JPH0229319B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to α ₂ -plasmin inhibitor (hereinafter referred to as α ₂
-Abbreviated as PI. ) and a reagent for the measurement. Currently, patients with a genetic deficiency of _α2 -PI or patients whose concentration is pathologically reduced, intravascular coagulation syndrome, and liver disease are known to cause severe bleeding tendencies, and for the treatment of these patients, etc. α ₂ -PI measurements are being carried out. Among the previously thought plasmin inhibitors in the blood, α ₂ -PI is a substance that exists in the smallest amount but has the ability to inhibit plasmin most rapidly. Although several types of plasmin inhibitors other than α ₂ -PI exist in plasma, a simple and rapid method for enzymatically separating and quantifying them from α ₂ -PI has not yet been found. Conventional methods for measuring plasmin inhibitory activity include immunological methods (J. Clin. Invest., vol. 60, p. 361, 1977) and hematological methods (Thronbosis and Disorders, 391-
404 pages, Academic Press, USA 1971), but they require special reagents and complicated operations such as the preparation of specific antibodies against α ₂ -PI, the creation of immunoagar plates, and the creation of human plasminogen-free fibrin agar plates. and does not reflect only the plasmin inhibitory activity inherent to α ₂ -PI,
The method was unsatisfactory as it 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 in blood coagulation and fibrinolysis tests, and a method for measuring α ₂ -PI using this substrate is P.
Friberger et al. (Scando.J.Clin.Invest., vol. 37, 403
Page, 1977), and has attracted attention among researchers. However, this method performs measurements on the premise that only α ₂ -PI forms a complex with plasmin. The present inventors purified human α ₂ -PI and immunized rabbits to obtain an anti-human α ₂ -PI antibody, which was then added to Sepharose 4B using the Bromsian method (J. Biol. Chem., Vol. 245, p. 3059).
α ₂ -PI-depleted plasma was prepared by specifically removing only α ₂ -PI from normal plasma using an anti-human α ₂ -PI antibody-sepharose conjugated with 1970). Next, using normal plasma, purified α ₂ -PI separated from the same amount of normal plasma, α ₂ -PI-depleted plasma, and a buffer solution, these anti-plasmin activities were determined using the above-mentioned P. Friberger.
It was measured using the method of et al. That is, (a) analyte + constant excess amount of plasmin for a certain period of time――――――――→ Plasmin (inactive)・α ₂ -PI complex + residual plasmin (b) residual plasmin + peptide chromogenic substrate for a certain period of time--- ------→ Peptide + chromophore (c) The absorbance of the chromophore was measured. As a result, the anti-plasmin activity was clearly different between normal plasma and the purified α ₂ -PI, and between α ₂ -PI-depleted plasma and the buffer solution. This is α ₂ in this measurement method.
-This result is due to the simultaneous measurement of plasmin inhibitors other than PI. Furthermore, it was confirmed that the longer the reaction time in (a), the stronger this tendency was. Therefore, the present inventors completed the present invention as a result of various research into methods for accurately measuring only α ₂ -PI. The present invention involves adding plasmin and a peptidic chromogenic substrate or a peptidic fluorescent substrate that acts specifically on plasmin and hydrolyzing it to a test sample, carrying out a reaction, and measuring the generated chromophore or fluorophore _.
- In the method for measuring plasmin inhibitors, the following general formula is used before contacting the specimen with plasmin: (In the formula, R ₁ and R ₂ are the same or different and represent a lower alkyl group, a hydroxy lower alkyl group, an amino lower alkyl group, or a hydrogen atom, and R ₃ represents a lower alkyl group, a hydroxy lower alkyl group, or an amino lower alkyl group. A method for measuring α ₂ -plasmin inhibitor characterized by treatment with an organic amine represented by (shown) or a salt thereof; This is a reagent for measuring α ₂ -plasmin inhibitor in a specimen, which consists of a chromogenic substrate or a peptidic fluorescent substrate, and plasmin. To carry out the present invention, first, α ₂ -PI in a specimen is
The activity of other plasmin inhibitors is blocked with organic amines. Next, a peptide chromogenic substrate or a peptide fluorescent substrate (hereinafter simply referred to as peptide 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 with 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 ₂ -PI. The present invention can be expressed as follows. Analyte + Organic amine for a certain period of time――――――――→ α ₂ -PI + Plasmin inhibitor that has lost inhibitory activity α ₂ -PI + Constant excess plasmin for a certain period of time――――――――→ α ₂ -PI/plasmin Complex + residual plasmin Residual plasmin + peptidic substrate for a certain period of time ――――――→ Peptide + chromophore or fluorophore Measure the absorbance of the chromophore or the fluorescence intensity of the fluorophore. Note that each reagent 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. This is because the reaction of plasmin α ₂ -PI is extremely fast, so when plasmin is added to a system where α ₂ -PI, an organic amine, and a peptide substrate coexist, plasmin first reacts with α ₂ -PI.
