JP2673688B2 - Plasma protein assay - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for quantifying protein C (hereinafter abbreviated as PC). More specifically, without separating PC from plasma,
PC directly by enzymatically measuring PC activity in plasma
The quantification method. (Prior Art) PC is one of vitamin K-dependent plasma proteins, and Ca ²⁺
In the presence of ions, activated protein C (hereinafter abbreviated as PCa) is activated by the thrombin-thrombomodulin complex. PCa selectively inactivates factor V and factor VIII of coagulation system coenzyme and shows a strong anticoagulant action, and
It is attracting attention as one of the plasma proteins that release vascular plasminogen activator and have a fibrinolytic promoting action. Blood PC level is known to decrease in liver diseases such as generalized intravascular coagulation (DIC), liver cirrhosis, and chronic hepatitis, and PC deficiency with multiple thrombosis has recently been reported. . Therefore, in diagnosing the above-mentioned PC deficiency, DIC, liver disease and the like, and for early detection of these diseases, an accurate and simple PC quantitative method is required. At present, several PC quantification methods are used, and one of them is the enzymatic activity measurement method. This assay method uses a synthetic peptide substrate specific for PCa. However, there are PC inhibitors in blood and interfering substances that act on synthetic substrates without being inhibited by inhibitors such as antithrombin III. The activity is difficult to quantify. Therefore, using plasma such as antibody column or adsorbent,
A pre-treatment of separating is required. However, these methods require a large amount of plasma to be used, are time-consuming and troublesome, and are disadvantageous when rapidly processing a large number of samples. Furthermore, the pretreatment using an adsorbent has a drawback in the recovery rate of PC, and when an antibody column is used, the recovery rate is good, but the column is very expensive. . In addition to using a synthetic substrate, there is a biological activity measurement method that utilizes the anticoagulant effect of PC. This method involves activating PC with snake venom and then adding it to the plasma coagulation system to measure its coagulation time-prolonging action, but it cannot be said to be a highly sensitive method. There is also an immunological method for quantifying PC. This method is
A method for quantitatively quantifying PC by an antigen-antibody reaction using a polyclonal or monoclonal antibody against PC, a radioimmunoassay method, an enzyme immunoassay method,
The Laurell method etc. are implemented. Although the immunological quantification method can be quantitatively quantified, the antibody used is very expensive,
The operation time is long, and it cannot be confirmed by this method whether the PC has normal activity. As a result of intensive research to solve the problems of the conventional PC quantification method as described above, the present inventors have no pretreatment for separating PC from plasma, are easy to operate, and can handle a large amount of sample in a short time. The present invention has been completed by finding out a method for measuring an enzymatic activity capable of measuring the. (Problems to be Solved by the Invention) An object of the present invention is to provide a novel and useful PC assay method. (Means for Solving the Problems) The present invention is characterized by specifically inhibiting the action of an interfering substance on a substrate in a method of enzymatically measuring PC activity using a synthetic peptide substrate. This is a quantitative method for PC. That is, the present invention relates to PCa acting on a synthetic peptide substrate.
By specifically inhibiting the interfering substances in plasma other than PC, the activity of PCa in plasma could be directly measured without separating PC from plasma. The PC quantification method of the present invention can be performed as follows. PC by diluting plasma and adding PC activator
Activate. After completion of PC activation, an inhibitor against an interfering substance in plasma other than PCa that acts on the synthetic substrate is added. PC is quantified by adding a synthetic peptide substrate and reacting, and measuring the color development or fluorescence of the degradation product by PCa. The quantification method of the present invention described above will be described in more detail. 1) Plasma Dilution Ratio Since there are PC inhibitors that inhibit PCa in plasma, PCa activity cannot be accurately measured without any treatment. In the quantification method of the present invention, the effect of the PC inhibitor could be ignored by diluting the plasma about 10 times or more. The plasma may be prepared by a conventional method, and for example, citrated plasma obtained by collecting blood in the presence of citric acid and then centrifuging may be used. 2) Buffer solution As a buffer solution for diluting plasma, it is preferable to use a buffer solution prepared by a buffering agent such as Tris-hydrochloric acid to have a pH close to in-vivo conditions. Similar to the commonly used buffer, salts such as sodium chloride may be added in order to match the in vivo conditions. In order to suppress the action of various proteases in plasma, protease inhibitors such as soybean trypsin inhibitor (SBTI) and aprotinin can be added to the extent that PCa activity is not affected. Further, a stabilizer such as bovine serum albumin (BSA) may be added to prevent precipitation of insoluble protein under weak acidity when the final reaction is stopped. 3) PC Activator Examples of the PC activator include thrombin-thrombomodulin complex and snake venom. Considering the original physiological conditions, the thrombin-thrombomodulin complex is preferable. Thrombin and thrombomodulin may be added to the reaction system after forming a complex, or may be added individually. In addition, since Ca ²⁺ ions are required for activation of PC, the optimum concentration should be set in the reaction system for PC activation.
