JP2009198506A - Activity measuring method and activity measuring reagent for blood-clotting reaction inhibitory plasma protein - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an activity measuring method and an activity measuring reagent for blood-clotting reaction inhibitory plasma protein in a sample. <P>SOLUTION: The activity measuring method for blood-clotting reaction inhibitory plasma protein in the sample obtains an activity value of blood-clotting reaction inhibitory plasma protein in the sample by (a) bringing sample-underived activated protein C or protein C activator, phospholipid, calcium ion, activated blood coagulation factor V, activated blood coagulation factor X, prothrombin and thrombin substrate into contact with the sample, (b) measuring a signal amount produced from the thrombin substrate as the result of reaction by each component in (a), and (c) determining the signal amount suppressed in formation according to the activity of the blood-clotting reaction inhibitory plasma protein contained in the sample, thus acquiring the activity value of the blood-clotting reaction inhibitory plasma protein contained in the sample. The activity measuring reagent for blood-clotting reaction inhibitory plasma protein in the sample contains the activated protein C or protein C activator, phospholipid, calcium ion, activated blood coagulation factor V, activated blood coagulation factor X, prothrombin and thrombin substrate. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

In the present invention, the measurement value is not affected by the abundance (concentration) of the component (factor) involved in the blood coagulation reaction (concentration) in the sample or its variation, and can be measured with high sensitivity. The present invention relates to a method and a reagent for measuring the activity of a blood coagulation reaction-inhibited plasma protein.
The present invention is particularly useful in the field of life science such as clinical examination, molecular biology, and medicine.

  A schematic diagram of the control mechanism of the blood coagulation system in the living body is shown below.

  In this in vivo blood coagulation system control mechanism, protein C, protein S, and the like are known as plasma proteins that suppress blood coagulation reaction, that is, blood coagulation reaction suppression plasma proteins.

In the living body, protein C is limitedly decomposed by the thrombin / thrombomodulin complex and activated by releasing the activated peptide to become activated protein C.
Activated protein C is a serine protease that plays a role in suppressing blood coagulation by degrading activated blood coagulation factor V and activated blood coagulation factor VIII that promote blood coagulation in humans.
Protein S is a prosthetic factor (cofactor) of activated protein C. Due to the presence of protein S, activated blood coagulation factor V decomposition reaction and activated blood coagulation factor VIII decomposition reaction by activated protein C. Is promoted.

A decrease or deficiency in the activity of blood coagulation reaction-inhibited plasma proteins such as protein C and protein S, which have a function of suppressing the blood coagulation reaction, can cause thrombosis in vivo.
In fact, people with congenital deficiency of blood coagulation reaction-suppressing plasma protein frequently have deep vein thrombosis, venous thrombosis such as superficial phlebitis or pulmonary infarction, or coronary artery thrombosis causing heart failure This will cause arterial thrombosis of the patient.
In addition, in disseminated intravascular coagulation syndrome (DIC), vitamin K deficiency or hepatic hypofunction, a decrease or deficiency in the activity of blood coagulation reaction-inhibited plasma proteins is also observed.
That is, by measuring the activity of the blood coagulation reaction-suppressing plasma protein in a sample such as plasma, it is possible to grasp the decrease or deficiency in the activity of the blood coagulation reaction-suppressing plasma protein. It plays an important role in predicting the onset, early detection, and determining therapeutic effects.

  As a method of measuring the activity of protein C or protein S in a sample, the sample is incubated with protein C-activator from snake venom under the formation of activated protein C or protein S, and blood coagulation factor XII Adding a blood coagulation factor VII or blood coagulation factor II (prothrombin) activator and reducing the formation of thrombin from prothrombin mediated by the blood coagulation factor and its activator, a chromogenic thrombin substrate Has disclosed a method for photometric measurement using a laser beam (see Patent Document 1).

  As a method for measuring the protein S activity in another sample, a protein C activator or protein C activating protein C is added to a plasma sample, and then activated blood coagulation factor IX is added and incubated. Then, the amount of thrombin produced is measured by a known method, and this is compared with the value obtained by measuring a sample with known protein S activity to measure the activity of protein S in the sample. It is disclosed (see Patent Document 2).

  In addition, as a method for measuring the activity of protein C in other samples, a protein C activator or protein S that activates protein C is added to a plasma sample, and then activated blood coagulation factor IX is added and incubated. Then, the amount of thrombin produced is measured by a known method, and this is compared with the value obtained by measuring a sample with known protein C activity to measure the activity of protein C in the sample. It is disclosed (see Patent Document 2).

  Further, as a method of measuring the activity of protein S in another sample, the sample is incubated with activated protein C, blood coagulation factor VIII, phospholipids and calcium ions, and then this mixture is activated with blood coagulation. Incubate with factor II (thrombin), activated blood coagulation factor IX and activated blood coagulation factor X, then add activated blood coagulation factor X specific substrate to the incubation mixture and cleave this substrate Has disclosed a method for measuring the activity of protein S in a sample, which comprises measuring the amount of signal generated by (see Patent Document 3).

  In these conventional methods for measuring the activity of blood coagulation reaction-inhibited plasma proteins and reagents for measuring activity in these samples, at least one of the components (factors) of the blood coagulation reaction used in the measurement reaction is a component contained in the sample. (Factor) is used as it is. In other words, it depends on the components contained in the sample.

However, when a component (factor) contained in this sample is used for a measurement reaction, when that component (factor) [eg, activated blood coagulation factor X] is deficient or decreased in the sample provider, Since at least one of the reaction components (factors) of the measurement reaction does not exist in a sufficient amount, the measurement reaction does not proceed sufficiently, and the obtained measurement value (activity value of blood coagulation reaction-suppressed plasma protein) may be It is far from the original value and includes errors.
That is, the measured value may be influenced by the amount (concentration) in the sample of the component (factor) involved in the blood coagulation reaction or its variation.
This is because a patient who is trying to measure blood coagulation reaction-suppressed plasma protein activity is likely to have an abnormality in the blood coagulation / fibrinolysis system, and thus the reaction component (factor) is deficient or decreased. Because the possibilities are great, the above problems are not uncommon.

  Further, the conventional methods for measuring the activity of blood coagulation reaction-suppressed plasma proteins and reagents for measuring blood coagulation in a sample are complicated because the measurement reaction undergoes many reactions, and many components are required for the measurement. . For this reason, this measurement reaction was easily influenced by various factors and various conditions.

  Furthermore, the conventional methods for measuring the activity of blood coagulation reaction-suppressed plasma proteins and reagents for measuring activity in samples cannot be said to have sufficiently high sensitivity for measurement.

JP-A-62-212569 Japanese National Patent Publication No. 4-506603 JP-T 6-504682

  The problem of the present invention is that the measurement value is not affected by the abundance (concentration) in the sample of the component (factor) involved in the blood coagulation reaction or its variation, and the number and measurement of reactions used in the measurement reaction. The blood coagulation reaction-suppressed plasma in a sample is simpler, requires fewer components, is less susceptible to various factors and conditions, and provides high measurement sensitivity. It is to provide a protein activity measuring method and an activity measuring reagent.

