JPS63237797A - Separation and determination of isozyme - Google Patents

Abstract

PURPOSE:To permit the separation and determination of isozyme in a short time by inhibiting the isozyme with the reactions which specifically inhibits the isozyme in a specific fraction by action of protease in the presence of protein. CONSTITUTION:Using the reaction system which specifically inhibits isozyme in a specific fraction by the action of protease in the presence of protein, the isozyme is inhibited and the other remaining isozymes not inhibited is determined. Thus, as a result, the isozyme is determined. When an isozyme in a specific fraction is inhibited, it is recommended that a reaction system deactivating protease with a protease inhibitor is always added. Glutamic acid-oxalacetic acid transaminase isozyme is suitable for the isozyme.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a method for differentially quantifying isozymes, and mainly relates to a method for differentially quantifying isozymes for use in the field of clinical testing.

(Prior Art) Today, some of the isozymes whose existence is known in enzyme research have come to be used in daily clinical tests. A typical example is:
Glutamate oxaloacetate transaminase (hereinafter referred to as
It's called GOT. ) isozyme and lactate dehydrogenase (hereinafter referred to as LDH) isozyme.

There are two types of GOT isozymes that have different intracellular localizations: an isozyme that exists in the l1lf fraction on the cell (referred to as S-GOT), and an isozyme that exists in the mitochondrial fraction (referred to as S-GOT); -G
It's called OT. ) is known to exist in f1. By separately quantifying these, it is said that f1 can be used for clinical analysis of pathological conditions as qualitative differences in cell damage. The D I-1 isozyme contains L D II
There are isozymes consisting of five fractions: LDII5, ~LDII5, and the composition pattern differs depending on the organ. Therefore, clinically, differential quantification of serum LI)H isozymes is considered to be a clue for organ diagnosis. for example,
The activity in normal serum is LDH,<[,DI-1, but in the serum of patients with myocardial infarction, LDH and LDH
As H2 increases, their activity increases as L D H, >
It is known that L D l-1 is reversed.

Electrophoresis is a well-known method for measuring such isozers and immu. For example, in the case of L D 11 isozyme, LD is determined in descending order of mobility by electrophoresis.
II, -LD[(5) are separated. In addition to electrophoresis, ion exchange methods and immunochemical methods are also known.

(Problem to be solved by the invention) However, such electrophoresis method, ion exchange method,
Conventional methods such as immunochemical methods require complicated operations for determining d111 and require a long time, so there is a problem that they cannot be applied to automated analyzers that are routinely used in clinical tests. Furthermore, methods such as electrophoresis,
It has also been pointed out that the accuracy of isozyme separation is low, which poses a problem in the separation and quantification of isozymes.

The purpose of the present invention is to solve these problems and provide a method for differentially quantifying isozymes that can be useful in daily clinical tests.

(Means for Solving the Problems) In order to solve these problems and achieve the above-mentioned objectives, the present inventors have carried out intensive research and have found that, in the presence of proteins, specific We have discovered that isozymes can be fractionated by using a reaction system that specifically inhibits isozymes in fractions.

Furthermore, by using a reaction system that inactivates excess protease through the action of a protease inhibitor,
It was found that the fractional 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 separate and quantify isozymes contained in samples, particularly blood samples used in clinical tests, plasma, etc., by the action of proteases in the presence of proteins. The invention consists in a method for fractionating and determining isozymes, which is characterized by using a reaction system that specifically inhibits isozymes in a specific fraction of the isozymes.

The reaction system related to protease action in the present invention consists of a reaction that specifically inhibits a specific fraction of isozyme caused by protease action, but all known proteases can be used as such proteases as long as they have the action of the present invention. Otsu's can be used.

Examples of such proteases include a-chymotrypsin, try-psin, and thrombin.
in), elastase, endoprotease, subtilisin, brom
a la in), papain (papa in),
Carpoquine pevsidase (carboxy peptase)
idase), etc.

The fraction of isozymes that are specifically inhibited by protease action may differ depending on the protease used, and in such cases, the present invention can be carried out more effectively by appropriately selecting the type of protease.

A reaction system related to protease action is constituted by a reaction solution containing such protease and protein. The content of protease in this reaction solution can be changed depending on the measurement conditions, but for example, when using α-chymotrypsin, t
What is necessary is just to contain an o-too unit.

In the reaction system related to protease action of the present invention, the specificity of the inhibition reaction can be improved by proceeding with the reaction in the presence of a protein. As the protein used here, any known protein can be used as long as it does not produce such a result, but albumin, globulin, gelatin, etc. are particularly preferred. The content of such proteins in the reaction solution can vary depending on the measurement conditions, but for example, when albumin is used, it is 0.1
Contains ~10.0%! All you have to do is make it i1.

Furthermore, the present invention provides a method for fractionating and determining isozymes, which adds a reaction system for inactivating excess protease by the action of a protease inhibitor, in addition to the reaction system related to the action of 7-tease as described in iFj.