Ca ²⁺ ions are preferably added to the plasma dilution buffer or the solution of the PC activator. 4) Inhibitors of interfering substances Thrombin added to activate PC acts on the synthetic substrate used to measure PC activity and interferes with the measurement. Inhibitors need to be added. Although this inhibitor inhibits thrombin, it is available that has no effect on PCa,
Antithrombin III or the like can be used. Since the antithrombin III inhibitory reaction is significantly promoted by the presence of heparin, coexistence of heparin is practical and preferable. However, antithrombin III alone cannot suppress the activity of other interfering substances. In fact, as a result of measuring the PC activity using a synthetic substrate only by adding antithrombin III-heparin, the activity was several hundred times that of the PC amount by the immunological assay, Was measured. As described above, when the PC activity is measured by adding only antithrombin III, there is a problem that the activity of PCa cannot be accurately measured due to the existence of a substance acting on the synthetic substrate other than PC, and thus, conventionally, the column etc. It required complicated processing such as separating the PC at. The present inventor, as a result of various studies of methods for suppressing the activity of interfering substances acting on a synthetic substrate used for measuring PC activity, a substance having a low molecular weight, not a substance having a large molecular weight such as antithrombin III, By using a thrombin inhibitor, it was clarified that the activity of interfering substances other than thrombin was suppressed, and we succeeded in measuring only PCa activity without the need for operations such as separation and purification of PC. Low molecular weight thrombin inhibitors that can be used in the present invention, it is preferable to use those that do not affect the activity of PCa, a thrombin inhibitor having a molecular weight of 2,000 or less, preferably a substance having a molecular weight of 1,000 or less, for example, (2R, 4R ) −
1- [N ² - (3- methyl-1,2,3,4-tetrahydro -8
-Quinolinesulfonyl) -L-arginyl] -4-methyl-2-piperidinecarboxylic acid (Compound 1), dansylarginine N- (3-ethyl-1,5-pentanediyl) amide (Compound 2) and other N-arylsulfonyls −
L-arginine derivative [J. Med. Chem., 23, 827-836, 12
93-1299, (1980), etc.], N- (2-naphthylulphonylglycyl) -4-amidinophenylalanine peperidino (Compound 3) and other N-arylsulfonylglycyl-amidinophenylalanine derivatives (Thromb. Res. , 36, No.5,
457-165 (1984) etc.] and the like. 5) Synthetic peptide substrate As a synthetic peptide substrate for measuring PC activity, those used in conventional enzymatic activity measuring methods can be used. For example, pGlu-Pro-Arg-pNA (S-2366). : Pyroglutamyl-prolyl-arginyl-p-nitroanilide), D-Val-Leu-Arg-pNA (S-2266: D-valyl-leucyl-arginyl-p-nitroanilide), D-
Phe-Pip-Arg-pNA (S-2238: D-phenylalanyl-
Colorimetric synthetic substrates such as piperidino-arginyl-p-nitroanilide) and Boc-Leu-Ser-Thr-Arg-MCA (3112-V:
t-butoxycarbonylleucyl-seryl-threonyl-arginyl-4-methylcoumarinamide) and the like. Since the above-mentioned synthetic peptide substrate is decomposed by PCa to produce a chromogenic or fluorescent substance, PC activity can be quantitatively determined by colorimetric or fluorescent quantification of these degradation products. (Example) -Operation- Human plasma 10μ buffer 140μ Thrombin-thrombomodulin complex 50μ-Human plasma: human citrate plasma-Buffer: 50mM Tris-HCl (pH8.0), 0.1M sodium chloride, 1mM calcium chloride, 25U / ml aprotinin / thrombin-thrombomodulin complex: 20 U / ml thrombin, 1.7 nmol / ml thrombomodulin The reaction solution having the above composition was reacted at 37 ° C for 30 minutes. -Operation- 25 U / ml antithrombin III 100 U / ml heparin 100 μg / ml Compound 1 100 μl of the mixed solution having the above composition was added to the reaction solution of the operation and reacted at 37 ° C. for 10 minutes. -Operation- 100 mM 3 mM solution of the synthetic substrate S-2366 is added to the reaction solution of the operation.
