JPH012600A - Protein C activity measurement kit - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

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

(Prior art) PC is one of the vitamin-dependent plasma proteins, and
Activated protein C (hereinafter abbreviated as PCa) is activated by the thrombin-thrombomodulin complex in the presence of toxins.
is activated. PCa is a plasma protein that exhibits a strong anticoagulant effect by selectively inactivating the coagulation system coenzymes Factor V and Convict II, and also releases vascular plasminogen activator and promotes fibrinolysis. It is attracting attention as

Blood PC value indicates disseminated angiotropic coagulation syndrome (DIC)
It is known that C deficiency is decreased in liver diseases such as liver cirrhosis, chronic hepatitis, etc., and I) C deficiency syndrome, which presents with multiple thrombosis, has also been reported in recent years. Furthermore, attention is being paid to how the PC content in various blood coagulation products such as factor (I) affects their quality.

Therefore, there is a need for a simple method for quantifying PC in various blood coagulation products, for diagnosis of PC deficiency disease and DIC1 liver disease, 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, PC inhibitors exist in the blood, and there are also interfering substances that act on the synthetic substrate during activity measurement without being inhibited by inhibitors such as antithrombin. Conventionally, it has been difficult to quantify PC activity.

Therefore, PC can be extracted from plasma using antibody columns, adsorbents, etc.
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 processing large numbers of samples quickly. , PC has a drawback in the recovery rate, and when an antibody column is used, the recovery rate is good, but 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 involves quantifying the amount of PC antigen by an antigen-antibody reaction using a polyclonal or monoclonal antibody against PC, and radioimmunoassay, enzyme immunoassay, Laurell method, etc. have been implemented. Immunological quantitative methods can accurately measure the amount of PC antigen, but the antibodies used are very expensive, the operation time is long, and whether or not PC has normal activity cannot be confirmed by the final method.

The present inventors conducted extensive research to solve the problems of the conventional PC quantification method as described above, and found that there is no need for pretreatment to separate PC from plasma, the operation is simple, and a large amount of sample can be prepared in a short time. We have discovered a method for measuring enzymatic activity that can measure the pc of the present invention.
We have completed a kit for measuring activity.

(Problems to be Solved by the Invention) An object of the present invention is to provide an accurate and simple kit for measuring PC activity.

(Means for Solving the Problems) The present invention specifically inhibits interfering substances in plasma other than PCa that act on synthetic peptide substrates, thereby directly injecting PC into five blood sacs without separating PC from plasma. This is a kit for measuring PC activity based on a new measurement method capable of measuring PCa activity.

The kit of the present invention is composed of the following components.

a) Protein C activator b) Antithrombin■ C) Low molecular weight thrombin inhibitor d) Synthetic peptide substrate The kit of the present invention will be explained in more detail below.

1) Dilution of plasma Since PCa inhibitors that inhibit PCa are present in plasma, P
In the present invention, Ca activity cannot be measured accurately.
By diluting the plasma approximately 10 times or more, the effect of the PC inhibitor could be ignored.

As a buffer solution for diluting plasma, it is preferable to use a buffer solution that has been adjusted to a pH close to in-vivo conditions using an 11-layer agent 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.

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

2) PC activating substance Examples of PC activating substances include thrombin-thrombomodulin complex, snake venom, etc. Considering the original physiological conditions, thrombin-thrombomodulin complex is preferable, and thrombin and thrombomodulin are used as a complex. They may be added to the reaction system, or they may be added individually.

In addition, since Ca'' ions are required for PC activation, Ca1 ions are added to the bloodstream diluting solution or as a PC activating substance to achieve the optimal concentration in the PC activation reaction system. Preferably, it is added to the solution.

3) 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, only by adding antithrombin ■-heparin, PC
As a result of measuring the activity, the apparent activity was several hundred times higher than that of PCfi 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 the synthetic substrate used to measure PC activity, the present inventor found that instead of using a substance with a large molecular weight such as antithrombin ■, a low-molecular type It was revealed that the activity of interfering substances other than thrombin was suppressed by using a thrombin inhibitor, and it was possible to measure only the activity of PCa without the need for operations such as fractional purification of PC. .