In this way, by inactivating excess protease after applying the protease reaction system,
The influence of proteases on subsequent reactions can be removed. The reaction system related to this protease inhibitor action is constituted by a reaction solution containing a protease inhibitor. As such a protease inhibitor, any known 4-hetenorano can be used as long as it has the effect of the present invention; examples thereof include aprotinin (a
prot 1nin), Anjito [lunbin! II (
antithrombinIn), a heanditrypnone (α+ trypsininhibitor(trypsininl+1bilor)
), leupeptin, α2-maeroglobu
l in), phenylmethylsulfonyl fluoride (phc
nyl methyl 5ulronyl fluor
ide), (p-amidinophenyl)methanesulfonyl fluoride hydrochloride [(p-amidinophenyl)
yl) metl+anesul "onyl fluo
ride hydroehloridaj and the generic name gabexate menluate ([ethyl -4-(G -g
uanidin.

hexanoyloxy)benzoate]meth
ane 5ulfonatc).

Contains pro-tease inhibitors in the reaction solution! If can be changed depending on the measurement conditions. For example, when using aprotinin, 100 to 500
It is sufficient to contain 0,000/WC.

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

When isozyme is differentially quantified by the method of the present invention, the known enzyme activity determination method 9 can be used to determine the isozyme J. of other fractions that remain uninhibited. There are two methods for measuring absorbance in the visible region, such as using a chromogenic chromogen or producing a pigment through a condensation reaction, and a method for measuring absorbance in the ultraviolet region using coenzyme NAD II. It can be classified into methods such as methods of measuring absorbance at . These methods are currently widely used in the field of clinical testing1.
1, and many of them are known to be easy to operate, have short reaction times, and have excellent performance such as measurement accuracy, sensitivity, and accuracy. Select the appropriate one from among these. can be 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.

A reaction solution prepared by adding If and serum albumin (1.0%) to 0.1 M phosphate buffer (pl-18, 0) containing saturated α-chymotrypsin (200 jl'/zI2), 0.1 mσ
To this, 0.02a (0.02a) of purified human-derived rtr-GOT (1 OOO trophic/■,) was added and reacted at 37°C for 5 minutes. After the reaction, 20mM asvelagic acid, 10mM
Add 30πa of an enzyme assay reagent consisting of O, 1M Tris-HCl buffer (pl, (8,0) containing 2-oxoglutaric acid, 0.2mM NADt1, and malate dehydrogenase (1000 units/Q). Heat to ゛C, wavelength 3.10
The amount of change in absorbance per minute in nm was determined.

The same operation was performed for S-GOT.

As a control, the same procedure was performed except that the protease was removed from the reaction solution, and the residual activity percentages of each were calculated in comparison. In addition, the same procedure was carried out when raw serum albumin was removed from the reaction solution.

The results are shown in Table 1, and 5-GOT is inhibited in the presence of albumin as a protein according to the method of the present invention by using a reaction system based on the action of α-chymotrypsin as J-tease. It was shown that m-GOT was not inhibited and could be differentially quantified.

Example 2 Example 1 using subtilisin (100 units/R(1))
When I did the same thing, the results were as shown in Table 2.

It has been shown that comparable results can be obtained using subtilisin as the protease used according to the invention.

Example 3 Using a reaction system based on protease action according to the method of the present invention using an automatic analyzer used in clinical tests.
m-GOT, an o'r isozyme, was quantified and compared with a conventional immunochemical method.

Take 0.02xQ of each of 10 serum samples as specimens and add α-chymotrypsin (200 units/L (1
) and 0.1M phosphate buffer (pT48.0) containing bovine serum albumin (1.0%).
After the reaction, 0.35 mQ of the enzyme quantitative reagent used in Example 1 was added to determine the amount of change in absorbance per minute. - Converted to activity from the standard curve of GOT.

On the other hand, m-GOT activity was determined for these 10 serum samples using a commercially available m-GOT activity measurement kit (company name: Eiken Co., Ltd.) using an immunochemical method. 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.

Table 3 Example 4 In order to investigate the influence of the protease used in the present invention on known enzyme quantitative reagents, malate dehydrogenase (MDI
, EC1,1,1,37), lactate dehydrogenase (LDH,
EC1,1,1,28), o-1-zaloacetate decarboxylate (OAC, EC4,1,1,3), pyruvate okindase (POP, EC1,2,3°3), peroxidase (POD, EC1, 11, 1゜7), the activity μII damage caused by Zuro protease was observed.

α-Chymotrypsin (200 units/I) was added to a 0.1M phosphate buffer containing a fixed amount of each enzyme, and after heating at 37°C for 30 minutes, the residual activity of each enzyme was measured.
As shown in the table, it was found that OAC and POP were significantly inhibited.

Table 4 Example 5 In Example 4, abrodinin (1000 units/+1112) was added in advance, and the residual activity of each enzyme determined in the same manner was 100%. Therefore, it was found that abrodinin as a protease inhibitor used in the present invention eliminates the influence of α-chymotrypsin.

Example 6 Using a reaction system based on protease action and a reaction system based on protease inhibitor action according to the method of the present invention, G
m-G, an OT isozyme.

'F was quantified and compared with a conventional immunochemical method.