The present invention includes the following inventions.
(1) A method for measuring an activity value of a blood coagulation reaction-suppressing plasma protein in a sample,
(A) Contacting a sample with activated protein C or a protein C activator, phospholipid, calcium ion, activated blood coagulation factor V, activated blood coagulation factor X, prothrombin and thrombin substrate not derived from the sample Let
(B) measuring the amount of signal generated from the substrate of thrombin as a result of the reaction by each component of (a),
(C) determining the amount of signal whose production is suppressed according to the activity of the blood coagulation reaction suppression plasma protein contained in the sample;
The blood coagulation reaction suppression plasma protein activity measuring method in the sample which obtains the activity value of the blood coagulation reaction suppression plasma protein contained in the sample.
(2) Consists of activated protein C or protein C activator, phospholipid, calcium ion, activated blood coagulation factor V, activated blood coagulation factor X, prothrombin and thrombin substrate not derived from the sample A method for measuring an activity value of a blood coagulation reaction-suppressing plasma protein in a sample using a reaction system,
(A) contacting the sample with at least activated protein C or protein C activator;
(B) When the sample contains blood coagulation reaction-suppressed plasma protein, the activated blood coagulation factor V degradation reaction catalyzed by the activated protein C produced or further activated in the presence of phospholipids and calcium ions Promoted,
(C) The reaction to generate thrombin from prothrombin catalyzed by activated blood coagulation factor X in the presence of phospholipids and calcium ions, promoted by activated blood coagulation factor V, is activated in (b) above. Suppressed by promoting the degradation reaction of blood coagulation factor V, reducing the production of thrombin,
(D) By reducing the generation of thrombin in (c), the reaction that generates a signal from the thrombin substrate catalyzed by thrombin is suppressed,
(E) measuring the amount of signal whose production is suppressed by the suppression of the reaction in (d),
Thus, the method for measuring the activity of the blood coagulation reaction-suppressing plasma protein in the sample according to (1), wherein the activity value of the blood coagulation reaction-suppressing plasma protein contained in the sample is obtained.
(3) The method for measuring the activity of the blood coagulation reaction-suppressed plasma protein in the sample according to (1) or (2) above, wherein the signal amount is measured by measuring absorbance, transmittance, or fluorescence intensity.
(4) The numerical value of the amount of change per unit time of the measured signal amount, the numerical value calculated by first derivative of the measured signal amount value with respect to time, or the numerical value calculated by secondary differentiation, The activity value of the blood coagulation reaction-suppressing plasma protein contained in the sample is obtained using a calibration curve representing the relationship with the activity value of the blood coagulation reaction-suppressing plasma protein in the sample. The method for measuring the activity of a blood coagulation reaction-suppressed plasma protein in any one of the samples.
(5) The amount of change per minute in absorbance obtained by measurement, or the rate of change in absorbance obtained by first derivative of the absorbance obtained by measurement with respect to time, or the absorbance obtained by measurement in time The blood coagulation reaction-suppressing plasma protein contained in the sample using a calibration curve representing the relationship between the acceleration of the absorbance change obtained by second-order differentiation and the activity value of the blood coagulation reaction-suppressing plasma protein in the sample The activity measurement method of the blood coagulation reaction suppression plasma protein in the sample in any one of said (1)-(4) which obtains the activity value of.
(6) Blood coagulation reaction in a sample containing activated protein C or a protein C activator, phospholipid, calcium ion, activated blood coagulation factor V, activated blood coagulation factor X, prothrombin and thrombin substrate Reagent for measuring inhibitory plasma protein activity.
(7) containing activated protein C or a protein C activator, phospholipid, calcium ion, activated blood coagulation factor V, activated blood coagulation factor X, prothrombin and thrombin substrates, The reagent for measuring the activity of blood coagulation reaction-suppressed plasma protein in a sample, which is used in the method for measuring the activity of blood coagulation reaction-suppressing plasma protein in a sample according to any one of (5).

  The blood coagulation reaction-suppressing plasma protein activity measuring method and activity measuring reagent in the sample of the present invention are obtained by measurement based on the abundance (concentration) of the component (factor) involved in the blood coagulation reaction in the sample or by variation thereof The activity value of the blood coagulation reaction-suppressed plasma protein is not affected. Further, the number of reactions used for the measurement reaction and the number of components necessary for the measurement are not large, and the reaction is simpler and hardly affected by various factors and various conditions. In addition, by finding the optimum conditions for the measurement reaction of the blood coagulation reaction-suppressed plasma protein in the sample, the measurement reaction can be promoted and high measurement sensitivity can be obtained.

It is the figure which showed the change by the time of the light absorbency in wavelength 405nm when three types of samples whose protein S activity value is known are measured.

It is the figure which showed the relationship between the inclination of the light-absorbance change of three types of samples with which protein S activity value is known, and time.

It is the figure which showed the calibration curve in the measurement of the protein S activity in a sample.

It is the figure which showed the effect of the composition of the phospholipid in the catalytic reaction of activated blood coagulation factor X.

1. General The method for measuring the activity of the blood coagulation reaction-suppressing plasma protein in the sample of the present invention comprises:
(A) Contacting a sample with activated protein C or a protein C activator, phospholipid, calcium ion, activated blood coagulation factor V, activated blood coagulation factor X, prothrombin and thrombin substrate not derived from the sample Let
(B) measuring the amount of signal generated from the substrate of thrombin as a result of the reaction by each component of (a),
(C) determining the amount of signal whose production is suppressed according to the activity of the blood coagulation reaction suppression plasma protein contained in the sample;
By this, it is the measuring method which obtains the activity value of the blood coagulation reaction suppression plasma protein contained in the sample.

  Here, “not derived from the sample” means that the sample is not contained in the sample, and is necessary for the measurement reaction in the blood coagulation reaction-suppressing plasma protein activity measurement method in the sample of the present invention. Components (factors) [activated protein C or protein C activator, phospholipid, calcium ion, activated blood coagulation factor V, activated blood coagulation factor X, prothrombin and thrombin substrate] It is not used depending on what is contained, but a sufficient amount of each component (factor) is used in the measurement reaction system by adding something other than that contained in the sample.

  Thereby, for example, even in a patient sample in which any of the above components (factors) is deficient or reduced, the measurement reaction proceeds without being affected by the deficiency or reduction of the components (factors). Thus, an accurate blood coagulation reaction-suppressing plasma protein activity value can be obtained.

  However, in the activity measurement method and the activity measurement reagent of the present invention, even if the components contained in the sample work in the measurement reaction, it is not a problem in the present invention and does not depart from the spirit of the present invention. Absent.

  In the present invention, each component (factor) necessary for the measurement reaction is present in an amount sufficient to cause the measurement reaction to proceed, so that it is not affected by the amount of the component (factor) contained in the sample. Even if the component (factor) contained in the sample is further worked, there is no problem in the measurement reaction, and it does not differ from the gist of the present invention.

Hereinafter, the method for measuring the activity of the blood coagulation reaction-suppressing plasma protein and the reagent for measuring the activity in the sample of the present invention will be described more specifically.
In the method for measuring the activity of blood coagulation reaction-suppressed plasma protein in the sample of the present invention,
(A) Activated protein C or protein C activator, phospholipid, calcium ion, activated blood coagulation factor V (Va), activated blood coagulation factor X (Xa) not derived from the sample as described above Prothrombin and thrombin substrate are contacted with the sample.
In addition, the contact between these components and the sample may be performed in one step by making all the components contact at one time and performing all the reactions of the reaction system composed of these components in one step (1 step). Method), the reaction of a reaction system composed of these components may be performed in a plurality of stages by dividing the components and performing the contact in a plurality of stages (multi-step method).

(B) Due to the contact in (a), the reaction of the reaction system composed of the above components proceeds.
Then, the amount of signal generated from the thrombin substrate by a series of reactions in this reaction system is measured.
That is, the thrombin substrate is subjected to an action such as degradation by thrombin by the series of reactions described above, and the amount of signal (for example, absorbance etc.) generated as a result is measured.

(C) If the sample contains blood coagulation reaction-suppressed plasma protein, activated protein C is generated or further activated in the above reaction, thereby promoting the degradation of activated blood coagulation factor V As a result, the reaction that generates a signal from the substrate of thrombin catalyzed by thrombin is suppressed, and the generation of the signal is suppressed.
The degree of inhibition of this signal generation increases according to the activity value of the blood coagulation reaction-suppressed plasma protein contained in the sample.
Therefore, by measuring the amount of signal in (b) above, the amount of signal whose production is suppressed is measured, thereby obtaining the activity value of the blood coagulation reaction-suppressed plasma protein contained in the sample. Can do.

The method for measuring the activity of blood coagulation reaction-suppressed plasma protein in the sample of the present invention will be described more specifically.
The method for measuring the activity of the blood coagulation reaction-suppressing plasma protein in the sample of the present invention is an activated protein C or protein C activator, phospholipid, calcium ion, activated blood coagulation factor V (Va) not derived from the sample. A method for measuring an activity value of a blood coagulation reaction-suppressing plasma protein in a sample using a reaction system comprising activated blood coagulation factor X (Xa), prothrombin and a substrate of thrombin,
(A) The sample is brought into contact with at least activated protein C or a protein C activator.

(B) When the sample contains a blood coagulation reaction-inhibited plasma protein, the activated protein C is generated or further activated so that the activated protein C catalyzes in the presence of phospholipids and calcium ions. The degradation reaction of blood coagulation factor V is promoted.

