JPH0965895A - Method and reagent for determining alpha2-plasmin inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a convenient and accurate method for determining an α2-plasmin inhibitor for clinical examinations, etc., by mixing and reacting a plasmin-containing sample with a plasmin reagent which contains an excess of a specific non-reducing sugar, and measuring activity of the remaining plasmin. SOLUTION: The objective convenient and accurate method for determining an α2-plasmin inhibitor to be used for e.g. clinical examinations is to mix an α2-plasmin containing sample (e.g. plasma specimen) with a plasmin reagent which contains an excess of specific non-reducing sugar to make the plasmin react with the α2-plasmin inhibitor, and then determine the α2-plasmin inhibitor remaining in the sample. The plasmin reagent to be used is a liquid one that contains 1-60% of a 4-24C non-reducing sugar such as sorbitol, trehalose or sucrose, 0.005-3.0mol of a salt and 0.001-100 units/ml of plasmin.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION The present invention is a clinical test alpha used in ₂ - plasmin inhibitor - How to measure and alpha ₂ - plasmin inhibitor - assay reagent, a method of storing plasmin, and a method of storing a protease.

[0002]

_2. Description of the Related Art α ₂ -plasmin inhibitor (hereinafter referred to as
(Also described as α ₂ -PI) immediately reacts with plasmin and inhibits plasmin enzyme activity by forming an α ₂ -PI · plasmin complex. Also, α ₂ −
PI is known as a fibrinolysis inhibitor having a property of inhibiting the binding of fibrinogen to plasmin, and its reduction in blood concentration is a problem. α ₂ -PI
Known as the diseases in which the concentration of α _2- decreases are α ₂ -PI birth defects, α ₂ -PI abnormalities, liver diseases, DIC (diffuse coagulation group) and the like. Therefore, α ₂ -PI has become an important index for diagnosis of these diseases.

Methods for measuring α ₂ -PI are roughly classified into immunological assay methods and activity assay methods. Immunoassays are the method of measuring the alpha ₂ -PI recognize the structural portion of the alpha ₂ -PI, inert alpha ₂ -PI · plasmin complex as well, similar to the active alpha ₂ -PI ,taking measurement. Therefore, this measurement method has a drawback in that data that accurately reflects active α ₂ -PI cannot be obtained. As a result, the activity measuring method is generally used as a method for measuring α ₂ -PI.

[0004] For activity assay, typically, to a sample containing alpha ₂ -PI, the addition of plasmin excess reagent is reacted with plasmin and its alpha ₂ -PI, then measured the residual plasmin activity Therefore, α ₂ -PI in the sample
The method of measuring is becoming mainstream. In particular, a constant excess of plasmin was added to the sample so that plasmin and its α ₂
After reacting with -PI and allowing the reaction to proceed, the residual plasmin and the synthetic substrate for plasmin are reacted to measure the residual plasmin activity. From the comparison between the plasmin activity of the reagent used and the residual plasmin activity, α ₂ -A measuring method for calculating PI activity is used. Α by such a synthetic substrate
_{The 2-} PI measurement method is, for example, Petter F
liberger, The Scandinavian
Journal of Clinical & La
laboratory Investigation, 42
Vol. 41-48 (1982).

The α ₂ -PI measuring reagent by such a synthetic substrate method comprises a plasmin reagent unit containing an excess amount of plasmin and a synthetic substrate reagent unit for plasmin,
It is configured. However, among these constituent reagents, particularly the plasmin reagent is very unstable because the main component is plasmin, which is a self-degrading protease. As a result, when these reagents are stored in a liquid state, the plasmin activity in the sample is lowered, so that there is a problem that α ₂ -PI in the sample cannot be accurately measured.

On the other hand, some methods for stably storing the plasmin reagent have been developed. As a method,
A method of making a freeze-dried product or a method of storing it in a liquid state is known.

In the method using a lyophilized product, plasmin is dissolved in advance to prepare a liquid plasmin reagent, the reagent is lyophilized and refrigerated as a lyophilized product, and further frozen at the time of measurement. The dry product must be redissolved and the α ₂ -PI in the sample must be determined. Therefore, it is inconvenient for the user side who measures α ₂ -PI to prepare the reagent by dissolving it, there is a possibility of preparation error at the time of dissolution, and the number of reagents is limited because a dedicated solution is attached. There is a problem of storage location due to the large number. In addition, the manufacturer needs a manufacturing process called freeze-drying, and as a result, there is a problem that preparation of the reagent takes a long time.

