JPS63185399A - Quantitative determination of plasma protein - Google Patents

Abstract

PURPOSE:To enable the quantitative determination of plasma protein for the diagnosis of disseminated intravascular coagulation syndrome, etc., without separating the plasma protein, by using a synthetic peptide substrate and enzymologically determining protein C activity while specifically inhibiting the action of inhibiting substance against said substrate. CONSTITUTION:Human plasma is diluted >=10 times with a tris-HCl buffer solution having a Ph close to the pH in living body to exclude the influence of protein C inhibitor. The plasma solution is added with a thrombin- thrombomodulin complex, etc., as an activation substance for protein C to effect the activation of protein C. The system is added with an inhibitor (e.g. antithrombin III) against the inhibitive substance in the plasma other than activated protein C reactive with synthetic peptide substrate for the determination of the protein C activity and then added with a synthetic peptide substrate (e.g. pyroglutamyl-prolyl-arginyl-p-nitroanilide). Protein C activity can be determined by measuring the absorbance of produced p-nitroaniline using light of 405nm wavelength.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a method for quantifying protein C (hereinafter abbreviated as pc). More specifically, the present invention relates to a method for quantifying PC by directly enzymatically measuring PC activity in plasma without separating PC from plasma.

(Prior art) PC is one of the vitamin-dependent plasma proteins, and
It is activated into activated protein C (hereinafter abbreviated as PCa) by the thrombin-thrombomodulin complex in the presence of 24 ions. PCa is attracting attention as one of the plasma proteins that selectively inactivates coagulation system coenzymes factor ① and factor VM and exhibits a strong anticoagulant effect, as well as releasing vascular plasminogen activator and promoting fibrinolysis. has been done.

Blood PC value indicates disseminated angiotropic coagulation syndrome (DIC)
PC deficiency is known to decrease in liver diseases such as liver cirrhosis, chronic hepatitis, etc., and PC deficiency disease, which causes multiple thrombosis, has been reported in recent years. Therefore, the above-mentioned PC deficiency and DI
Accurate and simple PC quantitative methods are required for diagnosing C. and liver diseases and for early detection of these diseases.

Currently, several PC quantitative methods are used, one of which is the enzymatic activity measurement method.

This measurement method uses a synthetic peptide substrate specific to PCa. However, since there are PC inhibitors in the blood and interfering substances that act on synthetic substrates without being inhibited by inhibitors such as antithrombin, current synthetic substrates can be used to directly inhibit PC in plasma. Activity is difficult to quantify.

Therefore, using antibody columns, adsorbents, etc., PC can be extracted from plasma.
Pretreatment is necessary to separate the However, these methods require a relatively large amount of plasma to be used, and are time-consuming and labor-intensive, which is disadvantageous when a large number of samples are processed quickly. Furthermore, pretreatment using an adsorbent has a disadvantage in the recovery rate of PC, and while the recovery rate is good when using an antibody column, the column is very expensive. .

In addition to using synthetic substrates, there are biological activity measurement methods that utilize the anticoagulant effect of PC. This method involves activating PC with snake venom, adding it to the blood coagulation system, and measuring its effect on prolonging the coagulation time, but it cannot be said to be a highly sensitive method.

There is also a method of immunologically quantifying PC. This method quantitatively quantifies PC by an antigen-antibody reaction using a polyclonal or monoclonal antibody against PC, and radioimmunoassay, enzyme immunoassay, Laurel method, etc. have been implemented. Immunological assays can quantitatively quantify the amount, but the antibodies used are very expensive, the operation time is long, and it cannot be confirmed whether PC has normal activity or not.

The present inventors have conducted intensive research to solve the problems of the conventional PC quantification method as described above, and have found that there is no pretreatment to separate PC from plasma, the operation is simple, and a large amount of sample can be prepared in a short time. The present invention was completed by discovering an enzymatic activity measurement method that can measure .

(Problems to be Solved by the Invention) An object of the present invention is to provide a new and useful PC quantitative method.

(Means for Solving the Problems) The present invention is a method for enzymatically measuring PC activity using a synthetic peptide substrate, which is characterized by specifically inhibiting the action of an interfering substance on the substrate. This is a method for quantifying PC. That is, the present invention provides P that acts on synthetic peptide substrates.
By specifically inhibiting interfering substances in plasma other than Ca, it has become possible to directly measure the activity of PCa in plasma without separating PC from plasma.