(C) The reaction of generating thrombin from prothrombin catalyzed by activated blood coagulation factor X in the presence of phospholipids and calcium ions, promoted by activated blood coagulation factor V, is activated in (b) above. Stimulation of blood coagulation factor V is promoted to suppress thrombin generation.

(D) The reaction that generates a signal from the substrate of thrombin catalyzed by thrombin is suppressed by reducing the generation of thrombin in (c).
The degree of inhibition of this signal generation increases according to the activity of the blood coagulation reaction-suppressed plasma protein contained in the sample.

(E) The amount of signal whose production is suppressed by the suppression of the reaction in (d) is measured.
As described above, by measuring the amount of signal generated by the above reaction, that is, by measuring the amount of signal with suppressed generation, the activity value of the blood coagulation reaction-suppressed plasma protein contained in the sample can be obtained. .

The method for measuring the activity of blood coagulation reaction-suppressed plasma protein in the sample of the present invention will be described below, taking the case where the blood coagulation reaction-suppressed plasma protein is protein S as an example.
When the blood coagulation reaction-suppressed plasma protein is protein S, it is not derived from a sample, but is activated protein C, phospholipid, calcium ion, activated blood coagulation factor V (Va), activated blood coagulation factor X (Xa) Using the reaction system composed of prothrombin and the thrombin substrate, the activity value of the blood coagulation reaction-suppressing plasma protein (protein S) in the sample is measured as follows using the following reaction system.

(A) The sample is brought into contact with at least activated protein C and phospholipid.
Thereby, when protein S is contained in the sample, activated protein C is further activated in the presence of phospholipid.

(B) Further activation of activated protein C in the above (A) promotes the degradation reaction of activated blood coagulation factor V that activated protein C catalyzes in the presence of phospholipids and calcium ions.

(C) The reaction of generating thrombin from prothrombin catalyzed by activated blood coagulation factor X in the presence of phospholipid and calcium ions, promoted by activated blood coagulation factor V, is activated in the above (B) Stimulation of blood coagulation factor V is promoted to suppress thrombin generation.

(D) The reduction in the generation of thrombin in (C) suppresses the reaction that generates a signal from the thrombin substrate catalyzed by thrombin.
The degree of inhibition of this signal generation increases according to the activity of protein S contained in the sample.

(E) The amount of signal whose production is suppressed by the suppression of the reaction in (D) is measured.
As described above, the activity value of protein S contained in the sample can be obtained by measuring the signal amount generated by the above reaction, that is, by measuring the signal amount in which the generation is suppressed.

Further, the method for measuring the activity of the blood coagulation reaction-suppressed plasma protein in the sample of the present invention will be described below, taking as an example the case where the blood coagulation reaction-suppressed plasma protein is protein C.
When the blood coagulation reaction-suppressing plasma protein is protein C, the protein C activator, phospholipid, calcium ion, activated blood coagulation factor V (Va), activated blood coagulation factor X (Xa) not derived from the sample ), The activity value of blood coagulation reaction-suppressed plasma protein (protein C) in the sample is measured as follows using a reaction system composed of prothrombin and a substrate of thrombin, using the following reaction system.

(I) A sample is brought into contact with at least a protein C activator. Thereby, when protein C is contained in a sample, protein C is activated and activated protein C is generated.

(Ii) The activated blood coagulation factor V decomposition reaction catalyzed by the activated protein C produced in (i) in the presence of phospholipids and calcium ions proceeds.

(Iii) The reaction in which thrombin is generated from prothrombin catalyzed by activated blood coagulation factor X in the presence of phospholipid and calcium ion, promoted by activated blood coagulation factor V, is activated in the above (ii) The production of thrombin is reduced by the suppression of the blood coagulation factor V degradation reaction.

(Iv) The reaction that generates a signal from the substrate of thrombin catalyzed by thrombin is suppressed by reducing the generation of thrombin in the above (iii).
The degree of inhibition of this signal generation increases according to the activity of protein C contained in the sample.

(V) A signal amount whose production is suppressed by the suppression of the reaction in (iv) is measured.
As described above, the amount of signal generated by the above reaction is measured, that is, the amount of signal in which production is suppressed is measured, thereby obtaining the activity value of protein C contained in the sample.

The reagent for measuring the activity of plasma protein for inhibiting blood coagulation reaction in the sample of the present invention is activated protein C or a protein C activator, phospholipid, calcium ion, activated blood coagulation factor V, activated blood coagulation factor X , Containing prothrombin and thrombin substrate.
You may contain components other than the said component as needed.
The activity measuring reagent of the present invention may be composed of a plurality of measuring reagents by separating the components to be contained.

The reagent for measuring the activity of blood coagulation reaction-suppressed plasma protein in the sample of the present invention will be described below by taking as an example the case where the blood coagulation reaction-suppressed plasma protein is protein S.
When the blood coagulation reaction-suppressed plasma protein is protein S, the reagent for measuring the activity of blood coagulation reaction-suppressed plasma protein (protein S) in the sample of the present invention is activated protein C, phospholipid, calcium ion, activated blood coagulation. It contains factor V, activated blood coagulation factor X, prothrombin and thrombin substrate.

In addition, the reagent for measuring the activity of the blood coagulation reaction-suppressed plasma protein in the sample of the present invention will be described below by taking as an example the case where the blood coagulation reaction-suppressed plasma protein is protein C.
When the blood coagulation reaction-suppressed plasma protein is protein C, the reagent for measuring the activity of the blood coagulation reaction-suppressed plasma protein (protein C) in the sample of the present invention is a protein C activator, phospholipid, calcium ion, activated blood. Coagulation factor V, activated blood coagulation factor X, prothrombin and thrombin substrate.

2. Sample In the blood coagulation reaction-suppressing plasma protein activity measurement method and activity measurement reagent in the sample of the present invention, the sample is a substance that may contain blood coagulation reaction-suppressing plasma protein such as protein S or protein C. .

  Examples of this sample include biological samples (samples derived from humans or animals).

  Examples of biological samples include blood, plasma, saliva, sweat, urine, tears, cerebrospinal fluid, ascites, amniotic fluid, and other biological fluids; liver, heart, brain, bone, hair, skin, nails, muscle, nerve tissue, etc. And extracts of organs, tissues, cells and the like.

3. Activated protein C
The activated protein C used in the activity measurement method and activity measurement reagent of the present invention can be used without particular limitation regardless of its origin (origin) and preparation method.
For example, the thing derived from mammals, such as a human, a cow, or a pig, etc. can be mentioned. Moreover, what was refine | purified and prepared from body fluids or organs, such as plasma, or what was prepared by genetic engineering operation, cell engineering operation, cell culture operation, etc. can be mentioned.
In addition, activated protein C may be generated from protein C using a protein C activator or the like during the measurement reaction in the activity measurement method and activity measurement reagent of the present invention and used as activated protein C in the present invention. Good.

In the activity measuring method and activity measuring reagent of the present invention, when activated protein C is brought into contact with the sample and protein S, which is a blood coagulation reaction-suppressing plasma protein, is contained in the sample, In the presence, its activity is further activated.
Activated protein C serves as a catalyst for a reaction that decomposes activated blood coagulation factor V in the presence of phospholipids and calcium ions.
Thus, the presence of protein S promotes the reaction of activated protein C degrading activated blood coagulation factor V.

Contacting the sample with activated protein C or the like and contacting activated protein C with activated blood coagulation factor V or the like may be performed simultaneously.
However, after contacting activated protein C with the phospholipid with the sample and incubating at least 1 minute, preferably 5 minutes or more, at room temperature or 37 ° C., etc., contact with activated blood coagulation factor V and calcium ions, Incubation is preferably performed to cause a degradation reaction of activated blood coagulation factor V.

When this activated protein C is used, the concentration is usually 1 pM to 100 nM when contacting activated blood coagulation factor V and degrading activated blood coagulation factor V in the presence of phospholipids and calcium ions. It is preferable that it exists in.
However, in order to obtain high measurement sensitivity, it is preferable that the activated protein C concentration is high. Therefore, the activated protein C concentration is more preferably 10 pM to 10 nM, and more preferably 100 pM to 1 nM. Particularly preferred.

Moreover, in the activity measuring reagent of this invention, it is preferable to make it contain in the activity measuring reagent so that the activated protein C may become the said density | concentration at the time of the said decomposition reaction.
For example, the activated protein C is preferably contained in an amount of 1 pM to 200 nM, more preferably 10 pM to 20 nM, and particularly preferably 100 pM to 2 nM.