[0008] As a method of storing and using plasmin in a liquid state, it is suspended in 3.2M ammonium sulfate and stored under refrigeration, and 0.05M tris (hydroxymethyl) aminomethane-0.02M containing 25% glycerol. Lysine-0.1M sodium chloride-0.001M ethylenediaminetetraacetic acid (disodium salt), pH 9.0 as a concentrated solution in a buffer solution, chilled at -20 ° C to -30 ° C [M
methods in ENZYMOLOGY, Volume 19, 1
84-199] and the like are known. However, in these methods, any of the plasmin reagents cannot be used as it is for the measurement of α ₂ -PI, and redissolved, diluted, or subjected to some pretreatment, and then α
It was necessary to measure _2- PI. Further, when glycerol is used, there is a problem that the plasmin reagent is likely to become viscous, which easily causes an error in the measured value.

[0009]

SUMMARY OF THE INVENTION The above problems of the α ₂ -PI measuring reagent have been solved, and they have not been redissolved or diluted,
That is, the preparation of reagents does not take time and labor, and
A long period of time, be sure that you save the measurement reagent, exactly α ₂ -P
It is an object of the present invention to provide a method for measuring I and a reagent therefor. Another object of the present invention is to provide a plasmin reagent in which plasmin activity does not decrease even when stored in a liquid state for a long time, and a method for storing plasmin.

[0010]

[Means for Solving the Problems] In view of these problems,
As a result of the inventors' earnest research, surprisingly,
Plasmin, which has hindered the liquefaction of the reagent due to its instability, is retained for a long period of time by the presence of certain non-reducing sugars in the reagent composition, and as a result,
Using this, it was found that α ₂ -PI can be accurately measured, and the present invention has been completed.

In the invention according to claim 1, plasmin and its α ₂ -plasmin inhibitor are reacted by mixing a sample containing α ₂ -plasmin inhibitor and an excess amount of the plasmin reagent, and then the residual plasmin is left behind. A method for measuring α ₂ -plasmin inhibitor in a sample by measuring plasmin activity, wherein the plasmin reagent is a plasmin reagent containing a non-reducing sugar having 4 to 24 carbon atoms It is a method for measuring α ₂ -plasmin inhibitor in the medium.

According to the first aspect of the present invention, i) the plasmin reagent is stable even when stored in a liquid state at an appropriate temperature, and is not viscous. Therefore, α ₂ -P
I can be accurately measured; ii) Even if a reagent is prepared and stored in a liquid state, there is no need for re-dissolution, dilution, or other pretreatment, and there is an effect that it can be measured as it is. The appropriate temperature is usually 1 to 30 ° C.

Various kinds of samples containing α ₂ -PI can be used, and there is no particular limitation as long as it is desired to measure α ₂ -PI. Examples thereof include body fluids such as plasma and serum, dilute solutions thereof, and treatment solutions.

In the invention according to claim 1, the plasmin reagent is in an excessive amount and contains a non-reducing sugar having 4 to 24 carbon atoms. Excessive amount of plasmin is necessary to convert α ₂ -PI in the sample to α ₂ -PI.plasmin complex by plasmin, and to allow measurable residual plasmin to exist in the liquid after the completion of the first step. This is to secure a sufficient amount of plasmin.

The excess amount of plasmin reagent means the α in the sample.
It is a reagent that can eliminate the plasmin inhibitory activity of _2- PI and, after the elimination, contains an amount of plasmin that allows the presence of measurable residual plasmin.

The plasmin concentration in the plasmin reagent varies depending on the α ₂ -PI concentration of the sample, but is usually 0.001.
To 100 units / ml, preferably 0.01 to 10 units / ml. In the present specification, 1 unit of plasmin means the amount of enzyme that hydrolyzes the substrate NS-2200 by 1 μmole per minute.

