referred to as CK) in a biological fluid.
And creatine kinase-isoform (hereinafter abbreviated as CK-iso) activity simultaneous measurement reagent.

(Prior Art) Measurement of CK activity in a biological fluid, particularly in serum, is useful for diagnosis of muscular disease, particularly important for diagnosis of myocardial infarction, and is measured on a daily basis. It is known that CK has three isozymes by combining two subunits, which are CK-MM, CK-MB and CK-BB, respectively, and CK-MM is mainly composed of skeletal muscle and cardiac muscle. CK-MB is present in myocardium, CK-BB is mainly present in organs such as brain and kidney.
Among them, since CK-MB is specifically present in myocardium, its specific measurement has been revealed to be a particularly useful marker in the diagnosis of myocardial infarction.

Therefore, several reagents for specifically measuring CK-MB have been proposed, and among them, the immunoinhibition method is most often used. That is, an antibody that specifically inhibits the M subunit in CK-MM and MB was completely inhibited by adding an antibody.
A method for measuring the activity of the remaining B subunits
-19239, JP-B-58-20274).
The present inventors have further improved the above-mentioned method by proposing the use of a monoclonal antibody as an inhibitory antibody (see JP-A-63-49095).

However, although the measurement of CK-MB is specific and useful for myocardial infarction, it is said that the measured value exceeds the normal range after 10 hours [eg, Wu et al., Clinical Chemistry (Clin, chem.) 33, 358 (1987)], and a method capable of measuring in a shorter time has been desired.

Recently, M subunits in CK-MM and MB that have escaped into the blood from myocardial infarction or muscular disease from the heart or skeletal muscle, etc., are degraded by carboxypeptidase N in plasma,
It has been revealed that the M ′ subunit is modified [for example, George et al., Journal of Biological Chemistry (J. Biol, Chem.) 259, 2667].
(1984). That is, CK-MM is MM ₃ (organization type,
From MM) to MM ₂ (hybrid type, MM ′) and then to MM ₁ (serotype, M′M ′) (M ′ indicates a modified subunit). Furthermore, in normal times, the value of MM ₃ / MM ₁ is 0.3
In the case of myocardial infarction, 3-6
It was found that the value of MM ₃ / MM ₁ increased with time and showed a value around 2. [For example, Hashimoto et al., Circulation, Vol. 71, p. 363 (1985)
Year)). That is, by measuring the value of MM ₃ / MM ₁ , early diagnosis of myocardial infarction is possible. It is said that the success or failure of intracoronary thrombolysis, which is an effective treatment method for myocardial infarction, depends on how early it is performed after onset [Isobe et al., Recent Medicine, 42, 1418 (1987) )). In addition, early detection of recurrent cases becomes easier.

As a method for measuring this MM ₃ / MM ₁ (hereinafter referred to as isoform ratio) in a short time, a CK quantitative reagent containing creatine phosphate as described in JP-A-62-104598 is used. To measure the total CK activity.
Activity (CK-iso activity) by an immunoinhibition method using a reagent for measuring CK activity containing an antibody that inhibits only the M subunit and does not inhibit the M 'subunit described in
There is a method of measuring and calculating by calculation.

(Problems to be Solved by the Invention) The measurement of the isoform ratio is significant if the diagnosis can be made at an early stage as described above, and if seizures have already occurred, surgical treatment should be performed as soon as possible. Since each measurement must be performed, the measurement of each item must be performed very quickly.
However, in the above-described method, the total CK activity and the CK-iso activity are measured separately, which requires time and effort. In addition, there are many items for the purpose of diagnosing myocardial infarction, such as lactate dehydrogenase, oxybutyrate dehydrogenase, aldolase, and myoglobin, and at least one item must be measured for comprehensive judgment. This requires a relatively large amount of sample (serum). However, large volumes of blood from patients with myocardial infarction attacks can be painful and serious, requiring the use of less serum per item.

(Means for solving the problem) As a result of intensive studies to solve such problems, the present inventors found that immediately after measuring the total CK activity, only the M subunit was inhibited and the M 'subunit was inhibited. It was found that the total CK activity and CK-iso activity could be measured at the same time by adding an antibody that did not inhibit the isoform ratio, and the isoform ratio could be measured more quickly and with a small amount of serum. Was.

That is, the present invention inhibits creatine kinase-M subunit and does not inhibit creatine kinase M 'subunit after adding a first reagent containing at least creatine phosphate to a sample and measuring total creatine kinase activity. A method for measuring total creatine kinase and creatine kinase isoform activities, comprising adding a second reagent containing an antibody to the same sample and measuring creatine kinase and creatine kinase isoform activities.

 The principle of measuring the total CK and CK-iso in the present invention is shown below.

(1) Total CK activity measurement principle (In the reaction formula, CrP is creatine phosphate, ADP is adenosine diphosphate, Cr is creatine, HK is hexokinase, GlcK is glucokinase, G6PDH is glucose 6-phosphate dehydrogenase, and NAD (P) is β-nicotinamide Adenine dinucleotide (phosphate) and NAD (P) H indicate reduced β-nicotinamide adenine dinucleotide (phosphate), respectively, and are abbreviated below.) (2) Principle of measuring CK-iso activity CK-M A reagent containing an antibody that inhibits the subunit but does not inhibit the CK-M 'subunit is added, and the measurement is performed using the same principle as after the inhibition of the CK-M subunit.