After the reaction was carried out at 37 ° C. for 20 minutes, 100 μ of 50% acetic acid was added to stop the reaction, and colorimetry was performed at 450 nm. A dilution calibration curve of this example was prepared using normal human pool plasma and PC-depleted plasma, and this is shown in FIG. As is clear from FIG. 1, the calibration curve shows a good linearity in the range of 0 to 100%, and the PC quantification method of the present invention has excellent quantification. Next, the results of examining various conditions for the quantification method of the present invention will be shown. The basic operation method was performed according to the method of the above-described embodiment. Plasma Dilution Rate As a result of examining the effect of PC inhibitor and plasma dilution rate,
In the assay method of the present invention, a linear relationship was obtained between the dilution rate and the PC activity by diluting plasma approximately 10 times or more.
As the dilution rate becomes lower than this, the action of the PC inhibitor is further expressed, and the PC activity is suppressed, resulting in a shift from the linear relationship, which is not preferable. Therefore, in the example of the present invention, a buffer solution was added to plasma 10 μm.
140 μm was added and diluted 15-fold for use. Ca ²⁺ ion concentration As a result of examining the optimum concentration of Ca ²⁺ ions during PC activation,
Ca ²⁺ ion has an optimal concentration of 0.1 to 4 mM, 0.5 to 2.5
mM is more preferred. Addition amount of thrombin In the reaction system of this example, PC was able to be activated almost 100% by adding 1 U or more of thrombin. In order to reduce the amount of antithrombin III-heparin added after PC activation as much as possible, the amount of thrombin added is 1 U in this example. Thrombomodulin addition amount As a result of variously changing the addition amount of thrombomodulin, in this example, PC activation was sufficiently performed with an addition amount of 0.04 nmol or more, and it is preferable to perform it with 0.06 to 0.16 nmol. Addition amount of compound 1 As a result of examining variously changing the concentration of compound 1 in the mixed solution added in the operation of this example, when the concentration of compound 1 in the mixed solution was about 75 μg / ml or more, it acted on the synthetic substrate S-2366. It was able to inhibit the interfering substances other than PCa that had a significant effect. In addition, the inhibitory effect of Compound 1 on PCa was examined using separated and purified PC, and it was found that 150 μg / ml in the mixed solution of the operation.
Since the PC activity is inhibited by the above, it is preferable to use it at a concentration lower than that in this example. As a result of using Compound 2 instead of Compound 1, the present invention could be carried out using a solution having the same concentration as that of Compound 1. In the case of Compound 3, the solution concentration is about 25 μg / ml.
It was possible to suppress the activity of interfering substances. Although the quantification method of the present invention has been described in detail by the above examples, for example, the following kit for protein C activity measurement can be prepared based on the essential requirements characterized by this method. -Kit Components-a) Protein C Activator b) Antithrombin III c) Low Molecular Weight Thrombin Inhibitor d) Synthetic Peptide Substrate A more specific example of the kit is shown. The amounts in each vial are shown. a) Thrombin-thrombomodulin complex (freeze-dried product) 10U / ml thrombin-0.85n mol / ml thrombomodulin-Dissolved in 4ml of distilled water for use. b) Antithrombin III-heparin complex (lyophilized product) 25 U / ml antithrombin III-100 U / ml heparin. c) Compound 1 aqueous solution 5 ml (100 μg / ml) d) Synthetic substrate S-2366 (freeze-dried product) -Dissolved in 4 ml of distilled water for use (3 mM aqueous solution). The kit comprising the above-mentioned components includes, for example, 50 ml of a buffer for plasma dilution (50 mM Tris-HCl (pH 8.0) -0.1 M sodium chloride-1 mM calcium chloride-0.1% BSA-0.1 mg / ml SBT.