The low-molecular 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 12,000 or less, preferably a substance with a molecular weight of 1.000 or less, e.g. ,(
2R,4R)-1-(N”-(3-methyl-1,2,3
,4-tetrahydro-8-quinolinesulfonyl)-L-
4-methyl-2-piperidinecarboxylic acid (compound 1), dandylarginine N-(3-ethyl-
N- such as 1,5-pentanediyl)amide (compound 2)
Arylsulfonyl-arginine derivatives (J, M
ed, Chem, 23.827-836, 12
93-1299. (1980) etc.), N-arylsulfonylglycyl-amidinophenylalanine derivatives (Thr
omb.

Res, 36. No, 5.457-165 (198
4) etc.].

4) Synthetic peptide substrate As the synthetic peptide substrate for measuring PC activity, those used in conventional enzymatic activity assay methods can be used, such as Pyr-Pro-Arg-p.
NA (S-2366: Pyroglutamyl-prolyl-
arginyl rule-nitroanilide), D-Val
-Leu-Arg pNA (S-2266:
D-valyl-leucyl-arginyl-p-nitroanilide), D-Phe-Pip-Arg-pNA (S
-2238: chromogenic synthetic substrates such as D-phenylalanyl-piperidino-arginyl-p-nitroanilide) and B
oC-Leu-Ser-Thr-Arg-? l
Examples include fluorescent synthesis substrates such as C^ (3112-V: 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.

Note that the enzymatic reaction between the synthetic peptide substrate and PCa can be stopped by making the reaction system weakly acidic. Therefore, it is preferable to use a weak acid such as citric acid or acetic acid as a reaction terminator.

5) How to use the kit Next, how to use the kit for measuring PC activity of the present invention will be explained. The outline is as follows.

■ Activate PC by diluting plasma and adding PC activating substance.

(2) After completion of pc activation, an inhibitor (antithrombin m, N''-arylsulfonyl-L-argininamide compound) against interfering substances in plasma other than PCa that act on synthetic 5'H is added.

(2) A synthetic peptide substrate is added to carry out an enzymatic reaction, and after the reaction is stopped, the PC-5 property is measured by measuring the color development or fluorescence of the degradation product caused by PCa.

In the following examples, the key for measuring the pcc property of the present invention/
Here is an example of how to use it and how to use it.

(Example) ~Kit components (each in one vial) ~a)) Thrombin-thrombomodulin? ! +combined<'tB dry product) 10U/mff1) Longbin-〇, 85 n ++ol
/ Use vd Toro 7 Pomodyurin 4- dissolved in distilled water.

b) Antithrombin m-heparin complex (lyophilized product) 25 U/- antithrombin lll - 100 U/- heparin - Used by dissolving in solution 4- in d below.

C) Compound l aqueous solution 5 ml (100 Ijg/+
d) d) S-23(i6 (lyophilized product)・4-
Use by dissolving in distilled water (3mM aqueous solution).

For example, a kit containing the above-mentioned components may include plasma dilution buffer 50+d (50mM), lithium salt tl (pH 8,
0)-0,1M sodium chloride-1mM calcium chloride-0,1% n5A-0,1w/dSBT I), a reaction terminator 2l-(2% citric acid 1 volume Y&,), etc. may be added.

~Measurement operations~ ■Dilute the test plasma 21 times with plasma dilution buffer.

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

■Thrombin-thrombomodulin? 1 Add 100 tpl of combined fJ solution and incubate at 37°C for 30 minutes.

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

(2) Add 100 μl of S-2366 solution and incubate at 37° C. for 30 minutes.

Add 02% citric acid solution 500I11 and measure the absorbance at 405rn.

According to the present embodiment described above, one set of the kit provides 40
Although a specimen can be measured, it is possible to create various kits suitable for the purpose by changing the component content per 1 vial, the dilution rate of plasma, etc., and changing the scale of the entire system as appropriate.

~Test FIt curve~ Figure 1 shows a calibration curve prepared using normal human pooled plasma at various dilutions.

As is clear from FIG. 1, the calibration curve shows good linearity in the range from 0 to 100%, and the calibration curve shows good linearity in the range from O to 100%.

PC activity measurement using this method has excellent quantitative properties.

Next, the results of examining various conditions for the kit 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.

+11 Plasma dilution rate As a result of investigating the effects of PC inhibitors and plasma dilution rate,
In the measurement method using the kit of the present invention, a linear relationship between the dilution rate and PC activity was obtained by diluting plasma approximately 10 times or more. As the dilution rate becomes lower than this, P
As the action of the C inhibitor becomes more pronounced, PC activity is suppressed and a deviation from the linear relationship occurs as the temperature increases.