The invention according to claim 1 is characterized in that the plasmin reagent is a plasmin reagent containing a non-reducing sugar having 4 to 24 carbon atoms. Therefore, the plasmin reagent of the present invention is difficult to reduce the plasmin activity when stored in a liquid state under appropriate conditions.
It has a preferable effect that α 2 -PI in a sample can be accurately measured even after a long period of time. In this specification,
The non-reducing sugar refers to a saccharide having no free reducing group, and includes non-reducing oligosaccharide, sugar alcohol, glycoside, and polysaccharide. Preferably non-reducing oligosaccharides, sugar alcohols,
It is. In the invention according to claim 1, the number of carbon atoms is 4 to 24.
The non-reducing sugar of is preferably a non-reducing disaccharide or sugar alcohol having 5 to 18 carbon atoms. Examples of the non-reducing disaccharide having 5 to 18 carbon atoms include trehalose and sucrose. Examples of the sugar alcohol having 5 to 18 carbon atoms include sorbitol and inositol.

In the present invention, the concentration of the non-reducing sugar having 4 to 24 carbon atoms contained in the plasmin reagent is preferably 1 to 60%, more preferably 5 to 45%, and particularly preferably 10 to 30%. is there. If the concentration is too low, the stability of plasmin deteriorates, and if the plasmin reagent is left for a long time, α ₂ in the sample is measured by the measuring method according to claim 1.
-It is difficult to measure PI accurately. On the other hand, if the concentration is too high, the reagent becomes viscous and it is difficult to measure α ₂ -PI accurately.

The pH of the plasmin reagent is plasmin and α
_From the optimum point of reaction with _2- PI, pH of 6.
The pH is 0 to 10.0, and more preferably pH 7.0 to 9.0. The plasmin reagent can contain a buffering agent for pH adjustment and the like. The buffer concentration of the plasmin reagent is preferably 0.005 to 1.000 M, more preferably 0.
It is 02-0.30M.

As the pH adjusting buffer, tris, phosphoric acid, barbital, imidazole, veronal, glycylglycine, BES, MOPS, TES, HEPES,
DIPSO, TAPSO, POPSO, HEPPSO,
EPPS, Tricine, Bicine, TAPS, etc. can be used. Also in the reagent containing the synthetic substrate for plasmin according to claim 2, these various pH adjusting buffers can be added at appropriate times.

It is preferable that the plasmin reagent further contains a salt for stability. In this case, the salt is a water-soluble salt, preferably a water-soluble organic salt or halogenated inorganic salt. As the water-soluble organic salt, sodium acetate,
An example is potassium acetate. Examples of the water-soluble halogenated inorganic salt include sodium chloride and potassium chloride. The salt concentration is usually 0005 to 3.0 M, preferably 0.
05-1.0M, more preferably 0.2-0.8M.

In the invention according to claim 1, in addition to the requirement that the plasmin reagent is: i) in excess and ii) contains a non-reducing sugar having 4 to 24 carbon atoms; It depends on the amount of α 2 -PI, but usually iii) the plasmin concentration is 0.001 to 100 units /
ml) iv) the concentration of the non-reducing sugar is 1-60%, v) the pH is 6.0-10.0, and vi) a buffer having a concentration of 0.005-1.000 M,
In addition, vii) preferably contains a salt having a concentration of 0005 to 3.0M.

In addition to such a plasmin reagent, a chelating agent such as EDTA or EGTA, a heparin inhibitor such as polybrene or protamine, or Triton, from the viewpoint of reagent performance or the like.
Various substances such as surfactants such as X-100 and Tween-20, saccharides, amino acids, proteins such as albumin, amine compounds such as monoethylamine, polyethylene glycol, glycerol and the like can be appropriately added. .
In addition, these additives can be appropriately added to the reagent containing the synthetic substrate for plasmin of the invention according to claim 2.

In the first step of the invention according to claim 1, α ₂
Plasmin and its α ₂ -plasmin inhibitor are reacted by mixing a sample containing -plasmin inhibitor with an excess amount of plasmin reagent. Thereby, the plasmin inhibitory activity of α ₂ -PI in the sample can be lost. At this time, the sample may be added to the plasmin reagent, or the plasmin reagent may be added to the sample.

In the second step of the invention according to claim 1, the residual plasmin activity is measured. As the method, a method of reacting the residual plasmin with a synthetic substrate for plasmin and measuring the residual plasmin is preferable. This is because residual plasmin can be measured accurately in a short time. When the residual plasmin is reacted with the synthetic substrate for plasmin, the synthetic substrate is cleaved. The residual plasmin can be measured by measuring the substance produced by cleavage.