4). Phospholipid used for catalytic reaction by activated protein C In the activity measuring method and activity measuring reagent of the present invention, further activity of activated protein C by protein S, which is a blood coagulation reaction-inhibited plasma protein, contained in the sample During activation and during the degradation reaction of activated blood coagulation factor V by this activated protein C, phospholipids necessary for the activation and degradation reaction are present.

This phospholipid can be used without particular limitation regardless of its origin (origin) and preparation method.
Examples thereof include those derived from mammals such as humans, cows and pigs, those derived from other animals, those derived from plants, those derived from microorganisms, and those synthesized artificially.
Examples of the phospholipid include glycerophospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, and diphosphatidylglycerol, and sphingophospholipids such as sphingomyelin.

As the phospholipid used at the time of further activation of the activated protein C and the decomposition reaction of activated blood coagulation factor V, it is possible to use phosphatidylcholine alone or a mixture of phosphatidylcholine and phosphatidylserine. The activation and decomposition reaction promotion effects are enhanced, which is preferable.
More preferably, the composition ratio of phosphatidylcholine is 85% to 70%, and the composition ratio of phosphatidylserine is 15% to 30%.
In particular, it is preferable that the composition ratio of phosphatidylcholine is 85% to 80% and the composition ratio of phosphatidylserine is 15% to 20%.

The concentration at the time of using this phospholipid is usually preferably 0.1 μM to 100 μM, and preferably 0.5 μM to 10 μM in the degradation reaction of activated blood coagulation factor V by activated protein C. It is particularly preferred.
And in the activity measuring reagent of this invention, it is preferable to make it contain in an activity measuring reagent so that a phospholipid may become the said density | concentration at the time of the said activation and degradation reaction.
For example, it is preferable to contain 0.1 μM to 200 μM of phospholipid, and it is particularly preferable to contain 0.5 μM to 20 μM.

5. Protein C Activating Substance The protein C activating substance used in the activity measurement method and activity measuring reagent of the present invention can be used without particular limitation as long as it can generate activated protein C from protein C.
Examples of the protein C activator include snake venom enzymes derived from Southern copperhead snacks, Mokasin snacks, and the like. Moreover, what was prepared by genetic engineering operation, cell engineering operation, or cell culture operation etc. can be mentioned.

In the activity measuring method and activity measuring reagent of the present invention, the protein C activator is brought into contact with the sample, so that protein C, which is a blood coagulation reaction-inhibited plasma protein, is contained in the sample. To activate activated protein C.
Activated protein C serves as a catalyst for a reaction that decomposes activated blood coagulation factor V in the presence of phospholipids and calcium ions.
Therefore, due to the presence of protein C in the sample, a reaction in which activated protein C decomposes activated blood coagulation factor V occurs and proceeds.

The contacting of the protein C activator and the sample with the activated blood coagulation factor V or the like may be simultaneously performed.
However, after contacting the protein C activator with the sample and incubating at room temperature or 37 ° C. for at least 1 minute, preferably 5 minutes or more, it is contacted with activated blood coagulation factor V, phospholipids and calcium ions. Incubation is preferably performed to cause a degradation reaction of activated blood coagulation factor V.

6). Protein S
In the activity measurement method and activity measurement reagent of the present invention, when the blood coagulation reaction-suppressed plasma protein is protein C, the produced activated protein C may be contacted with protein S in the presence of phospholipid, or the activity Protein S may be included in the measurement reagent.

Protein S used here can be used without particular limitation regardless of its origin (origin) and preparation method.
For example, the thing derived from mammals, such as a human, a cow, or a pig, etc. can be mentioned. Moreover, what was refine | purified and prepared from body fluids or organs, such as plasma, or what was prepared by genetic engineering operation, cell engineering operation, cell culture operation, etc. can be mentioned.

In the activity measurement method and activity measurement reagent of the present invention, protein S further activates activated protein C by contacting with the produced activated protein C in the presence of phospholipid.
Therefore, when the generated activated protein C and protein S are brought into contact with each other in the presence of phospholipid, the reaction in which activated protein C degrades activated blood coagulation factor V in the presence of phospholipid and calcium ions is Further promoted.

It should be noted that contacting activated protein C and protein S in the presence of phospholipid and contacting activated protein C with activated blood coagulation factor V or the like may be performed simultaneously.
However, activated protein C is brought into contact with protein S together with phospholipid, and is incubated for at least 1 minute or more, preferably 5 minutes or more at room temperature or 37 ° C., and then contacted with activated blood coagulation factor V and calcium ions. Incubation is preferably performed to cause a degradation reaction of activated blood coagulation factor V.

7). Calcium Ion In the activity measurement method and activity measurement reagent of the present invention, prothrombin by activated protein C during the degradation reaction of activated blood coagulation factor V and by activated blood coagulation factor X and activated blood coagulation factor V Calcium ions are present during the reaction to generate more thrombin.

  As this calcium ion, not only calcium ion itself but also a compound containing calcium ion such as calcium salt can be used without particular limitation.

  Examples of the compound containing calcium ions include calcium fluoride, calcium chloride, calcium bromide, calcium iodide, calcium sulfate, calcium nitrate, calcium acetate, calcium lactate, and calcium cyanide.

The concentration of this calcium ion is the same as that of the activated blood coagulation factor V decomposition reaction by activated protein C, and the generation of thrombin from prothrombin by activated blood coagulation factor X and activated blood coagulation factor V. In the reaction to be carried out, it is usually preferably 0.1 mM to 100 mM, particularly preferably 1 mM to 10 mM.
And in the activity measuring reagent of this invention, it is preferable to make it contain in an activity measuring reagent so that calcium ion may become the said density | concentration at the time of the said decomposition reaction and thrombin formation reaction.
For example, it is preferable to contain 0.1 mM to 200 mM of calcium ions or a compound containing calcium ions, and it is particularly preferable to contain 1 mM to 20 mM.

8). Activated blood coagulation factor V The activated blood coagulation factor V used in the activity measuring method and activity measuring reagent of the present invention can be used without particular limitation regardless of its origin (origin) and preparation method.
For example, the thing derived from mammals, such as a human, a cow, or a pig, etc. can be mentioned. Moreover, what was refine | purified and prepared from body fluids or organs, such as plasma, or what was prepared by genetic engineering operation, cell engineering operation, cell culture operation, etc. can be mentioned.

In the activity measurement method and activity measurement reagent of the present invention, activated blood coagulation factor V is degraded by activated protein C in the presence of phospholipids and calcium ions.
When the blood coagulation reaction-suppressing plasma protein is contained in the sample, activated protein C is generated or further activated, and this decomposition reaction is promoted.

  Activated blood coagulation factor V promotes three reactions that generate thrombin from prothrombin, which is catalyzed by activated blood coagulation factor X in the presence of phospholipids and calcium ions.

  Therefore, when the blood coagulation reaction-suppressing plasma protein is contained in the sample, the activated protein C is further activated, the decomposition reaction of the activated blood coagulation factor V is promoted, and the activated blood coagulation factor V is promoted. The abundance (concentration) of the factor is reduced. Then, since the effect of activating blood coagulation factor V to the reaction of generating thrombin from prothrombin catalyzed by activated blood coagulation factor X is reduced, the reaction of generating thrombin from prothrombin is suppressed.

Contacting the activated blood coagulation factor V with activated protein C and the like, and contacting the activated blood coagulation factor V with activated blood coagulation factor X and prothrombin, etc. are performed simultaneously. Also good.
However, after contacting activated blood coagulation factor V with activated protein C together with phospholipids and calcium ions and incubating at room temperature or 37 ° C. for at least 1 minute or more, preferably 5 minutes or more, the activated blood coagulation factor It is preferable to contact with factor X, phospholipid, prothrombin, etc. and incubate to carry out a reaction for generating thrombin from prothrombin.

The concentration at which this activated blood coagulation factor V is used is usually 0.5 pM when contacting activated protein C and degrading activated blood coagulation factor V in the presence of phospholipids and calcium ions. Preferably it is ˜5 nM.
However, when the concentration of this activated blood coagulation factor V is high, the blood coagulation reaction proceeds due to a component (factor) derived from the sample, and fibrin is precipitated or a great amount of color is produced, so that accurate measurement is possible. The concentration of the activated blood coagulation factor V is more preferably 5 pM to 500 pM, and particularly preferably 20 pM to 200 pM.