The method of the present invention uses a reagent consisting of a two-reagent system.

The first reagent only needs to contain CrP, which is a substrate of CK, and preferably contains substances and enzymes necessary for the detection system. An example of such a first reagent is GlcK
Alternatively, HK is 0.1 to 40 units / ml, G6PDH is 0.1 to 40 units / ml, CrP is 0.8 to 28 mM, ADP is 0.1 to 20 mM, nicotinamide adenine dinucleotide (hereinafter abbreviated as NAD ⁺ ) or nicotinamide adenine diene. Nucleotide phosphate (hereinafter abbreviated as NADP ⁺ ) 0.05 to 20 mM, glucose 1 to 2
00 mM adenosine monophosphate (hereinafter abbreviated as AMP)
0.2-20 mM, diadenosine pentaphosphate (hereinafter abbreviated as Ap5A) is 0.001-0.1 mM, N-acetylcysteine (hereinafter, NAC)
Abbreviation) or other thiol compound 0.5 ~ 50m
M, magnesium salts (eg, magnesium acetate)
One containing 5 to 30 mM, 0.5 to 50 mM sodium azide, 0.1 to 20 mM ethylenediaminetetraacetic acid (hereinafter abbreviated as EDTA), and 5 to 500 mM buffer (pH 6.7).

More preferably, GlcK or HK is 0.2 to 20 units / ml,
G6PDH 0.2-20 units / ml, CrP 5-40 mM, ADP 0.2-
10 mM, NAD ⁺ or NADP ⁺ 0.1 to 10 mM, glucose 2 to 10
0 mM, AMP 0.5 to 15 mM, Ap5A 0.002 to 0.050 mM, NAC 2-3
The concentration may be adjusted to 0 mM, magnesium salts to 2 to 15 mM, sodium azide to 1 to 30 mM, EDTA to 0.2 to 10 mM, and buffer to 10 to 250 mM.

The second reagent inhibits the CK-M subunit and
-It is only necessary to include an antibody that does not inhibit the M 'subunit. Examples of such a second reagent include those containing 0.001 to 5 mg / ml of an M subunit inhibitory antibody (hereinafter abbreviated as Ab), 1 to 30 mM of sodium azide, and 5 to 50 mM of a buffer, More preferably, it may be adjusted to contain 0.005 to 2 mg / ml Ab, 1 to 30 mM sodium azide, and 10 to 250 mM buffer.

The antibody used for the second reagent can be used irrespective of polyclonal or monoclonal antibodies, but monoclonal antibodies are preferably used. An example of its physicochemical properties will be shown.

(1) Action It acts on CK to cause an antigen-antibody reaction and inhibits CK-M subunit activity.

(2) Reaction specificity Among the CK-M subunits, they inhibit the original M subunit and do not inhibit the modified M 'subunit.

(3) Optimum pH: 6 to 9 (4) Stable pH range: 3 to 11 (5) Measuring method of titer The titer is measured by a dilution factor that can be detected by inhibition rate measurement.

(6) Range of suitable temperature for operation: 0 to 40 ° C (7) Deactivation conditions Deactivation occurs by heating at 100 ° C for 30 minutes.

(8) Molecular weight: 130,000-210,000 To obtain the monoclonal antibody of the present invention, first, anti-CK
-Obtain a hybridoma cell line capable of producing a monoclonal antibody inhibiting M activity. Examples of the hybridoma cell line include a cell line having the following cytological properties.

(1) Origin This is a fusion cell created by fusing an anti-CK-M antibody-producing lymphocyte with a myeloma cell.

(2) Morphology It shows almost the same morphology as myeloma cells.

 For example, the size is 10 to 20 μm.

(3) Function It constantly produces an anti-CK-M activity-inhibiting monoclonal antibody that recognizes a single antigenic determinant.

(4) Proliferative property Proliferation property is almost the same as myeloma cells.

 That is, it grows about 10 times in 72 hours.

(5) Storage property It can be stored very easily at -120 ° C or lower for a long time.

(6) Optimum growth conditions: temperature 37 ° C, pH 7.2 (7) Growth range: It is possible to grow at a temperature of 32 to 42 ° C and pH 6.5 to 7.8.

In order to obtain such a cell line, for example, JP-A-63-49
A hybridoma producing an anti-CK-M activity-inhibiting monoclonal antibody can be obtained by the method described in JP-A No. 955, and a hybridoma that does not inhibit the M 'subunit can be selected by the same measurement method.

By the above method, inhibits CK-MM _3, and hybridomas that produce antibodies that do not inhibit CK-MM ₁ is obtained.
One of the hybridomas created according to this method was CKH
-5, and deposited on January 26, 1988 at the Research Institute of Microorganisms and Industrial Technology of the Ministry of International Trade and Industry of Japan, and accepted as Microbial Laboratories No. 9839 (FERM P-9839). . This CKH-5 can be frozen and stored almost permanently at -120 ° C or lower, and is in a state of being continuously distributed.