The PC quantitative method of the present invention can be carried out as follows.

■ Activate the PC by diluting the plasma and adding a PC activator.

(2) After completion of PC activation, an inhibitor for interfering substances in plasma other than PCa that act on the synthetic substrate is added.

(2) A synthetic peptide substrate is added and reacted, and PC is quantified by measuring the color development or fluorescence of the degradation product caused by PCa.

The above-mentioned quantitative method of the present invention will be explained in more detail.

1) Plasma dilution rate Since PCa inhibitors that inhibit PCa are present in plasma, PCa activity cannot be accurately measured unless some treatment is performed. In the assay method of the present invention, the influence of PC inhibitors could be ignored by diluting plasma approximately 10 times or more.

Incidentally, plasma prepared by a conventional method can be used, and for example, citric acid oil plasma obtained by collecting blood in the presence of citric acid and then centrifuging it can be used.

2) Buffer Solution As a buffer solution for diluting plasma, it is preferable to use one whose pH is adjusted to be close to in-vivo conditions using a buffer such as Tris-HCl. As with commonly used buffers, salts such as sodium chloride may be added to match in vivo conditions.

Furthermore, in order to suppress the actions of various proteases in plasma, protease inhibitors such as soybean trypsin inhibitor (SBTI) and aprotinin can be added to an extent that does not affect PCa activity. Furthermore, a stabilizer such as bovine serum albumin (BSA) may be added in order to prevent precipitation of insoluble proteins under mildly acidic conditions at the time of final reaction termination.

3) PC activating substance Examples of the PC activating substance include thrombin-thrombomodulin complex, snake venom, etc., but in consideration of the original physiological conditions, thrombin-thrombomodulin complex is preferable. Thrombin and thrombomodulin may be added to the reaction system as a complex, or each may be added individually.

In addition, since Cat'' ions are required for PC activation, Ca2'' ions are added to the plasma dilution buffer or PC activating substance in order to reach the optimal concentration in the PC activation reaction system. It is preferable to add it to the solution.

4) Inhibitor of interfering substances Thrombin added to activate PC acts on the synthetic substrate used to measure PC activity and interferes with the measurement, so after activating PC, It is necessary to add an inhibitor. This inhibitor can be used to inhibit thrombin but has no effect on PCa, such as antithrombin ■.

Since the inhibition reaction of antithrombin (III) is significantly promoted by the presence of heparin, it is practical and preferable to coexist with heparin.

However, antithrombin ■ alone cannot suppress the activity of other interfering substances. In fact, by simply adding antithrombin-heparin, PC
As a result of measuring the activity, the apparent activity was several hundred times higher than the PC amount determined by immunoassay. When measuring PC activity by adding only antithrombin ■, there is a problem in that there are substances other than PC that act on the synthetic substrate, making it impossible to accurately measure PCa activity. This required complicated processing such as separating the PC.

As a result of examining various methods for suppressing the activity of interfering substances that act on synthetic substrates used to measure PC activity, the present inventor found that instead of using substances with a large molecular weight such as antithrombin ■, a substance with a low molecular weight We revealed that the activity of interfering substances other than thrombin can be suppressed by using a thrombin inhibitor, and succeeded in measuring only the activity of PCa without requiring operations such as separation and purification of PC.

The low molecular weight thrombin inhibitor that can be used in the present invention is preferably one that does not affect the activity of PCa, and is preferably a thrombin inhibitor with a molecular weight of 2,000 or less, preferably a substance with a molecular weight of 1,000 or less, such as (
2R,4R)-1-(N”-(3-methyl-1,2,3
,4-tetrahydro-8-quinolinesulfonyl)-L-
arginyl]-4-methyl-2-piperidinecarboxylic acid (compound 1), dandylarginine N-(3-ethyl-
N- such as 1,5-pentanediyl)amide (compound 2)
ArylsulfonyJL/-L-arginine derivative (J,
Med, Chem, 23.827-836, 1293-1299. (1980) etc.), N −(
Thromb.

Res,, 36. No, 5.457465 (19
84) etc.].