Moreover, in the activity measuring reagent of this invention, it is preferable to make it contain in an activity measuring reagent so that the activated blood coagulation factor V may become the said density | concentration at the time of the said decomposition reaction.
For example, the activated blood coagulation factor V is preferably contained at 0.5 pM to 10 nM, more preferably 5 pM to 1 nM, and particularly preferably 20 pM to 400 pM.

9. Activated blood coagulation factor X The activated blood coagulation factor X used in the activity measuring method and activity measuring reagent of the present invention can be used without particular limitation regardless of its origin (origin) and preparation method.
For example, the thing derived from mammals, such as a human, a cow, or a pig, etc. can be mentioned. Moreover, what was refine | purified and prepared from body fluids or organs, such as plasma, or what was prepared by genetic engineering operation, cell engineering operation, cell culture operation, etc. can be mentioned.

In the activity measurement method and activity measurement reagent of the present invention, activated blood coagulation factor X catalyzes a reaction for generating thrombin from prothrombin in the presence of phospholipid and calcium ion.
This reaction of generating thrombin from prothrombin by activated blood coagulation factor X is promoted by the presence of activated blood coagulation factor V.

  Therefore, as described above, when the blood coagulation reaction-suppressing plasma protein is contained in the sample, activated protein C is generated or further activated, thereby causing the degradation reaction of activated blood coagulation factor V. Because of this, the promoting effect of activated blood coagulation factor V on the reaction that generates thrombin catalyzed by activated blood coagulation factor X is reduced, so that the reaction that generates thrombin is suppressed.

When this activated blood coagulation factor X is used, the concentration is usually in contact with activated blood coagulation factor V, prothrombin, etc., and when thrombin is produced from prothrombin in the presence of phospholipid and calcium ion, It is preferable that it exists in 0.1 pM-300 pM.
However, if the concentration of this activated blood coagulation factor X is high, the blood coagulation reaction by the component (factor) derived from the sample proceeds as in the case of the above-mentioned activated blood coagulation factor V, and fibrin Or a large amount of color develops and accurate measurement cannot be performed. Therefore, the activated blood coagulation factor X concentration is more preferably 1 pM to 100 pM, and particularly preferably 10 pM to 50 pM. .

Moreover, in the activity measuring reagent of the present invention, it is preferable to contain the activated blood coagulation factor X in the activity measuring reagent so as to have the above concentration during the thrombin generation reaction.
For example, the activated blood coagulation factor X is preferably contained in an amount of 0.1 pM to 600 pM, more preferably 1 pM to 200 pM, and particularly preferably 10 pM to 100 pM.

10. Phospholipid used for catalytic reaction by activated blood coagulation factor X. In the activity measurement method and activity measurement reagent of the present invention, the reaction is carried out in the reaction of generating thrombin from prothrombin catalyzed by activated blood coagulation factor X. The necessary phospholipids are present.

This phospholipid can be used without particular limitation regardless of its origin (origin) and preparation method.
Examples thereof include those derived from mammals such as humans, cows and pigs, those derived from other animals, those derived from plants, those derived from microorganisms, and those synthesized artificially.
Examples of the phospholipid include glycerophospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, and diphosphatidylglycerol, and sphingophospholipids such as sphingomyelin.

As the phospholipid to be present in the reaction for generating thrombin from the prothrombin, it is preferable to use a mixture of phosphatidylethanolamine, phosphatidylserine and phosphatidylcholine because the effect of promoting the reaction is enhanced.
More preferably, the composition ratio of phosphatidylethanolamine is 40% or more and the composition ratio of phosphatidylserine is 25% or more.
In particular, it is preferable that the composition ratio of phosphatidylethanolamine is 50% or more and the composition ratio of phosphatidylserine is 30% or more.

The concentration at the time of using this phospholipid is usually preferably 0.1 μM to 100 μM, particularly preferably 0.5 μM to 10 μM, in the reaction for generating thrombin from prothrombin.
And in the activity measuring reagent of this invention, it is preferable to make it contain in an activity measuring reagent so that it may become the said density | concentration in the reaction which produces | generates thrombin from the said prothrombin.
For example, it is preferable to contain 0.1 μM to 200 μM of phospholipid, and it is particularly preferable to contain 0.5 μM to 20 μM.

As the phospholipid in the reaction for generating thrombin from prothrombin, the phospholipid used at the time of further activation of the activated protein C and the decomposition reaction of activated blood coagulation factor V should be used as it is. Can do.
However, as described above, the composition of a suitable phospholipid differs depending on each reaction, and therefore it is preferable to use a phospholipid having a composition suitable for each reaction in the presence of the reaction.
In the reaction for generating thrombin from this prothrombin, a phospholipid having a composition suitable for the further activation of the activated protein C and the decomposition reaction of the activated blood coagulation factor V previously added is obtained. Even if it coexists with a phospholipid having a composition suitable for the reaction for generating thrombin from prothrombin, there is no problem in the activity measurement method and activity measurement reagent of the present invention.

11. Prothrombin The prothrombin used in the activity measurement method and activity measurement reagent of the present invention can be used without particular limitation regardless of its origin (origin) and preparation method.
For example, the thing derived from mammals, such as a human, a cow, or a pig, etc. can be mentioned. Moreover, what was refine | purified and prepared from body fluids or organs, such as plasma, or what was prepared by genetic engineering operation, cell engineering operation, cell culture operation, etc. can be mentioned.

In the activity measurement method and activity measurement reagent of the present invention, prothrombin becomes a substrate for the reaction catalyzed by activated blood coagulation factor X in the presence of phospholipids and calcium ions, and becomes thrombin.
This reaction of generating thrombin from prothrombin by activated blood coagulation factor X is promoted by the presence of activated blood coagulation factor V.

  Therefore, as described above, when the blood coagulation reaction-suppressing plasma protein is contained in the sample, activated protein C is generated or further activated, thereby causing the degradation reaction of activated blood coagulation factor V. Therefore, the effect of activating blood coagulation factor V on the reaction of generating thrombin from prothrombin catalyzed by activated blood coagulation factor X is reduced, so that the reaction of generating thrombin from prothrombin is suppressed. The amount (concentration) of thrombin produced is reduced.

  When this prothrombin is used, the concentration when it is brought into contact with activated blood coagulation factor V to produce thrombin from prothrombin in the presence of phospholipids and calcium ions is usually preferably 1 nM to 50 μM, More preferred is 50 nM to 5 μM, and particularly preferred is 100 nM to 1 μM.

Moreover, in the activity measuring reagent of this invention, it is preferable to contain in the activity measuring reagent so that prothrombin may become the said density | concentration at the time of the said thrombin production reaction.
For example, prothrombin is preferably contained at 1 nM to 100 μM, more preferably 50 nM to 10 μM, and particularly preferably 100 nM to 2 μM.

12 Thrombin Substrate The thrombin substrate used in the activity measurement method and activity measurement reagent of the present invention is one that generates some signal upon receiving a catalytic action as thrombin protease (as a thrombin substrate), or in addition to the catalytic reaction by thrombin. Furthermore, any signal can be used without particular limitation as long as other signals are generated by continuing other reactions.

  This kind of signal is generated, but it is possible to detect the generation or change of a signal (signal) in optical, electrical, magnetic, or other energy by being catalyzed by thrombin. Means that.

  For example, a substance whose absorbance, transmittance, or fluorescence intensity changes due to the catalytic action of thrombin, or a substance whose light absorption curve changes can be exemplified.

An example of this is a peptide or protein to which a compound whose absorbance, transmittance, fluorescence intensity, or light absorption curve changes when released is bound to the peptide or protein by thrombin catalysis. Mention may be made of substances that are liberated from proteins.
In such substances, thrombin is catalyzed and can be detected as a change in absorbance, transmittance or fluorescence intensity or light absorption curve when the compound is liberated, so that thrombin catalysis, ie thrombin enzyme activity Can be determined by measuring the amount of signal produced (absorbance, transmittance or fluorescence intensity or change in light absorption curve).