It has been determined that the substance produced by such cleavage is a dye, a chromogen such as a substance that reacts to produce a dye, a fluorescent substance, a substance having an absorbance at a wavelength of 270 to 340 nm, and the like. It is preferable in terms of ease. 5 of them
Chromogens such as AB and pNA are preferred. Note that 5AB represents a 5-amino-2-nitrobenzoic acid residue, and pNA
Represents a p-nitroaniline residue. In addition, the synthetic substrate for plasmin refers to a synthetic substrate that can be cleaved selectively by the enzymatic action of plasmin and the substance produced by the cleavage can be measured.

After measuring the residual plasmin activity, the α ₂ -PI activity in the sample can be determined from the calibration curve prepared in advance. It is also possible to measure by adding the plasmin reagent after mixing the sample with the synthetic substrate for plasmin.

The synthetic substrate for plasmin is preferably stable in solution. As such a synthetic substrate, NS-
2200 (H-D-Gln ( C 6 H 11) -Phe-Ly
s-5AB), S-2251 (HD-Val-Leu)
-Lys-pNA), Chromozyme (Tos-
Any known substrate such as Gly-Pro-Lys-pNA) can be used. In the present specification, C ₆
H ₁₁ represents a cyclohexyl group.

As the reagent containing the synthetic substrate for plasmin, the reagent containing the synthetic substrate for plasmin of the ordinary synthetic substrate method for α ₂ -PI measurement can be used as it is. For example, when measuring α ₂ -PI in plasma, the concentration of the synthetic substrate for plasmin is usually 0.01 to 50 mM.

In the measuring method according to claim 1, the measurement using such a synthetic substrate for plasmin can be performed by using the measuring reagent of the invention according to claim 2. The invention according to claim 2 is a kit for measuring α ₂ -plasmin inhibitor in a sample; a kit comprising a plasmin reagent unit containing a non-reducing sugar having 4 to 24 carbon atoms and a reagent unit containing a synthetic substrate for plasmin. , An α ₂ -plasmin inhibitor-measuring reagent containing as an essential unit. As the plasmin reagent of the invention according to claim 2, the synthetic substrate for plasmin, the reagent containing the synthetic substrate for plasmin, and the non-reducing sugar having 4 to 24 carbon atoms, those of the invention according to claim 1 can be used as they are. .

The invention according to claim 3 is a plasmin reagent containing a non-reducing sugar having 4 to 24 carbon atoms; a plasmin reagent containing a non-reducing sugar having 4 to 24 carbon atoms at a concentration of 1 to 60%. The reagent for measuring α ₂ -plasmin inhibitor according to claim 2.

The invention according to claim 4 provides the reagent for measuring α ₂ -plasmin inhibitor according to claim 2 or 3, wherein the non-reducing sugar having 4 to 24 carbon atoms is a non-reducing sugar having 6 to 18 carbon atoms. is there.

The invention according to claim 5 is the reagent for measuring α ₂ -plasmin inhibitor according to claim 2 or 3, wherein the non-reducing sugar having 4 to 24 carbon atoms is selected from sorbitol, trehaloose and sucrose. .

In the inventions according to claims 2 to 5, i) the plasmin reagent is stable and the reagent is not viscous if the reagent is dissolved and liquefied and then stored at an appropriate temperature. Therefore, it is possible to accurately measure α ₂ -PI in the sample; ii) even if the reagent is prepared and stored, redissolution, dilution,
It is a simple α ₂ -PI measuring reagent that has the effect of not requiring any other pretreatment.

The invention according to claim 6 is the α ₂ -plasmin inhibitor-measuring reagent selected from claims 2, 3, 4, and 5, wherein the plasmin reagent is liquid. The α ₂ -plasmin inhibitor-measuring reagent of the invention according to claim 6 has the effect of the inventions of claims 2, 3, 4, and 5, and since the plasmin reagent is stable even in a liquid state, it does not dissolve the reagent,
Even if the reagent is left for a long time at an appropriate temperature, it can be used for the measurement of α ₂ -PI.

The invention according to claim 7 is a plasmin reagent containing a non-reducing sugar having 4 to 24 carbon atoms. The invention according to claim 7 is stable in that i) it is stable even if it is dissolved and made into a liquid and then stored at an appropriate temperature. Ii) Once prepared, re-dissolution, dilution and other pretreatments are not particularly necessary. It is a simple plasmin reagent that has the effect of. This plasmin reagent can be used for the measurement of α ₂ -PI.