I) or a reaction terminator 21 ml (2% citric acid solution) may be added. The operation of this kit can be performed easily and in a short time as follows. The test plasma is diluted 21-fold with the plasma dilution buffer. This solution is incubated at 56 ° C. for 5 minutes, the produced fibrin is removed by centrifugation, and 200 μm is collected. 100 μm of thrombin-thrombomodulin complex solution
In addition, incubate at 37 ° C for 30 minutes. Add 100 μl of the mixed solution of antithrombin III-heparin-compound 1 and incubate at 37 ° C. for 5 minutes. Add 100 µ of S-2366 solution and incubate at 37 ° C for 30 minutes. Add 500 μ of 2% citric acid solution and measure the absorbance at 405 nm. About 40 samples can be measured with the kit of 1
Various kits suitable for the purpose can be prepared by changing the content of components per vial, the dilution rate of plasma, etc., or by appropriately changing the scale of the system. (Effect) Quantitative method of the present invention and immunological quantification method [EIA method: JC Giddings
et al, Brit. J. Haematol., 52, 495-502 (1982)] is used to quantify and compare the PC in plasma of healthy subjects, liver disease patients and DIC patients. The PC activity in the assay method of the present invention was determined using the calibration curve in FIG. 1 and expressed as a percentage. From FIG. 2, it is clearly shown that there is a good correlation between the PC activity by the assay method of the present invention and the amount of PC antigen by the immunological assay method. Moreover, this correlation is well suited not only for healthy individuals but also for patients with liver diseases and DIC patients, who are considered to have low plasma PC content. (Effect) According to the PC assay method of the present invention, it is possible to perform the assay using plasma itself without the pretreatment of separation and purification of PC, which is indispensable in the conventional method for measuring enzymatic activity. became. As is clear from the above-mentioned test results, the assay method of the present invention uses only 10μ of plasma as a sample, and it is possible to quantify PC in the plasma by a short-time operation, and the operation is very simple. is there. Further, in the comparison test with the immunological quantification method, it is clear that the PC activity quantified by this method has a good correlation with the amount of PC antigen, and thus the method of the present invention is extremely excellent in quantification. You can see that Thus, the PC quantification method of the present invention is much simpler, more accurate, and faster than the conventional quantification methods, and is particularly useful for treating a large amount of sample. Therefore, the assay method of the present invention is easy to prepare as a kit, and is a highly useful assay method for PC for diagnosis of PC deficiency, DIC, liver disease and the like.

BRIEF DESCRIPTION OF THE DRAWINGS 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 assay method of the present invention and the immunoassay method (EIA method).

Claims (1)

(57) [Claims] In a method for activating protein C in plasma by thrombin and thrombomodulin and measuring protein C activity using a synthetic peptide substrate, antithrombin III and low A method for quantifying protein C using a molecular weight thrombin inhibitor. 2. Thrombin and thrombomodulin are added to activate protein C in plasma and then antithrombin
The protein C activity according to claim 1, wherein the activity of the interfering substance on the synthetic peptide substrate is specifically inhibited by adding III and a low molecular weight thrombin inhibitor, and then the protein C activity is measured using the synthetic peptide substrate. Quantitative method. 3. The low molecular weight thrombin inhibitor is an N-arylsulfonyl-L-arginine derivative.
Item 2. A method for quantifying protein C according to item 2 or item 2. 4. Low molecular weight thrombin inhibitors are (2R, 4R) -1- [N
^2- (3-Methyl-1,2,3,4-tetrahydro-8-quinolinesulfonyl) -L-arginyl] -4-methyl-2-
The method for quantifying protein C according to claim 3, which is piperidinecarboxylic acid. 5. The method for quantifying protein C according to claim 3, wherein the low-molecular weight thrombin inhibitor is dansylarginine N- (3-ethyl-1,5-pentanediyl) amide. 6. The method for quantifying protein C according to claim 1 or 2, wherein the low molecular weight thrombin inhibitor is an N-arylsulfonylglycyl-amidinophenylalanine derivative. 7. The method for quantifying protein C according to claim 6, wherein the low-molecular-weight thrombin inhibitor is N- (2-naphthylulphonylglycyl) -4-amidinophenylalanine peperidino. 8. The method for quantifying protein C according to any one of claims 1 to 7, wherein heparin is used together with antithrombin III.