Examples of such a substance include “HD-phenylalanyl-L-pipecolyl-L-arginyl-p-nitroanilide dihydrochloride” [Test Team (registered trademark)].
Chromogenic substrate S-2238] (CHROMOGENIX, Daiichi Chemicals), "HD-hexahydrotyrosyl-L-alanyl-L-arginyl-p-nitroanilide diacetate" [SPECTROZYME (registered trademark)
TH] (AMERICAN DIAGNOSTICA, Cosmo Bio), “Benzoyl-phenylalanyl-valinyl-arginyl-p-nitroanilide hydrochloride” [Thrombin
Substrate I, Colorimetric] (CALBIOCHEM, Cosmo Bio), “Tosyl-glycyl-prolyl-arginyl-p-nitroanilide” [CHROMOZYME
TH] (PENTAPHARM), “HD-phenylalanyl-prolyl-arginyl-3-carboxy-4-hydroxy-aniline” (Sankyo), “benzoyl-phenylalanyl-valinyl-arginyl-AMC / hydrochloride” [Thrombin
Substrate III, Fluorogenic] (CALBIOCHEM, Cosmo Bio), “t-butoxycarbonyl-asparagyl (O-benzyl) -prolyl-arginyl-MCA” (Peptide Institute), or “t-butoxycarbonyl-valinyl-prolyl” -Arginyl-MCA "(Peptide Institute). In particular, “HD-phenylalanyl-L-pipecolyl-L-arginyl-p-nitroanilide dihydrochloride” is preferable.

  Contacting the activated blood coagulation factor V, activated blood coagulation factor X and prothrombin, etc., and contacting the generated thrombin with the substrate of this thrombin may be performed simultaneously or separately. The steps may be performed separately.

  In any case, the thrombin produced is brought into contact with a thrombin substrate and incubated at least at least 1 minute, preferably at least 5 minutes at room temperature or 37 ° C. to generate a signal from the thrombin substrate.

The concentration when this thrombin substrate is used is usually preferably 5 μM to 100 mM, particularly preferably 50 μM to 10 mM, when the thrombin substrate is catalyzed by thrombin.
And in the activity measuring reagent of this invention, when receiving the catalytic action of thrombin, it is preferable to contain in this activity measuring reagent so that this thrombin substrate may become the said density | concentration.
For example, the thrombin substrate is preferably contained in an amount of 5 μM to 200 mM, particularly preferably 50 μM to 20 mM.

13. Measurement of signal amount The substrate of thrombin in the activity measurement method and activity measurement reagent of the present invention generates a signal by being catalyzed by thrombin generated from prothrombin.
In the activity measurement method and activity measurement reagent of the present invention, the amount of signal generated from the thrombin substrate is measured.
When the blood coagulation reaction-suppressed plasma protein is contained in the sample, the amount of this signal is the amount of the signal whose generation is suppressed according to the activity value of the blood coagulation reaction-suppressed plasma protein in the sample.

Measurement of the generated signal amount (signal amount in which generation is suppressed) may be appropriately performed according to the signal.
For example, when the signal is a change in absorbance, transmittance or fluorescence intensity or light absorption curve, the measurement is performed by measuring the absorbance, transmittance or fluorescence intensity.
More specifically, the substrate of thrombin is the aforementioned “HD-phenylalanyl-L-pipecolyl-L-arginyl-p-nitroanilide dihydrochloride”, “HD-hexahydrotyrosyl- “L-alanyl-L-arginyl-p-nitroanilide diacetate”, “benzoyl-phenylalanyl-valinyl-arginyl-p-nitroanilide hydrochloride” or “tosyl-glycyl-prolyl-arginyl-p-” In the case of a substance in which p-nitroaniline is bound to a peptide such as “nitroanilide”, the absorption of light that p-nitroaniline liberated by the catalytic action of thrombin has in the vicinity of a wavelength of 405 nm has a wavelength of 405 nm or the vicinity thereof. By measuring absorbance at a wavelength of.
In this case, the absorbance measurement may be a single wavelength measurement of only the main wavelength, or a two-wavelength measurement measured at the main wavelength and the sub wavelength.
The absorbance measurement may be performed by the end point method or the rate method.

14 Activity value of blood coagulation reaction-inhibited plasma protein contained in sample In the activity measurement method and activity measurement reagent of the present invention, when the sample contains blood coagulation reaction-inhibited plasma protein, activated protein C is When generated or further activated, the decomposition reaction of activated blood coagulation factor V is promoted, and the abundance (concentration) of activated blood coagulation factor V is reduced.
Then, since the effect of activating blood coagulation factor V to the reaction of generating thrombin from prothrombin catalyzed by activated blood coagulation factor X is reduced, the reaction of generating thrombin from prothrombin is suppressed.
Thereby, since the production | generation of thrombin reduces, the reaction which produces a signal from the substrate of thrombin which this thrombin catalyzes is also suppressed, and the quantity of the signal to generate | occur | produce is suppressed.
That is, the degree of inhibition of this signal generation increases according to the activity value of the blood coagulation reaction-suppressed plasma protein contained in the sample.
Therefore, in the activity measurement method and activity measurement reagent of the present invention, the amount of signal generated by contacting the sample with activated protein C or a protein C activator and causing the reaction as described above is measured. That is, the activity value of the blood coagulation reaction-suppressed plasma protein contained in the sample is obtained by measuring the amount of signal whose production is suppressed.

  Obtaining the activity value of the blood coagulation reaction-suppressed plasma protein contained in the sample after measuring the signal amount in which this generation is suppressed may be performed as appropriate, but can be performed as follows, for example. .

About at least one sample whose blood coagulation reaction inhibitory plasma protein activity value is known and a sample whose blood coagulation reaction inhibitory plasma protein activity value is known to be “zero” (such as physiological saline or pure water) Then, the measurement operation is performed as described above, and the amount of generated signal (the amount of signal with suppressed generation) is obtained.
Then, a relationship between the generated signal amount (signal amount whose generation is suppressed) and the activity value of the blood coagulation reaction-suppressed plasma protein contained in the sample is represented by a mathematical formula or a graph to create a calibration curve.
This calibration curve represents the relationship between the degree of inhibition of signal generation and the activity value of the blood coagulation reaction-suppressed plasma protein contained in the sample.
Next, for the sample whose activity value of the blood coagulation reaction-suppressed plasma protein is unknown, the measurement operation is performed in the same manner as described above to determine the amount of signal generated (the amount of signal whose generation is suppressed).
This signal amount is applied to the calibration curve, and the activity value of the corresponding blood coagulation reaction-suppressed plasma protein is determined.
Thereby, the activity value of the blood coagulation reaction-suppressing plasma protein of the sample can be obtained from the amount of signal generated in the sample whose activity value of the blood coagulation reaction-suppressing plasma protein is unknown (the signal amount whose generation is suppressed).

  The calibration curve represents the relationship between the amount of signal (the amount of signal in which production was suppressed), that is, the degree of inhibition of signal generation, and the activity value of blood coagulation reaction-suppressed plasma protein contained in the sample. However, the signal amount (signal amount whose generation is suppressed) in the calibration curve may be the measured signal amount itself, or may be a numerical value calculated based on the value of the signal amount.

  That is, the calibration curve may represent a relationship between a numerical value calculated based on the value of the measured signal amount and the activity value of the blood coagulation reaction-suppressed plasma protein contained in the sample. Even in this case, the numerical value calculated based on the value of the measured signal amount corresponds to the signal amount whose generation is suppressed.

  Examples of the numerical value calculated based on the measured signal amount value include, for example, a numerical value of a change amount of the measured signal amount per unit time, or a first derivative of the measured signal amount value with respect to time. Or a numerical value calculated by second-order differentiation.

  For example, the amount of change per minute in the absorbance obtained by measurement, or the rate of change in absorbance obtained by first derivative of the absorbance obtained by measurement with respect to time, or the absorbance obtained by measurement in time On the other hand, the acceleration of the change in absorbance obtained by second-order differentiation can be exemplified.

15. Protein In the activity measurement method and activity measurement method of the present invention, proteins such as human serum albumin (HSA), bovine serum albumin (BSA), albumin such as ovalbumin, casein, or gelatin are present during the activity measurement reaction. It is preferable that it is contained in the activity measuring reagent.

The concentration at which this protein is present is usually preferably 0.1 μM to 1 mM.
And in the activity measuring reagent of this invention, it is preferable to make this protein contain in an activity measuring reagent so that it may become the said density | concentration.
For example, this protein is preferably contained at 0.1 μM to 2 mM.