The invention according to claim 8 has a concentration of 0.001-
100 units / ml of plasmin, a concentration of 1 to 60%, a non-reducing sugar having 4 to 24 carbon atoms, and a concentration of 0.005 to 3.0
A liquid plasmin reagent having a pH of 6 to 10 containing M salt. In addition to the effect of the invention according to claim 7, the invention according to claim 8 is a liquid plasmin reagent containing a low concentration of 0.001 to 100 units / ml of plasmin, which is usually difficult to maintain plasmin activity in liquid form. Is a liquid plasmin reagent that has the effect of being able to be stored while retaining sufficient activity.

The invention according to claim 9 has a concentration of 0.001 to 0.001.
100 units / ml of plasmin, a concentration of 1 to 60%, a non-reducing sugar having 4 to 24 carbon atoms, and a concentration of 0.005 to 3.0
This is a method of storing plasmin in a plasmin solution having a pH of 6 to 10 and containing M salt. The invention according to claim 10 is the method for preserving plasmin according to claim 9, wherein the non-reducing sugar having 4 to 24 carbon atoms is selected from sorbitol, trehaloose and sucrose. Claim 9 or 1
The invention according to 0 has an effect that it is possible to store a low concentration of 0.001 to 100 units / ml of plasmin, which is usually difficult to retain the plasmin activity, in a liquid state. The invention according to claim 11 is a method of preserving a protease by allowing a non-reducing sugar having 4 to 24 carbon atoms to be present in the protease. The invention according to claim 11 can stably store a protease such as plasmin.

In the invention of claim 1, 2 or 7, for example, when α ₂ -PI in plasma is measured by an automatic analyzer, it can be performed as follows.

The plasmin reagent is: i) 1.1 to 100 relative to α ₂ -PI in plasma of healthy subjects.
Containing an equivalent amount of plasmin, ii) containing a non-reducing sugar having 4 to 24 carbon atoms, iii) having a concentration of 1 to 60%, and iv) having a plasmin concentration of 0.001 to 100 units / m 2.
1) v) pH is 6.0 to 10.0, vi) contains a buffer having a concentration of 0.005 to 3.0 M, and vii) contains a salt having a concentration of 0.005 to 3.0 M , Using the plasmin reagent unit.

The synthetic substrate for plasmin has a concentration of 0.01-
A synthetic substrate reagent unit for 50 mM plasmin is used. The plasmin reagent and the synthetic substrate reagent for plasmin are stored at 1 to 10 ° C for 0 to 180 days and used in the following operations.

1 to 20 μl of plasma and 50 to 50 of plasmin reagent
Add 500 μl and react at 30-38 ° C. for 1-10 minutes. Reagent 5 containing synthetic substrate for plasmin, which can be measured at an appropriate wavelength after enzymatic cleavage in the resulting mixed solution
0 to 500 μl was added, the enzyme reaction was performed at 30 to 38 ° C., the change in absorbance at an appropriate wavelength per minute before the end of the reaction after addition of the synthetic substrate was determined as an index of plasmin activity, and these reagents were prepared in advance. The amount of α ₂ -PI in the sample can be accurately determined from the calibration curve prepared by using.

The invention according to claim 9 has a concentration of 0.001 to
100 units / ml of plasmin, concentration of 1 to 60%, non-reducing sugar having 4 to 24 carbon atoms, and concentration of 0005 to 3.0M
1 to 30 of a plasmin solution of pH 6 to 10 containing the salt of
Can store plasmin at ℃ for a long time.

[0044]

【Example】

Examples 1 to 3 and Comparative Examples 1 to 5 A. Stability of plasmin reagent up to 14 days 200 mM Tris-hydrochloric acid buffer pH 7.8 containing 80 mM monomethylamine and 200 mM sodium chloride as a basic composition, (a) basic composition (Comparative Example 1), (b) basic composition + 20% Sorbitol (basic composition containing 20% concentration of sorbitol, Example 1), (c) basic composition + 20%
Trehalose (Example 2), (d) basic composition + 20% sucrose (Example 3), (e) basic composition + 20% glucose (Comparative Example 2), (f) basic composition + 20% lactose ( Comparative Example 3), (g) basic composition + 20% maltose (Comparative Example 4), (h) basic composition + 20% maltotriose (Comparative Example 5) in 8 buffers and human plasmin (IIC).
(Manufactured by Japan) was added to Examples 1, 2, and
3 and the plasmin reagents of Comparative Examples 1 to 5. The plasmin reagent is 0.08 unit / ml. The plasmin reagent was stored at 5 ° C. and 25 ° C. for 14 days, and stability was examined.

