JPH06104074B2 - Fractional quantification method of isozyme - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for fractionating and quantifying isozymes, and particularly to a method for fractionating and quantifying isozymes mainly for use in the field of clinical examination.

(Prior Art) Some of the known existences of isozymes in enzyme research have come to be used in daily clinical tests. As a typical example,
Glutamate oxaloacetate transaminase (hereinafter,
It is called GOT. ) Isozymes and lactate dehydrogenase (hereinafter L
Called DH. There is an isozyme.

As GOT isozymes, two types of isozymes having different intracellular localizations, that is, an isozyme present in the cell supernatant fraction (referred to as S-GOT) and an isozyme present in the mitochondrial fraction (referred to as m-GOT). .) Is known to exist. It is said that it is clinically useful for pathological analysis as a qualitative difference in cell damage by separately quantifying these. In addition, LDH isozymes include isozymes consisting of ₅ fractions, LDH _{1 to} LDH ₅ , and the constitutional patterns differ depending on the organ. Therefore, it is clinically thought that the quantification of serum LDH isozymes can be used as a clue for organ diagnosis. There is. For example, the activity in normal serum is LDH ₁ <LDH ₂ , but in the serum of patients with myocardial infarction, LDH
As H ₁ and LDH ₂ increased, their activity was LDH ₁ > LD
It is known to reverse with H ₂ .

A well known method for measuring such isozymes is electrophoresis. For example, in the case of the LDH isozyme, LDH ₁ to LDH ₅ are separated by electrophoresis in the order of increasing mobility. Besides the electrophoresis method, the ion exchange method and the immunochemical method are also known.

(Problems to be Solved by the Invention) However, such an electrophoresis method, an ion exchange method,
Conventional methods such as immunochemical methods have a problem that they cannot be applied to automatic analyzers that are routinely used in clinical tests because scanning for measurement is complicated and the time required for the operation is long. Furthermore, it has been pointed out that methods such as electrophoresis have low accuracy in isozyme separation.
This is a problem in the differential quantification of isozymes.

An object of the present invention is to solve such problems and provide the method as a method for fractionating and quantifying isozymes that can be used as a useful one in daily clinical tests.

(Means for Solving Problems) The inventors of the present invention have made intensive studies to solve these problems and achieve the above-mentioned object, and as a result, identified by the action of protease in the presence of protein. It was found that the isozyme can be fractionated and quantified by using a reaction system that specifically inhibits the isozyme in the fraction.

Furthermore, by using a reaction system that inactivates excess protease by the action of a protease inhibitor,
It was found that the differential quantification of isozymes can be carried out more effectively, and the present invention has been completed.

That is, the gist of the present invention is to identify a specific isozyme group in the isozyme group by the protease action in the presence of a protein in the differential quantification of an isozyme contained in a sample, in particular, a sample such as serum or plasma used in a clinical test. A method for fractionating and quantifying isozymes is characterized by using a reaction system that specifically inhibits the isozymes in the fraction.

The reaction system relating to the protease action in the present invention comprises a reaction which specifically inhibits an isozyme of a specific fraction due to the protease action, and as such a protease, all known ones having the action of the present invention are known. Things can be used. Examples of such proteases include α-chymotrypsin (α-chymotrypsin), trypsin (try-psin), thrombin, elastase, endoproteases.
se), subtilisin, bromelain (br)
omelain), papain, carboxy peptidase, and the like.

The fraction of the isozyme that is specifically inhibited by the action of protease may vary depending on the brothase used, and in such a case, the present invention can be more effectively carried out by appropriately selecting the type of protease.
The reaction system relating to the protease action is composed of a reaction solution containing such protease and protein. The content of protease in this reaction solution can be changed depending on the measurement conditions. For example, when α-chymotrypsin is used, 10-1000 to inhibit S-GOT1 unit.
Units may be included.

In the reaction system relating to the protease action of the present invention, the specificity of the inhibition reaction can be improved by advancing the reaction in the presence of the protein. As the protein used here, any known protein can be used as long as it can bring about such a result, but albumin, globulin, gelatin and the like are particularly preferable. The content of such a protein in the reaction solution can be changed depending on the measurement conditions. For example, when albumin is used, 0.1 to 10.0% may be contained.

Further, the present invention provides a method for fractionating and quantifying isozymes, which comprises, in addition to the above-mentioned reaction system relating to protease action, a reaction system which inactivates excess protease by a protease inhibitor action. In this way, the excess protease is inactivated after the reaction system by the action of the protease is allowed to act, so that the influence of the protease in the subsequent reaction can be removed. The reaction system relating to this protease inhibitor action is composed of a reaction solution containing a protease inhibitor. As such protease inhibitors, all known ones can be used as long as they have the action of the present invention, and examples thereof include aprotinin and antithrombin I.
II (antithrombin III), α ₁ -antitrypsin (α ₁
-Antitrypsin), trypsin inhibitor (trypsin i
nhibitor), leupeptin (leupeptin), α ₂ - macroglobulin (α ₂ -macroglobulin), phenylmethylsulfonyl fluoride (phenyl methyl sulfonyl fluorid
e), (p-amidinophenyl) methanesulfone hydrochloride [(p-amidinophenyl) methane sulfo
nyl fluoride hydrochloride] and generic name gabexate mesylate {[ethy1-4- (6-guanidino hexanoylox
y) benzoate] methane sulfonate} and the like.