16. Salt In the activity measurement method and activity measurement method of the present invention, it is preferable that a salt such as a halogen element and alkali metal salt or a halogen element and alkaline earth metal salt is present during the activity measurement reaction, or activity measurement. It is preferable to make it contain in a reagent.

Examples of the halogen element and alkali metal salt include sodium chloride, potassium chloride, sodium fluoride, potassium fluoride, sodium bromide, and potassium bromide.
In addition, examples of the salt of a halogen element and an alkaline earth metal include magnesium chloride, magnesium fluoride, and magnesium bromide.

The concentration at which this salt is present is usually preferably from 5 mM to 1 M, particularly preferably from 50 mM to 250 mM.
And in the activity measuring reagent of this invention, it is preferable to contain in this activity measuring reagent so that this salt may become the said density | concentration.
For example, this salt is preferably contained in an amount of 5 mM to 2 M, particularly preferably 50 mM to 500 mM.

17. pH
In the activity measurement method of the present invention, this activity measurement reaction is preferably performed in the range of pH 6.0 to pH 10.0 (20 ° C.), and is performed in the range of pH 6.5 to pH 8.5 (20 ° C.). Is particularly preferred.
In addition, the activity measuring reagent of the present invention preferably has a pH at which the activity measuring reaction is performed in the above pH range.
For example, the pH of the activity measuring reagent of the present invention is preferably in the range of pH 6.0 to pH 10.0 (20 ° C.), particularly preferably in the range of pH 6.5 to pH 8.5 (20 ° C.). .

  In addition, in the activity measurement method and activity measurement reagent of the present invention, in order to keep the pH in the activity measurement reaction or the pH of the activity measurement reagent in the above pH range, a buffering agent having a buffer capacity is appropriately present in the above pH range. Or it is preferable to contain.

18. Other Components In the activity measuring method and activity measuring reagent of the present invention, components other than the above-described components such as preservatives, stabilizers, activators, saccharides or surfactants are appropriately added as necessary. It can be present during the activity measurement reaction or can be contained in the activity measurement reagent.

19. Measurement method In the activity measurement method and the activity measurement reagent of the present invention, the activity of the blood coagulation reaction-suppressed plasma protein in the sample may be measured by a conventional method or using an apparatus such as an automatic analyzer. Also good.

20. Measurement Stage In the activity measurement method of the present invention, the activity measurement reaction described in detail above may be carried out in one stage by bringing all components into contact with the sample at one time (one step). Method), and the activity measurement reaction may be performed in a plurality of stages by dividing the components and performing the contact in a plurality of stages (multi-step method).
When dividing into a plurality of stages, there is no particular limitation, but for example, it can be performed as follows.

・ Two-step method-1
(1) A sample, activated protein C or protein C activator, phospholipid, calcium ion, and activated blood coagulation factor V are contacted.
(2) Next, activated blood coagulation factor X, prothrombin, a substrate of thrombin, and a phospholipid are brought into contact with the above-mentioned (1).

・ Two-step method-2
(1) A sample, activated protein C or protein C activator, and phospholipid are contacted.
(2) Next, calcium ion, activated blood coagulation factor V, activated blood coagulation factor X, prothrombin, thrombin substrate and phospholipid are brought into contact with the above-mentioned (1).

-Three-step method (1) A sample, activated protein C or a protein C activator, and a phospholipid are contacted.
(2) Next, calcium ion and activated blood coagulation factor V are brought into contact with the above (1).
(3) Further, activated blood coagulation factor X, prothrombin, thrombin substrate and phospholipid are brought into contact with those of (2) above.

21. Configuration of Activity Measuring Reagent In the activity measuring reagent of the present invention, all the components of the activity measuring reaction described in detail above may be contained in one reagent (one reagent system), or each component may be a plurality of components. The reagent may be contained separately (multiple reagent system).
When each component is contained separately in a plurality of reagents, there is no particular limitation, but for example, it can be contained as follows.

2 reagent system-1
(1) Activated protein C or protein C activator, phospholipid, calcium ion and activated blood coagulation factor V.
(2) Activated blood coagulation factor X, prothrombin, thrombin substrate and phospholipid.

2 reagent system-2
(1) Activated protein C or protein C activator and phospholipid.
(2) Calcium ions, activated blood coagulation factor V, activated blood coagulation factor X, prothrombin, thrombin substrate and phospholipid.

3 reagent system (1) Activated protein C or protein C activator and phospholipid.
(2) Calcium ions and activated blood coagulation factor V.
(3) Activated blood coagulation factor X, prothrombin, thrombin substrate and phospholipid.

4 reagent system (1) activated protein C or protein C activator and phospholipid.
(2) Calcium ions and activated blood coagulation factor V.
(3) Activated blood coagulation factor X and phospholipid.
(4) Prothrombin and thrombin substrate.

  The present invention will be further described below with reference to examples. In addition, this invention is not limited by these.

[Example 1] (Preparation of a calibration curve for measuring protein S activity in a sample)
A calibration curve was prepared for measuring the activity value of protein S which is a blood coagulation reaction-suppressed plasma protein in a sample by the activity measurement method and activity measurement reagent of the present invention.

(1) Reagent for measuring protein S activity (a) First reagent The following components were dissolved in pure water so as to have the respective concentrations described above to prepare a reagent having a pH of 7.5 (20 ° C).

Activated protein C 344pM
Phospholipid (phosphatidylcholine 80%, phosphatidylserine 20%) 10 μM
Bovine serum albumin (BSA) 15 μM
Sodium chloride 150 mM
Tris (hydroxymethyl) aminomethane 50 mM

(B) Second reagent The following components were dissolved in pure water so as to have the respective concentrations described above to prepare a reagent having a pH of 7.5 (20 ° C).

Activated blood coagulation factor V 331pM
Phospholipid (phosphatidylcholine 80%, phosphatidylserine 20%) 10 μM
Calcium chloride 5 mM
Bovine serum albumin (BSA) 15 μM
Sodium chloride 150 mM
Tris (hydroxymethyl) aminomethane 50 mM

(C) Third reagent Each of the following components was dissolved in pure water so as to have the indicated concentration to prepare a reagent having a pH of 7.5 (20 ° C).

Activated blood coagulation factor X 44pM
Phospholipid (phosphatidylethanolamine 50%, phosphatidylserine 30%, phosphatidylcholine 20%) 7.5 μM
HD-Phenylalanyl-L-pipecholyl-L-arginyl-p-nitroanilide dihydrochloride (thrombin substrate) 750 μM
Prothrombin 695nM
Calcium chloride 5 mM
Bovine serum albumin (BSA) 15 μM
Sodium chloride 150 mM
Tris (hydroxymethyl) aminomethane 50 mM

(2) Sample Purified protein S (purity 99% or more) was added to human plasma without protein S so that each of the following activity values was obtained, and the following three types of protein S activity values were known. Samples were prepared.
In addition, the human plasma not containing protein S was obtained by passing human plasma through a column on which an anti-protein S antibody was immobilized, and separating the sample that passed through this column.

(A) 15 μg / mL equivalent (b) 10 μg / mL equivalent (c) 5 μg / mL equivalent

Further, human plasma not containing protein S was used as a sample having a protein S activity value of “zero”.
(D) Equivalent to 0 μg / mL

In the present specification and drawings, the protein S activity value of 1 μg / mL of a purified protein S product (purity 99% or more) obtained by purification from human plasma is defined as the protein S activity value equivalent to 1 μg / mL. .
Therefore, in the present specification and drawings, the activity value of protein S is represented by a value corresponding to the protein S concentration (μg / mL).

(3) Measurement of activity value of protein S in sample (a) For each of the four types of samples in (2), 4 μL of the sample and 96 μL of the first reagent were mixed and incubated at 37 ° C. for 10 minutes. .
(B) Thereafter, 100 μL of the second reagent was added thereto and mixed, and incubated at 37 ° C. for 10 minutes.
(C) Next, 180 μL of the mixed solution of (b) was taken, and 360 μL of the third reagent that had been incubated at 37 ° C. for 5 minutes was added and mixed, and the mixture was incubated at 37 ° C. .