Add 1 to 1 of plasmin reagent at 5 ° C and 25 ° C.
It was stored for 4 days, and changes in its plasmin activity were followed over time. The Hitachi 7050 automatic analyzer was used for the measurement, and the plasmin synthetic substrate NS-2200 (HD-Gln (C6H
11) -Phe-Lys-5AB) 8.9 mM aqueous solution was used as a synthetic substrate reagent for plasmin.

Plasmin reagent 250 in 3 μl of physiological saline
After adding μl and heating at 37 ° C. for 5 minutes, 150 μl of a synthetic substrate reagent for plasmin was added, and the absorbance at 405 nm from 60 seconds to 150 seconds after the addition was measured, and as an index of plasmin activity, 1 The change in absorbance per minute was determined. The change in absorbance is based on the change in absorbance derived from 5AB produced by cleavage of the synthetic substrate.

The time course of plasmin activity at 5 ° C. and 25 ° C. for each reagent composition is shown in FIGS. Tables 1 and 2 show the activity% values after 14 days when the activity on the first day of reagent preparation was 100%.

[0048]

[Table 1]

[0049]

[Table 2]

As a result, sorbitol, trehaloose,
The plasmin activity is highly maintained in sucrose,
It was found that these non-reducing sugars are greatly involved in stabilizing the plasmin reagent.

B. Stability of plasmin reagent for 180 days Among the plasmin reagents of A above, Comparative Example 1, Example 2,
The long-term stability of the plasmin reagent of Example 3 was confirmed. The measurement method was the same as in A above, and the stability was monitored for 180 days at 5 ° C and 25 ° C by the synthetic substrate method. Fig. 17 shows the results at 5 ° C and Fig. 18 shows the results at 25 ° C.
Shown in The plasmin reagent of Comparative Example 1 clearly shows a decrease in plasmin activity at 5 ° C. and 25 ° C., while the plasmin reagent of Example 2 or Example 3 showed 5
It was found that there was almost no decrease in activity at 0 ° C, and a small decrease in activity even at 25 ° C.

Example 4. Measurement of α ₂ -PI C. Calibration curve for α ₂ -PI measurement The plasmin reagent of Example 3 (with sucrose added) and the synthetic substrate reagent for plasmin used in A above were prepared and
Stored at 180 ° C for 180 days. Next, on the day of preparation and 180
A calibration curve for the reagent after day storage was created by the following method. The sample containing α ₂ -PI for preparing the calibration curve was prepared by diluting with standard commercially available plasma.

3 μl of sample containing known concentration of α ₂ -PI
To the above, 250 μl of the plasmin reagent of Example 3 was added, and plasmin in the reagent and α ₂ -PI in the sample were mixed with 3 μm.
Allow to react for 5 minutes at 7 ° C., then add to the resulting mixture,
150 μl of a synthetic substrate reagent for plasmin was added, the absorbance at 405 nm from 60 seconds to 150 seconds after the addition was measured, the change in absorbance per minute was determined as an index of plasmin activity, and a calibration curve was prepared.

The results of the calibration curve on the day of preparation are shown in FIG. 19, and the results of the calibration curve for the reagent 180 days after preparation are shown in FIG. The shape of the calibration curve hardly changed even after 180 days from the preparation of the reagent, and the performance of the α ₂ -PI measuring reagent was maintained for a long time after the preparation by adding the non-reducing saccharide to the plasmin reagent. It has been shown. Similar results were obtained by using plasmin reagent with trehalose or sorbitol instead of sucrose.

On the other hand, after 180 days of storage, an attempt was made to prepare a calibration curve in the same manner as above using the plasmin reagent without sugar of Comparative Example 1, but a sample containing no α ₂ -PI and 100% α _{2 was used.} With the sample containing -PI, the change in absorbance per minute showed almost the same value, and a calibration curve could not be created.

