JPS5898097A - Measurement of alpha2-plasmin inhibitor and reagent therefor - Google Patents

Abstract

PURPOSE:A reagent consisting of an organic amine, a color-developing peptide substrate or fluorescent substrate and plasmin is used to measure alpha2-plasmin inhibitor in a sample with high accuracy. CONSTITUTION:A reagent consisting of an amine of the formula (R1, R2 are same or different and are lower alkly, hydroxy-lower-alkyl, amino-lower-alkyl) or its salt, a peptide color-developing substrate specifically acting on plasmin, e.g., D-valyl-L-leucyl-L-lysyl-p-nitroanilide or a peptide fluorescent substrate, e.g., D-valyl-L-leucyl-L-lysyl-5-amido-isophthalic acid and plasmin is added to a sample and they are kept in a buffer solution of 6-9pH at 20-40 deg.C. Then, the formed chromophoric group or fluorescent groups is determined.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to α2-plasmin inhibitor (hereinafter referred to as α2-P
Abbreviated as I. ) and a reagent for the measurement.

Currently, it is known that patients with genetic deficiency of α2-PI or patients with pathologically reduced concentrations of α2-PI, vasotropic coagulation syndrome, and liver disease can lead to severe bleeding tendency, and for the treatment of α2-PI. , , -PI measurements are being carried out.

α,-PI is a substance that has the ability to inhibit plasmin most quickly, although it is present in the smallest amount among the plasmin blood inhibitors that were previously thought to be 0α2-PI.
Although several other plasmin inhibitors exist in plasma, a simple and rapid method for enzymatically separating and quantifying them from α2-PI has not yet been found.

The conventional method for measuring plasmin inhibitory activity is the immunological method (J
, C! Iin, Invest, vol. 60, 861.
page.

1977), coagulological methods (Thronbosis)
and Disorders, pp. 1391-404,
, Academic Press.

(T.J., S.A., 1971), etc., but they require special reagents and complicated procedures such as the preparation of specific antibodies against α, -PI and the creation of immunoagar plates or the creation of human plasminogen-free fibrin agar plates. This method was unsatisfactory because it required a lot of manipulation, did not reflect only the inherent plasmin inhibitory activity of α2-PI, had poor reproducibility, and required a long time for measurement. Recently, a synthetic peptide substrate that specifically acts on plasmin and hydrolyzes it has been developed and is used for blood coagulation and fibrinolysis tests. , J
, Cl1n, Invest, vol. 87, p. 403.

(1977), and has attracted attention among researchers. However, this method performs measurements on the premise that only α,-PI forms a complex with plasmin.

The present inventors purified human α2-PI and immunized rabbits to obtain an anti-human α,-PI antibody, which was then transferred to Sepharose 4B by the Bromsian method (J. Biol. Ohem, 1999).

245, p. 8059, 1970) was used to specifically remove α2-PI from normal plasma using anti-human α,-PI antibody-Sepharose.
Depleted plasma was prepared. Next, using normal plasma and α2-P-depleted plasma, the antiplasmin activity of both was determined by the above-mentioned P,
It was measured by the method of Friberger et al. That is, α, −P
I complex + residual plasmin Φ) residual plasmin + peptide chromogenic substrate -51i:I
Ir, Jl Peptide Ten Chromophore (C) The absorbance of the chromophore was measured.

As a result, the anti-plasmin activity was clearly different between normal plasma and α2-PI-depleted plasma. In this measurement method, this is α2-
This result is due to the simultaneous measurement of plasmin inhibitors other than PI.

Moreover, it was confirmed that (a) the longer the reaction time, the stronger the tendency. Therefore, the present inventors completed the present invention as a result of various research into methods for accurately measuring only α2-PI.

In the present invention, an organic amine, a peptide coloring substance that specifically acts on plasmin and is hydrolyzed, or a peptide fluorescent substrate and plasmin are added to the sample. α in the specimen kept at a temperature of °C.
A method for measuring α2-PI and organic amines determined by measuring the amount of chromophore or fluorophore that produces 2-PI.

Peptidic chromogenic substrates or monopeptide fluorescent substrates.

This is a reagent for measuring α2-PI in a specimen, which is composed of plasmin.

To carry out the present invention, first, the activity of plasmin inhibitors other than α2-PI in a test subject is inhibited using an organic amine. Next, a peptidic chromogenic substrate or a peptidic fluorescent substrate (hereinafter simply referred to as a peptidic substrate) and plasmin are added and maintained at a temperature of 20 to 40°C. These reactions are preferably carried out in a buffer solution having a pH of 6 to 9.

Next, the amount of chromophore or fluorophore released by the remaining plasmin is measured to determine the amount of α, -PI.

The present invention can be expressed as follows.

Sample 10 Organic amine ff11Mα2-PI0 Plasmin inhibitor that has lost inhibitory activity α2-PI 10 Constant excess plasmin α, -PI・
Plasmin complex 10 residual plasmin residual plasmin + peptide substrate! Measure the absorbance of the peptide decachromophore or fluorophore chromophore or the fluorescence intensity of the fluorophore.