Next, such a hybridoma may be cultured to obtain an antibody from the culture solution or the like. The method may be a commonly used method (for example, see JP-A-63-49095). Further, the obtained antibody can be used as it is, or further treated with a proteolytic enzyme such as pepsin or papain, and used in the form of a fragment of Fab, Fab ', F (ab)' ₂ .

As a method for determining the total CK activity and CK-iso activity using the reagent of the present invention, for example, first, 0.625 ml of the above-mentioned first reagent is kept at a constant temperature of 25 to 37 ° C. for 5 to 10 minutes. After that, 0.001 to 0.02 ml of an enzyme solution (or serum) containing CK is mixed, and the total CK activity is measured by a change in absorbance at 340 nm using a spectrometer keeping the cell chamber at the same temperature. Thereafter, 0.1 ml of the second reagent as described above is mixed, and at a constant temperature of 25 to 37 ° C.,
After incubating for 5 minutes, the CK-iso activity (residual activity) is measured in the same manner. From the total CK activity and CK-iso activity, the isoform ratio (MM ₃ / MM ₁ ) can be determined by the following equation.

(Action) The antibody used in the present invention inhibits CK-M subunits and does not inhibit CK-M 'subunits. CK that has escaped from the heart due to myocardial infarction has its M subunit modified with carboxypeptidase in the blood. That is, CK-M
M (MM ₃ ) changes with time through M ′ (MM ₂ ) to M′M ′ (MM ₁ ), and CK−MB (MB ₂ ) changes to M′B (MB ₁ ). To go. Therefore, immediately after departure from the heart, since the modification of the M subunit has not progressed, the isoform ratio according to the present invention increases, and thereafter decreases as the modification progresses. Thus, by measuring the isoform ratio with the reagent of the present invention, early diagnosis of myocardial infarction and estimation of the onset time can be made.

(Examples) The present invention will be specifically described with reference to examples, but the present invention is not limited to these examples.

Example 1 First, GlcK derived from Bacillus stearothermophilus
(Commercially available from Seikagaku Corporation) 1.4 unit / ml, G6PDH derived from Leuconostoc mesenteroides (purchased from Oriental Yeast Co., Ltd.) 1.2 unit / ml, ADP 1.2 mM, NADP 0.75
mM, glucose 25 mM, AMP 6.25 mM, Ap5A 12.5 μM, NAC 12.5 mM,
Magnesium acetate 12.5mM, CrP 100mM, sodium azide 10
mM, EDTA2.5mM, imidazole-acetate buffer (pH6.7) 150m
A first reagent consisting of M was prepared, and then a second reagent consisting of anti-CK-M activity-inhibiting monoclonal antibody CKA-5 0.5 mg / ml, sodium azide 10 mM, imidazole-acetate buffer (pH 6.7) 150 mM was prepared. Prepared.

0.625 ml of the first reagent was placed in a cell having an optical path length of 1 cm, and incubated for 3 minutes. Then, serum containing CK-MM was added, and the cell chamber was kept at a constant temperature of 30 ° C. with a spectrophotometer at 340 nm.
The total CK activity was measured by the change in absorbance of the sample. In addition, the second
0.1 ml of the reagent was added, the CK-iso activity was measured from the change in absorbance at 340 nm, and the isoform ratio was calculated from these measured values.

This procedure is repeated for 10 serum samples, and the total CK, CK-iso activity and isoform ratio are compared with the values measured by the conventional method for separately measuring total CK, CK-iso activity. Compared.

The results are shown in Table 1. The regression line of Y to X was determined by setting the measured value by the present method to X and the measured value by the conventional method to Y, and as a result, the equation Y = 0.95X + 0.03 was obtained. The value of 0.999 was obtained as a result of calculating the correlation coefficient. Both the regression coefficient (the slope of the regression line) and the correlation coefficient are close to 1, indicating that there is a very good correlation between the two methods. Was shown.

(Effect of the Invention) The reagent for simultaneous measurement of total CK and CK-iso activities of the present invention can measure the change of the M subunit of CK useful for early diagnosis of myocardial infarction in a simple and short time.

────────────────────────────────────────────────── ─── Continued on the front page (72) Inventor Naoki Suzuki 23 Uji Kozakura, Uji City, Kyoto Unitika Inside Central Research Laboratory Co., Ltd. (72) Inventor Hiroyuki Tsubota 1144 Owada Nitta, Yachiyo-shi, Chiba Pref. (58) Investigated field (Int.Cl. ⁷ , DB name) C12Q 1/50 G01N 33/573 G01N 33/577 BIOSIS (DIALOG) MEDLINE (STN)

Claims (1)

(57) [Claims] (1) adding a first reagent containing at least creatine phosphate to a sample, measuring total creatine kinase activity, and then inhibiting creatine kinase-M subunit;
A method for measuring total creatine kinase and creatine kinase isoform activity, comprising adding a second reagent containing an antibody that does not inhibit creatine kinase M 'subunit to the same sample, and measuring creatine kinase isoform activity.