This is because it reacts with PI, followed by a reaction between the remaining plasmin and the peptide substrate. Samples include plasma, serum, cerebrospinal fluid, joint fluid, etc. α ₂ -PI
It can be used as long as it contains. Note that, if necessary, the specimen may be diluted with a buffer before use. In the general formula representing the organic amine used in the present invention, lower alkyl is C ₁ to _{C 5} and is linear or branched, specifically methylamine,
ethylamine, propylamine, butylamine,
Examples include 1,2-diaminoethane, 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-Leucyl-L-Lysyl- manufactured by Kabi (Sweden).
P-Nitroanilide (Product name: Test Team,
S-2251), Tosyl-Glycyl-L-Prolyl-L-Lysyl-P- from Hentafarm (Switzerland)
Nitroanilide (trade name: Chromozyme PL), D-baryl-L-leucyl-L-lysyl-5-amido-isophthalic acid from Dade Corporation (USA), carbobenzoxy-L- from the Peptide Institute of the Protein Research Foundation. Glutamine-L-lysyl-L-lysyl-7
-Amido-4-methylcoumarin, carbobenzoxy-L-valyl-L-leucyl-L-lysyl-7
-Amido-4-methylcoumarin, etc. Buffer solutions to adjust the pH to 6 to 9 include trishydroxymethylaminomethane (Tris), phosphoric acid,
Boric acid, 5,5-diethylbarbital, etc. can be used. The reaction temperature is preferably 20 to 40°C, and more preferably around 37°C, which is a general enzyme reaction temperature. If this temperature is below 20°C, the plasmin substrate consumption rate decreases and the reaction takes time. Further, temperatures above 40°C are not preferred because plasmin tends to be deactivated. Note that the enzyme reaction can be measured using either the initial rate method or the end point method. When using the end point method, an organic acid such as acetic acid or citric acid can be used as a reaction stopper. Next, the present invention will be described with reference to Examples, but the present invention is not limited thereto. Example 1 Measurement reagent (a) Buffer; 50mM Tris buffer (PH7.4) (b) Organic amine; 0.1M monomethylamine (monomethylamine was dissolved in the buffer and the PH
(adjusted to 7.4) (c) Substrate solution; 4.9mM/D-baryl-L-leucyl-L-lysyl-P-nitroanilide aqueous solution (d) Plasmin solution; 0.3 casein units/ml human plasmin (human plasmin in 50% glycerin) -Dissolved in 1mM HCl) (e) Stop solution; 2% citric acid aqueous solution Measurement procedure (a) Dilute normal plasma with buffer solution and calculate the 30-fold dilution value.
Assuming 100%, create dilution series of 0, 25, 50, 75, 100, and 125%. (b) Add 0.1 ml of substrate solution to 0.2 ml of each diluted solution and heat at 37℃ for 5 minutes.
Warm for a minute. (c) After heating, add 0.1ml of plasmin solution to each diluted solution.
Further warm at 37°C for 10 minutes. (d) Add 2 ml of stop solution to stop the reaction. (e) Measure the amount of P-nitroaniline using a Hitachi Model 139 spectrophotometer at a wavelength of 405 nm. (F) On the other hand, dilute normal human plasma with organic amine and set the 30-fold dilution value as 100% to 0, 25, 50, 75,
Create dilution series of 100 and 125% and perform operations (b) to (e) in the same manner. The absorbance of both dilution series is summarized in Table 1.
This table shows that there are plasmin inhibitors that are inactivated by monomethylamine.

[Table] Next, dilution series were similarly created using (a) and (b) for the α ₂ -PI purified product, and operations (b) to (e) were performed for each dilution series. The results obtained are summarized in Table 2.

[Table] It can be seen from the table that it is not accepted.
Example 2 Reagent for measurement (a) Buffer; 50mM phosphate buffer (PH7.4) (b) Organic amine; 0.1M propylamine (propylamine was dissolved in buffer and adjusted to PH7.4) (c) Substrate solution: 4.9mM/chromozyme PL aqueous solution (d) Plasmin solution: 0.3 casein units/ml human plasmin (dissolve human plasmin in 50% glycerin-1mM HCl) Measurement procedure (a) Dilute normal human plasma with buffer solution. A dilution series similar to Example 1 is prepared. (b) Add 0.1 ml of substrate solution to 0.2 ml of each diluted solution and heat at 37℃ for 5 minutes.
Warm for a minute. (c) After heating, add 0.1ml of plasmin solution to each diluted solution.