For each of 10 serum samples, 0.02 skin Q was taken as a specimen, and 0.1M phosphate buffer (pH 8.0) containing α-chymotrypsin (200 units/paddy) and bovine serum albumin (1.0%) was added to each sample. Add 0.1yhQ of the reaction solution, 37
The reaction mixture was allowed to react at 37°C for 5 minutes, and 0 and 1 x Q of abrodinin (2000 units/RC) reaction solution were added, followed by reaction at 37°C for 5 minutes. After the reaction, 300mM aspartic acid, I
OnM2-oxoglutarate, 5mM PAD, ImM
N-ethyl-N-(2-hydroxy-3-sulfob[
1 pill) -m-1-luidine, 1mM 4-aminoantipyrine, ImM diamine birophosphate-1, 0.5
mM MnC (!t, P OP (500 units/[,),
0AC (300 units/I7), POIM 500 units/
200mM phosphate buffer containing (I-,) (p) 17.5
) was added to 1.0 mQ of the enzyme quantitative reagent consisting of 37
After warming for 20 min at °C, O, l containing 0.2 MEDTA
The reaction was stopped by adding M citrate buffer (pII5.0) 2.0J112, and the absorbance at a wavelength of 550 nn+ was measured. This was compared with a similar operation using m-GOT whose activity was known in advance and was converted into an activity value of 1+GOT.

On the other hand, using a commercially available mGOT activity measurement kit using an immunochemical method, m-GO
T activity was determined. The results are shown in Table 5. There was a good correlation between the method of the present invention and the conventional immunochemical method, and it was confirmed that m-GOT could be formed by the method of the present invention without being affected by proteases.

Table 5 Example 7 Regarding the L D I-I isozyme, L D H and LDH that are significant in the serum of patients with myocardial infarction as described by Moe
A differential quantification of t was performed.

0.0511!12 each for 8 serum samples as specimens
To each sample, add 0.05 g of a reaction solution of 0.1 M phosphate buffer (al 18.0) containing α-chymotrypsin (200 units/good) and bovine serum albumin (1%),
The reaction was carried out at 37°C for 5 minutes. After the reaction, 0 containing 16mM sodium pyruvate and 0.18mM NA, DI]
．． An enzyme quantitative reagent consisting of 1M phosphate buffer (pH 7, 8') was added for 3.0 RQ, and the change in absorbance per minute at a wavelength of 340 nm was determined.

This was converted into activity in advance from a calibration curve of LDI-(with known activity).

On the other hand, this serum sample was analyzed by electrophoresis, which is a known method, and the total activity of L D 11 and L D H was determined. When these two were compared, it was found that the inter-vehicle distance coefficient was 0.995, indicating a good correlation h<i. As a result, in the method of the present invention, protease
-When using chymotrybun, [, D H, and LD
It turns out that the sum of f(t) can be determined as a fractional constant q.

Example 8 Activity determined in the same manner as in Example 7 using subdirinone (100 units/village) in place of α-chymotrypsin, and L DH analyzed by electrophoresis.

When comparing the two in terms of their activity, the correlation coefficient was 0.9.
91, indicating a good correlation. As a result, it was found that when subtilisin was used as the protease in the method of the present invention, [, DI-1] could be determined separately.

(Effects of the Invention) The reaction system relating to protease action and the reaction system relating to protease inhibitor action, which are the methods of the present invention, are themselves easy to operate and can be carried out in a short time. In addition, because the specificity of the inhibition reaction is extremely high, it is also superior in terms of performance such as isozyme fractionation accuracy. As mentioned above, by appropriately selecting from known methods and adapting them to the present invention as a method for quantifying isozymes in other fractions that remained uninhibited, the present invention provides a method for differentially quantifying isozymes. A series of operations are simple and can be performed in a short time, and excellent performance can be provided as a method for any purpose. Therefore, the method of differentially quantifying isozymes according to the present invention can also be applied to automatic analyzers when used in the field of clinical testing. In particular, in recent years, clinical tests have become automated and automatic analyzers are used more frequently, so the present invention is highly effective as a tool useful in daily clinical tests.

Claims (1)

[Claims] 1. Inhibiting isozymes in a specific fraction using a reaction system that specifically inhibits isozymes in a specific fraction by the action of protease in the presence of protein, and quantifying other isozymes that remain uninhibited. A method for the fractional determination of isozymes, which is characterized by the following. 2. The method for differentially quantifying isozymes according to claim 1, wherein the protein is albumin, globulin, or gelatin. 3. The method for differentially quantifying isozymes according to claim 1 or 2, which comprises adding to the system a reaction system that inactivates protease by the action of a protease inhibitor when inhibiting a specific fraction of isozymes. 4. The method for differentially quantifying isozymes according to claim 3, wherein the protease inhibitor is aprotinin. 5. Claims 1 to 5, wherein the isozyme is a glutamate oxaloacetate transaminase isoenzyme.
4. The method for differentially quantifying isozymes according to any one of Item 4. 6. The method for differentially quantifying an isozyme according to any one of claims 1 to 4, wherein the isozyme is a lactate dehydrogenase isozyme.