The reagents may be added to the specimen in the order described above, or the organic amine and the peptide substrate may be added to the specimen at the same time. Any specimen containing α2-PI, such as plasma, serum, cerebrospinal fluid, and synovial fluid, can be used. Note that, if necessary, the specimen may be diluted with a buffer before use.

As the organic amine, those represented by the following general formula or salts thereof can be used.

General formula 1・＼ R2-NH (However, "R,, R,," are the same or different /
- R3 is lower alkyl, hydroxy lower alkyl, amino lower alkyl or H, and R3 represents lower alkyl, hydroxy lower alkyl, amino lower alkyl. ) Lower alkyl is 01 to C6, linear or branched, and specifically includes methylamine, ethylamine, propylamine, and butylamine.

Examples include 1,4-diaminobutane and monoethanolamine. Further, a concentration of about 0.1M is sufficient.

On the other hand, the peptide substrate is a peptide that specifically acts on plasmin and undergoes hydrolysis, and a peptide in which a chromophore or fluorophore is bound to the carboxyl group of a lysine residue via an amide bond can be used. A commercially available example is D-Baryl-L-L-L from Kabi (Sweden).
-Lysyl-P-nitroanilide (trade name: Test Team ■, S-2251), Tosyl-glycyl-L-prolyl-L-lysyl-P-nitroanilide (trade name: Chromofarm) from Hentafarm (Switzerland) PI, ), D-valyl-L-lysyl-L-lysyl-5-amido-isophthalic acid from Dito Inc. (USA), and carbobenzoxy-L-glutamyl-L from the Peptide Research Institute for Protein Research.
-Lysyl-L-lysyl-7-amido-4-methylcoumarin, carbobenzoxy-L-valyl-L-lysyl-L
-lysyl-7-amido-4-methylcoumarin and the like.

Trishydroxymethylaminomethane (Tris) is used as a buffer solution to adjust pH to 9.

Phosphoric acid, boric acid, 5,5-jethylbarbital.

etc. can be used. The reaction temperature is preferably 20-40'C, more preferably around 87°C, which is the general enzyme reaction temperature. If this temperature is below 0°C, the plasmin substrate consumption rate decreases and the reaction takes time. In addition, temperatures above 40°C are undesirable because they tend to cause plasmin deactivation.In addition, enzyme reactions can be measured using either the initial rate modified method or the endpoint method.When using the zero endpoint method, is acetic acid as a reaction stopper.

Organic acids such as citric acid can be used.

Next, nine embodiments of the present invention will be described.

The present invention is not limited to these.

Example 1 Measurement reagent (a) Buffer; 50mM Tris buffer (pH 7,4) (
b) Organic amine; 0.1M monomethylamine (monomethylamine was dissolved in a buffer solution and adjusted to pH 7.4) (C) Substrate solution; 4.9 mM/l D-valyl-L-
Lysyl-L-lysyl-P-nitroanilide aqueous solution (d) Plasmin solution; 03 zein units/ml human plasmin (human plasmin dissolved in 50% glycerin-1mMMC) (e) Stop solution; 2% citric acid Aqueous solution measurement procedure (a) Dilute normal human plasma with a buffer solution, and dilute the 80-fold dilution value to 1
Create dilution series of +'o, 25, 50, 75, 100° and 125%, with 00% as the dilution series.

(0) Add 0.1 ml of substrate solution to 0.2 ml of each diluted solution and heat at 87°C for 5 minutes.

(c) After heating, add 0.1 ml of plasmin solution to each diluted solution and further heat at 87°C for 10 minutes.

) Add 2 ml of stop solution to stop the reaction.

(e) The amount of P-nitroaniline is measured using a Hitachi Model 189 spectrophotometer at a wavelength of 405 nm.

(f) Meanwhile, normal human plasma was diluted with an organic amine.

Let's create each dilution series of 0.25, 50.75゜100 and 125% with the 80 times dilution value as 100%) - (E)
Perform the same operation.

The absorbance of both dilution series is summarized in Table-1.

This table shows that there is a plasmin inhibitor that is inactivated by monomethylamine.Table=1. Plasmin residual activity in normal human plasma is α, −
For the PI purified product, create dilution series in the same way using (a) and (b), and for each dilution series (0) ~
Perform the operations in (e)).

The results obtained are summarized in Table-2.

Table-2 α2-PI purified product plasmin residual activity α2-PI
It can be seen from the table that it is not affected at all by monomethylamine treatment.

Example 2 Measurement reagent (a) Buffer; 50mM phosphate buffer (pH 7,4) (
b) Organic amine; 0.1M propylamine (adjusted to pH 7.4 by dissolving propylamine in a buffer solution) (C) Substrate solution; 4.9mM/l Chromozyme PL aqueous solution (d) Plasmin solution; 0.8 casein units/me human plasmin (
Dissolve human plasmin in 50% glycerin-1mMHO/) Measurement procedure (a) Dilute normal human plasma with a buffer solution to create a dilution series similar to Example 1.