Example 5 Measurement of α ₂ -PI An actual plasma sample was measured by the measuring method of the present invention. The reagent used in Example 4 was used to measure α ₂ -PI in plasma samples, and the correlation between the measured values on the reagent preparation day and 180 days after the preparation was observed.

The plasmin reagent of Example 3 stored at 5 ° C. for 180 days after preparation was added to 3 μl of plasma containing α ₂ -plasmin inhibitor, and the plasmin in the reagent and its α ₂ -plasmin inhibitor were added. 5 minutes at 37 ℃,
The reaction is allowed to proceed, and then the resulting mixture is prepared at 5 ° C. for 1 hour.
150 μl of plasmin synthetic substrate reagent stored for 80 days
405n from 60 seconds to 150 seconds after addition
The absorbance at m was measured, the change in absorbance per minute was determined as an index of plasmin activity, and the amount of α ₂ -PI in the sample was determined from the calibration curve prepared in advance. The amount of α ₂ -PI in the same sample was similarly determined using the plasmin reagent and the plasmin synthetic substrate reagent on the day of preparation.

The α ₂ -PI in plasma samples was measured in the same manner for some plasmas, and the correlation was determined on the day of reagent preparation and on the day after 180 days of preparation. FIG. 21 shows the result. Regression formula Y = 0.96X + 3.82, correlation coefficient r = 0.99, showing good correlation, α ₂ -PI measuring reagent consisting of sucrose-containing plasmin reagent unit and plasmin synthetic substrate reagent unit is liquid It was shown that α ₂ -PI can be accurately measured even when stored at 5 ° C. for a long time. Even if trehalose or sorbitol-added plasmin reagent was used instead of sucrose, the correlation coefficient was r.
= 0.99.

[0059]

By stabilizing the plasmin reagent, it becomes possible to supply a liquid α ₂ -PI measuring reagent. The user can save the labor of reagent preparation. Further, it provides convenience such as avoidance of preparation error when preparing the reagent. In addition, it is possible to save space during storage. On the other hand, since the manufacturer does not need to go through the conventional freeze-drying process, the manufacturing period can be shortened and the facility cost can be greatly reduced.

[Brief description of drawings]

FIG. 1: 5 ° C. of plasmin reagent without sugar (Comparative Example 1)
The 14-day change in plasmin activity under storage is shown. The vertical axis represents the amount of change in absorbance per minute, and the horizontal axis represents the number of elapsed days (the vertical axis and the horizontal axis are the same in FIGS. 1 to 16).

FIG. 2: 25 of plasmin reagent without added sugar (Comparative Example 1)
The change in plasmin activity after 14 days of storage at 0 ° C is shown.

FIG. 3 shows a 14-day change in plasmin activity of plasmin reagent with sorbitol (Example 1) stored at 5 ° C.

FIG. 4 shows a 14-day change in plasmin activity of plasmin reagent with sorbitol (Example 1) stored at 25 ° C.

FIG. 5 shows a 14-day change in plasmin activity of a trehalose-added plasmin reagent (Example 2) under storage at 5 ° C.

FIG. 6 shows a 14-day change in plasmin activity of a plasmin reagent added with trehalose (Example 2) when stored at 25 ° C.

FIG. 7: Sucrose-added plasmin reagent (Example 3)
The change in plasmin activity after 14 days storage of 5 days is shown.

FIG. 8: Plasmin reagent with sucrose added (Example 3)
Figure 14 shows the change in plasmin activity after 14 days of storage at 25 ° C.

FIG. 9: Glucose-added plasmin reagent (Comparative Example 2)
The change in plasmin activity after 14 days storage of 5 days is shown.

FIG. 10 shows changes in plasmin activity of glucose-added plasmin reagent (Comparative Example 2) after storage at 25 ° C. for 14 days.

FIG. 11 shows a 14-day change in the plasmin activity of a plasmin reagent containing lactose (Comparative Example 3) stored at 5 ° C.

FIG. 12 shows changes in plasmin activity of a lactomin-added plasmin reagent (Comparative Example 3) stored at 25 ° C. for 14 days.

FIG. 13 shows changes in plasmin activity of maltose-added plasmin reagent (Comparative Example 4) stored at 5 ° C. for 14 days.

FIG. 14 shows changes in plasmin activity of maltose-added plasmin reagent (Comparative Example 4) stored at 25 ° C. for 14 days.