Further warm at 37°C for 10 minutes. (d) The reaction solution was measured by the initial velocity method using a Gilford stayr connected to a timer and printer. (e) Separately, dilute normal human plasma with an organic amine, prepare a dilution series similar to (a), and perform operations (b) to (d). On the other hand, dilution series are prepared for the α ₂ -PI purified product using a buffer solution and an organic amine, and operations (b) to (d) are performed for each dilution series. The results showed that there is a plasmin inhibitor that is inactivated by propylamine and that α ₂ -PI is not affected by propylamine treatment. Example 3 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 casein units/ml human plasmin (d) Stop solution; 2% citric acid aqueous solution Creation of standard curve Dilute normal human plasma with organic amine and dilute it 30 times. Assuming the dilution value to be 100% (equivalent to α ₂ -PI 6mg/100ml),
After creating dilution series of 0, 25, 50, 75, 100, and 125%, perform the following operations. Procedure: Add 0.1 ml of substrate solution to 60μ of each diluted solution, warm at 37°C for 5 minutes, then add 0.1 ml of plasmin solution to each diluted solution.
ml and further warmed at 37°C for 5 minutes, then 2 ml of stop solution was added to each to stop the reaction. The fluorescence intensity of each of the reaction-completed solutions was determined using a Hitachi MPF-4 fluorescence photometer (excitation wavelength: 335 nm, fluorescence wavelength: 430 nm), and a calibration curve was created. Measurement of specimen Dilute 0.1 ml of plasma with 2.9 ml of organic amine. 0.1 ml of the substrate solution was added to 60 µ of this diluted solution, heated at 37°C for 5 minutes, and the following operations during preparation of a calibration curve were performed to determine the fluorescence intensity, and α ₂ -PI was determined from the calibration curve. Example 4 Measurement reagent (a) Buffer; 50mM phosphate buffer (b) Organic amine; 0.15M 1,2-diaminoethane (1,2-diaminoethane was dissolved in Tris buffer and adjusted to pH 7.4) ) (c) Substrate solution; 4.9mM D-baryl-L-leucyl-L-lysyl-p-nitroanilide aqueous solution (d) Plasmin solution; 0.3 casein units/ml human plasmin (e) Stop solution; 2% citric acid aqueous solution Measurement procedure Normal human plasma was diluted with an organic amine and the same procedures as in Example 2 (a) to (e) were performed. Further, the same operation as in Example 2 was performed for the α ₂ -PI purified product.
The existence of a plasmin inhibitor that is inactivated by 1,2-diaminoethane and the fact that α ₂ -PI is 1,2
- The results showed that it was not affected by diaminoethane treatment.

Claims (1)

[Scope of Claims] 1. A reaction is carried out by adding plasmin and a peptidic chromogenic substrate or a peptidic fluorescent substrate that specifically acts on and hydrolyzes plasmin to a specimen,
In the α ₂ -plasmin inhibitor assay method that measures the generated chromophore or fluorophore, the following general formula is used before contacting the analyte with plasmin: (In the formula, R ₁ and R ₂ are the same or different and represent a lower alkyl group, a hydroxy lower alkyl group, an amino lower alkyl group, or a hydrogen atom, and R ₃ represents a lower alkyl group, a hydroxy lower alkyl group, or an amino lower alkyl group. A method for measuring α ₂ -plasmin inhibitor, which comprises treating with an organic amine represented by (shown in the figure) or a salt thereof. 2 Peptidic chromogenic substrates that act specifically on plasmin and are hydrolyzed are D-baryl-L-leucyl-L-lysyl-P-nitroanilide and tosyl-glycyl-L-prolyl-L-lysyl-P-nitroanilide. α ₂ - according to claim 1, which is
Method for measuring plasmin inhibitor. 3 Peptide fluorescent substrates that act specifically on plasmin and are hydrolyzed are D-baryl-L-leucyl-L-lysyl-5-amido-isophthalic acid and carbobenzoxy-L-glutamyl-L-lysyl-L.
-Lysyl-7-amido-4-methylcoumarin, carbobenzoxy-L-valyl-L-leucyl-L
A method for measuring α ₂ -plasmin inhibitor according to claim 1, which is -lysyl-7-amido-4-methylcoumarin. 4 General formula (In the formula, R ₁ and R ₂ are the same or different and represent a lower alkyl group, a hydroxy lower alkyl group, an amino lower alkyl group, or a hydrogen atom, and R ₃ represents a lower alkyl group, a hydroxy lower alkyl group, or an amino lower alkyl group. A reagent for measuring α ₂ -plasmin inhibitor in a specimen, which comprises an organic amine represented by (shown in the figure) or a salt thereof, a peptidic chromogenic substrate or a peptidic fluorescent substrate that specifically acts on and hydrolyzes plasmin, and plasmin. 5 Peptidic chromogenic substrates that act specifically on plasmin and are hydrolyzed are D-baryl-L-leucyl-L-lysyl-P-nitroanilide and tosyl-glycyl-L-prolyl-L-lysyl-P-nitroanilide. α ₂ - according to claim 4, which is
Reagent for measuring plasmin inhibitor. 6 Peptide fluorescent substrates that act specifically on plasmin and are hydrolyzed include D-baryl-L-leucyl-L-lysyl-5-amido-isophthalic acid and carbobenzoxy-L-glutamyl-L-lysyl-L.
-Lysyl-7-amido-4-methylcoumarin, carbobenzoxy-L-valyl-L-leucyl-L
The reagent for measuring α ₂ -plasmin inhibitor according to claim 4, which is -lysyl-7-amido-4-methylcoumarin. 7 Methylamine, D-valyl-L-leucyl-
The reagent for measuring α ₂ -plasmin inhibitor according to claim 4, which comprises L-lysyl-P-nitroanilide and plasmin.