(b) Add 0.1 ml of substrate solution to 0.2 rul of each dilution and heat at 37°C for 5 minutes.

(c) After heating, add 0.1 ml of plasmin solution to each diluted solution and further heat at 87°C for 10 minutes.

b) The reaction solution was measured using a modified initial velocity method using a Guilford Stisa single layer connected to a timer and printer. (e) Separately, normal human plasma was diluted with an organic amine.

Create a dilution series similar to (A) and perform operations (O) to).

On the other hand, a dilution series is prepared for the α2-PI purified product using a buffer solution and an organic amine, and operations (b) to (b) are performed for each dilution series.

The results showed that there is a plasmin inhibitor that is inactivated by propylamine and that α, -PI is not affected by propylamine treatment.

Example 8 Measurement reagent (a) Organic amine; 0.1M ethanolamine (ethanolamine was dissolved in Tris buffer and adjusted to pH 7.4) (b) Substrate solution; 0.8mM D-valyl-L- Leucyl-L-lysyl-5-amide-isophthalic acid aqueous solution (C) Plasmin solution: 0.07 zein units/ml human plasmin (d) Stop solution: 2% citric acid aqueous solution Preparation of standard curve Dilute with amine and make the 80x dilution value 10
0% (equivalent to a2-PI 619/100m1)
As, 0.25.50.75.100.125%
After creating each dilution series, perform the following operations.

Procedure Add 0.1 ml of substrate solution to 60 μl of each dilution and incubate at 87°C for 5 minutes.
After warming for minutes, each dilution was added with 0.1 liter of plasmin solution.
ml and further heated at 37°C for 5 minutes, and then 2ynl of stop solution was added to each to stop the reaction. Each of the reaction-completed solutions was measured using a Hitachi MPF-4 fluorophotometer (excitation wavelength 885
Determine the fluorescence intensity at 480 nm (fluorescence wavelength: 480 nm) and create a calibration curve.

Dilute 0.1 ml of the sample's plasma with 2.9 ml of organic amine.

Add 0.1 ml of substrate solution to 60 μl of this diluted solution and incubate at 87°.
After heating at C for 5 minutes, the following operations during preparation of a calibration curve were performed to determine the fluorescence intensity, and α2-PI was determined from the calibration curve.

17-

Claims (1)

[Scope of Claims] (1) A chromophore or a fluorophore produced after adding an organic amine, a peptidic chromogenic substrate or a peptidic fluorescent substrate that specifically acts on and hydrolyzes plasmin, or plasmin to a subject. 1. A method for measuring α2-plasmin inhibitor, which comprises measuring α2-plasmin inhibitor. (2) The organic amine is 1.\R, -NH (where R,, R2 are the same or different lower/Rs class alkyl, hydroxy lower alkyl, amino lower alkyl or H, and R, is lower alkyl, hydroxy lower alkyl (indicates amino lower alkyl) or a salt thereof. (3) A peptide coloring agent that specifically acts on plasmin and is hydrolyzed. The substrate is D-valyl-L-isoyl-L-I
J Silu-P-nitroanilide, Tosyl-glycyl-L
-Prolyl-L-lysyl-P-nitroanilide A method for measuring α2-plasmin inhibitor according to claim (1). (4) The peptide fluorescent substrate that specifically acts on and hydrolyzes plasmin is D-valyl-L-isoyl-L-lysyl-5-amido-isophthalic acid. Carbobenzoxy-L-glutamyl-L-lysyl-L-
Lysyl-7-amido-4-methylcoumarin, carbobenzoxy-L-valyl-L-leucyl-cy-lysyl-7-
Claim No. 1, which is amido-4-methylcoumarin (
The method for measuring α2-plasmin inhibitor according to item 11. (5) A reagent for measuring α2-plasmin inhibitor in a specimen consisting of an organic amine, a peptide chromogenic substrate or peptide fluorescent substrate that specifically acts on and hydrolyzes plasmin, and plasmin (6) An organic amine is or The reagent for measuring α2-plasmin inhibitor according to claim (5), which is a salt thereof. (The peptide chromogenic substrate that specifically acts on cabrasmin and is hydrolyzed is D-valyl-L-isoyl-L-IJ.
Sil-P-nitroanilide, Tosyl-glycyl-L-
The reagent for measuring α2-plasmin inhibitor according to claim (5), which is prolyl-L IJ lysyl P-nitroanilide. (8) The peptide fluorescent substrate that specifically acts on plasmin and is hydrolyzed is D-valyl-L-isoyl-L-lysyl-5-amido-isophthalic acid. Carbobenlaxy L-glutamyl-L-lysyl-L
-lysyl-7=amido-4-methylcoumarin, carbobenzoxy-L-valyl-L-lysyl-L-lysyl-7
Claim 1: A reagent for measuring α2-plasmin inhibitor, which is -amido-4-methyl(yellow)coumarin. (9) A reagent for measuring α2-plasmin inhibitor as set forth in claim (5), which comprises methylamine, D-valyl-L-leucylude L-lysyl-P-nitroanily, and plasmin.