5) Synthetic peptide substrate As the synthetic peptide substrate for measuring PC activity, those used in conventional enzymological activity measurement methods can be used, such as p Glu -Pro -Arg-
pH 8 (S-2366: biroglutamyl-brolyl-
arginyl-p-nitroanilide), D-Val-L
eu-Arg pNA (S-2266: D-valyl-leucyl-arginyl-p-nitroanilide), D
-Phe-P ip -Arg-pNA (S -22
38: Chromogenic synthetic substrates such as D-phenylalanyl-piperidino-arginyl-p-nitroanilide) and Boc-
Leu -Ser -Thr -Arg-phylum CA (3
112-V: Fluorescence synthesis substrates such as t-butoxycarbonylleucyl-cerylthreonyl-arginyl-4-methylcoumarinamide).

The above-mentioned synthetic peptide substrate is decomposed by PCa to produce a colored or fluorescent substance, so the PC activity can be quantitatively determined by colorimetrically or fluorescently determining these decomposed products.

(Example) ~Operation■~ Human plasma 10 μp buffer
140μl thrombin-thrombomodulin 50μl complex/human plasma: human citrate plasma/i buffer: 50mM) Lis-HCl (pH 8,0), 0.1
M sodium chloride, 1mM calcium chloride, 25U/-
Aprotinin-thrombin-thrombomodulin complex: 20 U/
d Thrombin, 1.7 nmol/mff1) Rhombomodulin A reaction solution having the above composition was allowed to react at 37°C for 30 minutes.

~Operation ■~ - 25 U/antithrombin ■ - 100 U/-heparin - 100 μg/- Compound 1 A mixed solution IQO11ffi having the above composition was added to the reaction solution of Operation (2) and reacted at 37° C. for 10 minutes.

~Operation ■~ Add 100 μm of 3mM solution of synthetic substrate S-2366 to the reaction solution of Operation ■, react at 37°C for 20 minutes, and then add 100μ
The reaction was stopped by adding 50% β acetic acid and colorimetrically determined at 450 nm.

A dilution standard curve for this example was prepared using normal human pooled plasma and PC removed blood, and is shown in FIG.

As is clear from FIG. 1, the calibration curve shows good linearity in the range from 0 to 100%, and the PC quantitative method of the present invention has excellent quantitative performance.

Next, the results of examining various conditions for the quantitative method of the present invention will be shown. Incidentally, the basic operating method was carried out in accordance with the method of the above-mentioned example.

As a result of investigating the effects of Koyo PC inhibitor and plasma dilution rate,
In the assay method of the present invention, by diluting plasma approximately 10 times or more, a linear relationship between dilution rate and PC activity was obtained. As the dilution rate is lower than this, the effect of the PC inhibitor becomes more pronounced, which suppresses the PC activity and deviates from the linear relationship, which is not preferable.

Therefore, in the Examples of the present invention, blood w1apβ was diluted 15 times by adding 140 μl of buffer.

Ca" ion concentration As a result of examining the optimal concentration of Ca" ions during PC activation, the optimal concentration of Ca" ions was 0.1 to 4 mM,
More preferably 0.5 to 2.5mM.

In the reaction system of this example, almost 100% of PC could be activated by adding 10 or more thrombin. Antithrombin added after PC activation -
In order to reduce the amount of heparin as much as possible, in this example, the amount of thrombin added is IU.

Amount of Thrombomodulin Added As a result of varying the amount of thrombomodulin added,
In this example, P was added in an amount of 0.04 nmol or more.
C is sufficiently activated and the thickness is 0.06 to 0.16 nm.
It is preferable to use ol.

Decoy additive ■1 Compound 1 in the mixed solution added in operation ■ of this example
As a result of examining various concentrations of compound 1, the concentration of compound 1 in the mixed solution was about 75 μg/- or more, and the synthetic substrate S-236
It was possible to inhibit interfering substances other than PCa that act on PCa to the extent that they had almost no effect.

In addition, as a result of investigating the inhibitory effect of compound 1 on PCa using isolated and purified PC, it was found that 150μ
Since pc activity is inhibited at concentrations higher than g/+d, it is preferable to use concentrations lower than this in this example.

As a result of using compound 2 instead of the above compound 10,
The present invention could be practiced using a solution with the same concentration as Compound 1. Furthermore, in the case of Compound 3, the concentration of the solution was approximately 25 μ/barrel, making it possible to substantially suppress the activity of the interfering substance.

Although the quantitative method of the present invention has been explained in detail through the above examples, the following kit for protein C activity measurement can be prepared, for example, based on the essential requirements featured in this study.