The content of the protease inhibitor in the reaction solution can be changed depending on the measurement conditions. For example, when aprotinin is used, the reaction solution may contain 100 to 5000 units / ml.

As described above, in the present invention, the isozyme of a specific fraction is specifically inhibited by the action of protease, and the excess protease can be further inactivated.

When the isozyme is fractionally quantified by the method of the present invention, a known enzyme activity quantification method can be used for quantifying the isozymes of other fractions that remain uninhibited. For that, a method of measuring the absorbance in the visible region by using a chromogenic chromogen, or by using the formation of a dye by a condensation reaction, and the absorbance in the ultraviolet region using coenzyme NADH, etc. Can be classified as a method of measuring These methods are currently widely used in the field of clinical examinations, and they are known to be simple in operation and have a short reaction time, and have excellent performances such as measurement accuracy, sensitivity and accuracy. However, an appropriate one can be selected from these and applied to the present invention.

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

Example 1 Purified human-derived m-GOT was added to 0.1 ml of a reaction solution prepared by adding bovine serum albumin (1.0%) to 0.1 M phosphate buffer (pH 8.0) containing α-chymotrypsin (200 units / ml). (1000 units /
L) was added in an amount of 0.02 ml and reacted at 37 ° C. for 5 minutes. After the reaction
To this, 3.0 ml of an enzyme quantification reagent consisting of 0.1 mM Tris-HCl buffer (pH 8.0) containing 20 mM aspartic acid, 10 mM 2-oxoglutarate, 0.2 mM NADH, malate dehydrogenase (1000 units / l) was added, and 37 ml was added. The mixture was heated to ° C, and the amount of change in absorbance per minute at a wavelength of 340 nm was determined.

The same operation was performed for S-GOT.

As a control, the same operation was performed when protease was removed from the reaction solution, and the residual activity ratio of each was calculated by comparison with this. The same procedure was performed when bovine serum albumin was removed from the reaction solution.

The results are shown in Table 1. S-GOT is inhibited by using the reaction system by the action of α-chymopsin as a protease in the presence of albumin as a protein according to the method of the present invention, but m-GOT It was shown not to be inhibited and to be quantified separately.

Example 2 When subtilisin (100 units / ml) was used in the same manner as in Example 1, the results are shown in Table 2. It has been shown that comparable results are obtained with subtilisin as the protease used according to the invention.

Example 3 GO using a reaction system by protease action according to the method of the present invention using an automatic analyzer used in clinical examination
The T isozyme, m-GOT, was quantified and compared with the conventional immunochemical method.

As samples, 0.02 ml of each of 10 serum samples was taken, and 0.1 ml of 0.1 M phosphate buffer (pH 8.0) containing α-chymotrypsin (200 units / ml) and bovine serum albumin (1.0%) was added to each. In addition, the mixture was reacted at 37 ° C for 5 minutes. After the reaction, 0.35 ml of the enzyme quantitative reagent used in Example 1 was added and the change in absorbance per minute was determined. This was converted into activity from the calibration curve of m-GOT with known activity in advance.

On the other hand, this serum sample 10 was prepared using a commercially available immunochemical method for measuring m-GOT activity (company name: Eiken Co., Ltd.).
The m-GOT activity was determined for the examples. The results are shown in Table 3.

It was found that there is a good correlation between the method of the present invention using an automatic analyzer and the conventional immunochemical method.

Example 4 In order to examine the effect of the protease used in the present invention on a known enzyme quantification reagent, malate dehydrogenase (MDH,
EC1.1.1.37), lactate dehydrogenase (LDH, EC1.1.1.28), oxaloacetate decarbonase (OAC, EC 4.1.1.3), pyruvate oxidase (POP, EC1.2.3.3), peroxidase ( P
The inhibition of the activity of each enzyme of OD, EC1.11.1.7) by protease was observed.

Α-Chymotrypsin (200 units / ml) was added to 0.1 M phosphate buffer containing a fixed amount of each enzyme, and the mixture was heated at 37 ° C for 30 minutes, and the residual activity of each enzyme was measured. The results are shown in Table 4, which shows that OAC and POP are significantly inhibited.

Example 5 In Example 4, aprotinin (1000 units / m
When l) was added, the residual activity of each enzyme obtained by the same operation was 100%. Therefore, it was found that aprotinin as a protease inhibitor used in the present invention eliminates the influence of α-chymotrypsin.

Example 6 Using the reaction system by the action of protease and the reaction system by the action of protease inhibitor according to the method of the present invention, GO
The T isozyme, m-GOT, was quantified and compared with the conventional immunochemical method.