Therefore, in the examples of the present invention, it is preferable to use plasma diluted 10 times or more.

+21Ca" ion concentration If the Ca" ion concentration when PC is activated! As a result of studying the temperature at 92 degrees, the optimal concentration of Ca' ions in the plasma dilution buffer was 0.1 to 4 mM, and 0.5 to 2.5
mM is more preferred.

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

(4) Thrombomodulin addition amount As a result of varying the addition amount of thrombomodulin,
In this example, PC was sufficiently activated with an addition amount of 0.04 nmol or more, and 0.06 to 0.16 nmol.
Preferably, it is carried out in mol.

(5) Amount of Compound 1 Added: 1 of Compound 1 in the mixed solution containing Compound 1 of this Example Kit; As a result of examining various concentrations, it was found that the concentration of Compound 1 in the mixed solution was about 75 n/- or more, and the synthesis 1! S? Ts-2
It was possible to inhibit interfering substances other than PCa that act on 366 to the extent that they had almost no effect.

Furthermore, as a result of investigating the inhibitory effect of compound 1 on PCa using separated and purified PC, it was found that PC activity was inhibited at concentrations of 150/1 or more in the mixed solution, so in this example, lower concentrations were used. It is preferable to use it in

As a result of using compound 2 instead of compound 1 above,
The present invention could be practiced using a solution with the same concentration as Compound 1. In addition, in the case of compound 3, the concentration of dissolved water is about 25/-
This made it possible to almost suppress the activity of interfering substances.

(Effect) Method using the kit for measuring PC activity of the present invention and immunological method CEIA method: J, C, Giddings et al.
al+ Br1t.

J, IIaemaLol,, 52.495-502
(1982)) were used for healthy subjects, patients with liver disease, and D.
Figure 2 shows the results of measuring and comparing IC, PC activity in patient plasma, and PC antigen amount.

The PC activity measured using the kit of the present invention was determined using the calibration curve shown in FIG. 1 and expressed as a percentage.

From FIG. 2, it can be seen that there is a difference between the PC activity measured using the kit of the present invention and the PC antigen amount quantified by the immunological method.
It is clearly shown that there is a good correlation. Furthermore, this correlation is well suited not only to healthy individuals but also to patients with liver disease or DIC, who are thought to have low plasma PC content.

(Effects) By using the kit for measuring PC activity of the present invention, plasma itself can be used without the pretreatment of separating and purifying PC, which is essential in conventional enzymatic activity measuring methods and is very time-consuming. It is now possible to measure PC activity.

As is clear from the above test results, the kit of the present invention:
Using a minute amount of plasma (less than 10 μl) as a sample, PC in the blood can be quantified in a short time, and the operation is very simple. In addition, in a comparative test of the PCC clearing kit using an immunological method, the PC activity measured using the kit of the present invention had a good correlation with the PC antigen star, indicating that the kit of the present invention has excellent stability. It is clear that there is.

In this way, compared to the conventional l)C activity measurement kit,
The kit of the present invention is very convenient, accurate and rapid, and is particularly useful for processing large amounts of samples.

Therefore, the kit of the present invention is useful for diagnosing PC deficiency II disease, DEC1 liver disease, etc., and for early detection of these diseases.
It is extremely useful for checking the quality of various blood products such as factor preparations and coagulation preparations.

[Brief explanation of drawings]

FIG. 1 shows a mouse line created according to an embodiment of the present invention.
Figure 2 shows the kit for measuring PC activity of the present invention and the immunological method (
These are the results of examining the correlation with the PC quantification kit using the EIA method. Agent (F1891) Patent attorney Sababe Murayama Figure 1 PC concentration

Claims (4)

[Claims] (1) A protein C activity measurement kit consisting of the following components. a) Protein C activator b) Antithrombin III c) Low molecular weight thrombin inhibitor d) Synthetic peptide substrate (2) The kit for measuring protein C activity according to claim 1, which contains a plasma dilution buffer and/or a reaction terminator as a component. (3) The kit for measuring protein C activity according to claim 2, wherein the protein C activator is a thrombin-thrombomodulin complex. (4) The kit for measuring protein C activity according to claim 3, wherein heparin is added to antithrombin III.