~Kit Components~ a) Protein C activator b) Antithrombin C) Low molecular weight thrombin inhibitor d) Synthetic peptide substrate A more specific example of the kit is shown below. The amount in each vial is indicated.

a) I-thrombin-thrombomodulin complex (lyophilized product) 100/d) Thrombin-〇, 85 nmol/ml
Dissolve Thrombomodulin/4WIQ in distilled water and use.

b) Antithrombin■-heparin complex (lyophilized product) 25U/-antithrombinllll-100U/W11
Henomarin - Use by dissolving in solution d below from 4 companies.

C) Compound 1 aqueous solution 5 ml (100R/+n1t
) d) Synthetic substrate S-2366 (lyophilized product) 4- Dissolved in distilled water and used (3mM aqueous solution).

For example, a kit containing the above components may include plasma dilution buffer 50mM (50mM) lithium-hydrochloric acid (pH 8.0).
) -0,1M Sodium Chloride -1mM Calcium Chloride -0,1% BSA 0.1mg/+IIQSBTI)
or reaction terminator 21, d (2% citric acid solution), etc. may be added.

This kit can be operated easily and quickly as follows.

■Test blood inclination is 5! Dilute 21 times with dilution buffer.

This solution is incubated at 56° C. for 5 minutes, and the generated fibrin is removed by centrifugation, followed by aliquoting 200 μl.

■ Thrombin-thrombomodulin complex solution 100%
μ! Add and incubate for 30 minutes at 37°C.

Add 100 pl of a mixed solution of (1) antithrombin (2)-heparin-compound 1, and incubate at 37°C for 5 minutes.

■Add S-2366 solution IQOp/ and heat at 37°C for 30 minutes.
Incubate for minutes.

Add 500 pf of 2% citric acid solution and measure the absorbance at 4 O5 nm.

The 1 cent kit can measure about 40 samples, but 1 cent.
By changing the component content per vial, the dilution rate of plasma, etc., and changing the scale of the system as appropriate, it is possible to create various kits suitable for the purpose.

(effect) Assay method of the present invention and immunoassay method CEIA method: J, C.

Giddings et al, Br1t, J.
) laematol, + 52+ 495-502 (
1982)) was used in healthy subjects, patients with liver disease, and DIC.
Figure 2 shows the results of quantifying and comparing PC in plasma of patients.

The pc activity in the assay method of the present invention was determined using the calibration curve shown in FIG. 1 and expressed as a percentage.

FIG. 2 clearly shows that there is a good correlation between the PC activity determined by the assay method of the present invention and the amount of PC antigen determined by the immunoassay method. Furthermore, this correlation is observed not only in healthy individuals but also in liver disease patients and DT patients, who are known to have low plasma PC content.
It also fits well in the case of patient C.

(Effects) The PC quantification method of the present invention makes it possible to use plasma itself without the need for the extremely time-consuming pretreatment of separating and purifying PC, which is essential in conventional enzymological activity assay methods. became.

As is clear from the above test results, the quantitative method of the present invention:
Using only 10 μl of plasma as a sample, PC in the plasma can be quantified in a short time, and the operation is very simple. In addition, in a comparative test with an immunological quantitative method, it is clear that the PC activity determined by nature has a good correlation with the amount of PC antigen.
It can be seen that the method of the present invention has excellent quantitative properties.

As described above, compared to conventional quantitative methods, the PC quantitative method of the present invention is extremely simple, accurate, and rapid, and is particularly useful for processing large amounts of samples. Therefore, the assay method of the present invention is easy to make into a kit, and is highly useful for diagnosing PC deficiency, DIG, liver disease, etc.
It is a quantitative method.

[Brief explanation of the drawing]

FIG. 1 is a calibration curve prepared according to an example of the present invention,
FIG. 2 shows the results of examining the correlation between the quantitative method of the present invention and the immunological quantitative method (EIA method). Agent (6891) Patent attorney Sabube Murayama Figure 2 50100 (Olo) Dρft111■- PU Zunoshi Jh'', 11 to: 1 Ordinary human blood improvement ×: Liver disease patient plasma analysis: DIC patient Hesaka

Claims (1)

[Claims] (1) A method for quantifying protein C, which is characterized by specifically inhibiting the action of interfering substances on the substrate, in a method for enzymatically measuring protein C activity using a synthetic peptide substrate.