FIG. 15 shows changes in plasmin activity of maltotriose-added plasmin reagent (Comparative Example 5) on storage at 5 ° C. for 14 days.

FIG. 16: 14 of plasmin activity of plasmin reagent added with maltotriose (Comparative Example 5) under storage at 25 ° C.
Indicates the change of day.

FIG. 17 shows the plasmin activity of plasmin reagent without sugar (Comparative Example 1), plasmin reagent with trehalothose (Example 2) and plasmin reagent with sucrose (Example 3) under storage at 5 ° C. Represents a change of 180 days.

FIG. 18 shows plasmin activity of plasmin reagent without sugar (Comparative Example 1), plasmin reagent with trehalothose (Example 2) and plasmin reagent with sucrose (Example 3) under storage at 25 ° C. Represents a change of 180 days.

FIG. 19 shows a calibration curve of α ₂ -PI on the day of preparation. The vertical axis represents the amount of change in absorbance (ΔE / min) per minute, and the horizontal axis represents the amount of α ₂ -PI.

FIG. 20 shows a calibration curve of α ₂ -PI after 180 days (6 months) after preparation. The vertical axis shows the change in absorbance per minute (△ E
/ Min), and the horizontal axis represents the amount (%) of α ₂ -PI.

FIG. 21 shows the correlation between the measurement on the day of reagent preparation and the measurement after storage for 180 days (6 months) after preparation in the measuring method of the present invention. The vertical axis represents the amount (%) of α ₂ -PI in plasma when using a plasmin reagent and a synthetic substrate reagent for plasmin after storage for 180 days (6 months) after preparation, and the horizontal axis represents the day of preparation. The amount (%) of α ₂ -PI in plasma when a reagent is used is shown.

Claims (11)

[Claims] 1. A method of reacting plasmin with its α ₂ -plasmin inhibitor by mixing a sample containing α ₂ -plasmin inhibitor with an excess amount of plasmin reagent, and then measuring the residual plasmin activity. According to the method for measuring α ₂ -plasmin inhibitor in the sample, the plasmin reagent is a plasmin reagent containing a non-reducing sugar having 4 to 24 carbon atoms, and the α ₂ -plasmin inhibitor in the sample is -A method of measuring. _2. An α ₂ -plasmin inhibitor in a sample
An α ₂ -plasmin inhibitor-measuring reagent, which comprises, as essential units, a plasmin reagent unit containing a non-reducing sugar having 4 to 24 carbon atoms and a reagent unit containing a synthetic substrate for plasmin. 3. The α according to claim 2, wherein the plasmin reagent containing a non-reducing sugar having 4 to 24 carbon atoms is a plasmin reagent containing a non-reducing sugar having 4 to 24 carbon atoms in a concentration of 1 to 60%.
₂ -plasmin inhibitor measuring reagent. 4. The α _{2 according to} claim 2 or 3, wherein the non-reducing sugar having 4 to 24 carbon atoms is a non-reducing sugar having 6 to 18 carbon atoms.
-Plasmin inhibitor-reagent. 5. The non-reducing sugar having 4 to 24 carbon atoms is selected from sorbitol, trehaloose and sucrose.
Or the α ₂ -plasmin inhibitor-measuring reagent described in 3 above. 6. The plasmin reagent is liquid, 2.
Α ₂ -plasmin inhibitor selected from 3, 4 and 5
Measuring reagent. 7. A plasmin reagent containing a non-reducing sugar having 4 to 24 carbon atoms. 8. A pH6 solution containing plasmin at a concentration of 0.001 to 100 units / ml, non-reducing sugar having a carbon number of 4 to 24 at a concentration of 1 to 60%, and a salt at a concentration of 0.005 to 3.0M.
10 liquid plasmin reagents. 9. A pH 6 solution containing plasmin at a concentration of 0.001 to 100 units / ml, non-reducing sugar having a carbon number of 4 to 24 at a concentration of 1 to 60%, and a salt at a concentration of 0.005 to 3.0M.
A method of storing plasmin in a plasmin solution of 10 to 10 in the solution. 10. The method for preserving plasmin according to claim 9, wherein the non-reducing sugar having 4 to 24 carbon atoms is selected from sorbitol, trehaloose and sucrose. 11. A method for preserving a protease by allowing a non-reducing sugar having 4 to 24 carbon atoms to be present in the protease.