As samples, 0.02 ml of each of 10 serum samples was taken, and 0.1 ml of 0.1 M phosphate buffer (pH 8.0) containing α-chymotrypsin (200 units / ml) and bovine serum albumin (1.0%) was added to each. In addition, the mixture was reacted at 37 ° C. for 5 minutes, 0.1 ml of aprotinin (2000 units / ml) was added, and the mixture was reacted at 37 ° C. for 5 minutes. After the reaction, 300 mM aspartic acid, 10 mM 2
-Oxoglutarate, 5 mM FAD, 1 mM N-ethyl-N-
(2-hydroxy-3-sulfopropyl) -m-toluidine, 1 mM 4-aminoantipyrine, 1 mM thiamine pyrophosphate, 0.5 mM MnCl ₂ , POP (500 units / L), OAC (30
0 unit / L), POD (500 unit / L) containing 200 mM phosphate buffer (pH 7.5) 1.0 ml of enzyme assay reagent, and add at 37 ℃
After warming for 20 minutes, 0.2M citrate buffer containing 0.1M EDTA (p
H5.0) 2.0 ml was added to stop the reaction, and the absorbance at a wavelength of 550 nm was measured. This is a known m-
The activity value of m-GOT was converted to that of the same operation using GOT.

On the other hand, the m-GOT activity of 10 serum samples was determined by using a commercially available immunochemical method for measuring m-GOT activity. The results are shown in Table 5. The method of the present invention and the conventional immunochemical method showed a good correlation, and it was confirmed that m-GOT can be quantified by the method of the present invention without being affected by protease.

Example 7 With respect to the LDH isozyme, the significant quantification of LDH ₁ and LDH ₂ was performed in the serum of patients with myocardial infarction as described above.

As specimens, take 0.05 ml of each of 8 serum samples and 0.05 ml of 0.1M phosphate buffer solution (pH 8.0) containing α-chymotrypsin (200 units / ml) and bovine serum albumin (1%). In addition, the mixture was reacted at 37 ° C for 5 minutes. After the reaction, 16
The amount of change in absorbance per minute at a wavelength of 340 nm was determined by adding 3.0 ml of a reagent for enzyme quantification consisting of 0.1 M phosphate buffer (pH 7.8) containing mM sodium pyruvate and 0.18 mM NADH. This was converted into activity from the calibration curve of LDH of known activity in advance.

On the other hand, this serum sample was analyzed by electrophoresis, which is a known method, to determine the total activity of LDH ₁ and LDH ₂ . When these two were compared, it was found that the correlation function was 0.995, which is a good correlation function. As a result, it was found that when α-chymotrypsin was used as the protease in the method of the present invention, the total sum of LDH ₁ and LDH ₂ can be separately quantified.

Example 8 Instead of α-chymotrypsin, subtilisin (100 units /
ml) and operating in the same manner as in Example 7,
When LDH ₁ activity analyzed by electrophoresis was compared with each other, the correlation function was 0.991, which was found to be a good correlation function. As a result, it was found that when subtilisin was used as the protease in the method of the present invention, LDH ₁ could be fractionally quantified.

(Effects of the Invention) The reaction system relating to the protease action and the reaction system relating to the protease inhibitor action, which are the methods of the present invention, can be easily operated by themselves and can be carried out in a short time. Further, since the specificity in the inhibition reaction is extremely high, it is also excellent in performance such as accuracy of isozyme separation. And as described above, by appropriately selecting from known methods as a method for quantifying the isozymes of other fractions that remain uninhibited and adapting to the present invention, the present invention provides a series of isozymes as a fractional quantification. The method is simple and can be performed in a short time, and can be provided as a method having excellent performance. Therefore, the method for differentially quantifying isozymes according to the present invention can be applied to an automatic analyzer when used in the field of clinical examination. Particularly in recent years, clinical tests have become more automated, and the frequency of use of automatic analyzers has increased, so that the effect of the present invention as being useful in daily clinical tests is great.

Claims (6)

[Claims] 1. A method of specifically inhibiting an isozyme of a specific fraction by the action of a protease in the presence of a protein, wherein the isozyme is inhibited, and other isozymes that remain uninhibited are quantified. And quantitative determination of isozymes. 2. The method for fractionating and quantifying isozyme according to claim 1, wherein the protein is albumin, globulin or gelatin. 3. The method for fractionating and quantifying isozymes according to claim 1 or 2, wherein a reaction system for inactivating a protease by a protease inhibitor action is added to the system when isozymes of a specific fraction are inhibited. 4. The method for differential quantification of isozyme according to claim 3, wherein the protease inhibitor is aprotinin. 5. The method for fractionating and quantifying isozymes according to any one of claims 1 to 4, wherein the isozyme is a glutamate oxaloacetate transaminase isozyme. 6. The method for fractionating and quantifying isozymes according to any one of claims 1 to 4, wherein the isozyme is a lactate dehydrogenase isozyme.