(D) After the addition and mixing in (c), the absorbance at a wavelength of 405 nm was measured.
A graph showing the change in absorbance is shown in FIG.
In this figure, the horizontal axis represents the time (minutes) after the addition and mixing in (c), and the vertical axis represents the absorbance (Abs.) At a wavelength of 405 nm.
In this figure, “◯” indicates the absorbance of a sample having a protein S activity corresponding to 0 μg / mL, “Δ” indicates the absorbance of a sample having a protein S activity corresponding to 5 μg / mL, and “□” indicates a protein S activity. The activity indicates the absorbance of the sample corresponding to 10 μg / mL, and “◇” indicates the absorbance of the sample corresponding to the protein S activity of 15 μg / mL.

(E) The absorbance change amount per minute in the graph of FIG. 1, that is, the slope of absorbance change in the graph of FIG. A graph was created with the time after mixing as the horizontal axis. The slope value of the absorbance change on the vertical axis is the first derivative value of absorbance, and represents the rate of absorbance change.
This graph is shown in FIG.
In this figure, “◯” indicates the value in the sample corresponding to the protein S activity of 0 μg / mL, “Δ” indicates the value in the sample corresponding to the protein S activity of 5 μg / mL, and “□” indicates the protein S activity. The value in the sample corresponding to 10 μg / mL is indicated, and “◇” indicates the value in the sample corresponding to protein S activity of 15 μg / mL.
For example, the value on the 18-minute scale on the horizontal axis is the amount of change in absorbance per minute from the 18th to 19th minute after the addition and mixing in (c), that is, the value of the slope of absorbance change (absorbance). 1st derivative value).

(F) Next, the vertical axis represents the slope value of each line of the graph (FIG. 2) in (e), and the horizontal axis represents the protein S activity value (corresponding to μg / mL) of each sample. Created. The value on the vertical axis is the second derivative value of absorbance, and represents the acceleration of absorbance change.
This graph is shown in FIG.
The graph of FIG. 3 is a graph showing a calibration curve in the measurement of the protein S activity value in the sample.

  For samples for which the protein S activity value is not known, the activity measurement operation is performed as described above, and the acceleration (second derivative of absorbance) of the absorbance change is determined from the absorbance obtained by the measurement (the amount of signal with suppressed production). The activity value of protein S contained in the sample can be obtained by obtaining this value and applying this value to the graph of FIG.

[Example 2] (Measurement of protein S activity value in sample)
The activity value of protein S, which is a blood coagulation reaction-suppressed plasma protein, in a human plasma sample was measured by the activity measurement method and activity measurement reagent of the present invention.

(1) Reagent for protein S activity measurement of the present invention The first reagent, the second reagent, and the third reagent prepared in Example 1 were used, respectively.

(2) Reagent for measuring protein S activity by clotting time method As a reagent for measuring protein S activity in conventional samples, a commercially available reagent for measuring protein S activity in plasma by the clotting time method “STACLOTT protein S” (Roche Diagnostics) Was used.

(3) Samples The following 6 types of human plasma were used as samples.
(A) Sample-1 (Plasma of healthy person)
(B) Sample-2 (perinatal female plasma)
(C) Sample-3 (perinatal female plasma)
(D) Sample-4 (perinatal female plasma)
(E) Sample-5 (perinatal female plasma)
(F) Sample-6 (perinatal female plasma)

(4) Measurement of the protein S activity value in the sample (a) Measurement with the protein S activity measurement reagent of the present invention For each of the six types of samples in (3), (a) (a) in Example 1 above The measurement operation was performed as described in () to (f), and the value of the acceleration of absorbance change (second derivative of absorbance) was determined.
By applying this absorbance change acceleration value (second-order absorbance differential value) to the calibration curve graph prepared in Example 1 (FIG. 3), the protein S activity value contained in the sample was determined. Obtained.
The protein S activity values obtained by this measurement are shown in Table 1.

(B) Reagent for measuring protein S activity by the coagulation time method For each of the six types of samples in (3) above, the above-mentioned “STACROT PROTEIN S” (Roche Diagnostics) was used, and the operation method of this package insert And the activity value of protein S by the coagulation time method was obtained.
In addition, the protein S activity value was obtained by the calibration curve prepared by using purified protein S (purity 99% or more) as a sample used in (2) of Example 1.
The measurement of the coagulation time was performed using a blood coagulation measuring device “Behring Fibrin Timer II” (Hoechst Japan).
The protein S activity values obtained by this measurement are also shown in Table 1.

(5) Summary As can be seen from Table 1, the protein S activity value of the plasma sample measured with the protein S activity measuring reagent of the present invention and the protein S activity value of the plasma sample measured with the protein S activity measuring reagent by the clotting time method. Was consistent in all samples.
From this, it was confirmed that the activity value of protein S contained in the sample obtained by the activity measurement method and the activity measurement reagent of the present invention is accurate.

[Example 3] (Examination of phospholipid used for catalytic reaction by activated protein C)
In the activity measurement method and activity measurement reagent of the present invention, the composition of phospholipid used for the catalytic reaction with activated protein C was examined.

(1) Reagent for measuring protein S activity (a) First reagent Six kinds of first reagents having different phospholipid compositions were prepared as follows.
The following components were dissolved in pure water so as to have the stated concentrations and adjusted to pH 7.5 (20 ° C.).

Activated protein C 344pM
Bovine serum albumin (BSA) 15 μM
Sodium chloride 150 mM
Tris (hydroxymethyl) aminomethane 50 mM
Phospholipid 10 μM
(I) Phosphatidylcholine 100%, Phosphatidylserine 0%
(B) Phosphatidylcholine 95%, Phosphatidylserine 5%
(C) Phosphatidylcholine 90%, Phosphatidylserine 10%
(D) Phosphatidylcholine 85%, Phosphatidylserine 15%
(E) 80% phosphatidylcholine, 20% phosphatidylserine, or
(F) 75% phosphatidylcholine, 25% phosphatidylserine

(B) Second Reagent Six types of second reagents having different phospholipid compositions were prepared as follows.
The following components were dissolved in pure water so as to have the stated concentrations and adjusted to pH 7.5 (20 ° C.).

Activated blood coagulation factor V 331pM
Calcium chloride 5 mM
Bovine serum albumin (BSA) 15 μM
Sodium chloride 150 mM
Tris (hydroxymethyl) aminomethane 50 mM
Phospholipid 10 μM
(I) Phosphatidylcholine 100%, Phosphatidylserine 0%
(B) Phosphatidylcholine 95%, Phosphatidylserine 5%
(C) Phosphatidylcholine 90%, Phosphatidylserine 10%
(D) Phosphatidylcholine 85%, Phosphatidylserine 15%
(E) 80% phosphatidylcholine, 20% phosphatidylserine, or
(F) 75% phosphatidylcholine, 25% phosphatidylserine

(C) Third reagent The third reagent prepared in (c) of (1) of Example 1 was used.

(2) Sample (a) The protein S prepared in (2) of Example 1 was used so that the activity value of protein S, which is a blood coagulation reaction-suppressed plasma protein, was equivalent to 0.2 μg / mL. A sample having a protein S activity value equivalent to 0.2 μg / mL was prepared by adding to human plasma not containing it.

(B) Human plasma containing no protein S was used as a sample having a protein S activity value equivalent to 0 μg / mL.

(3) Measurement of activity value of protein S in sample For each of the two types of samples of (2), each of the six types of first reagents prepared in (a) of (1) and (1 ) Of (b) of Example 1 and the third reagent prepared in (c) of (1) above, and (a) to (3) of (3) of Example 1 above. The measurement operation was performed as described in f), and the value of acceleration of absorbance change (second-order differential value of absorbance) was obtained.
In this measurement, the first reagent and the second reagent were used in combination with each other having the same phospholipid composition.

(4) Examination of effect by composition of phospholipid The value of acceleration of absorbance change (second derivative of absorbance) of a human plasma sample having a protein S activity value equivalent to 0.2 μg / mL, and a protein S activity value of 0 μg The ratio (α0.2 / α0) was determined by dividing by the value of the acceleration of the change in absorbance of the human plasma sample corresponding to / mL (second derivative of absorbance).
This is shown in Table 2.
The lower the value of this ratio, the difference in the amount of signal obtained between a human plasma sample with a protein S activity value equivalent to 0.2 μg / mL and a human plasma sample with a protein S activity value equivalent to 0 μg / mL. This is preferable in the measurement of the protein S activity value because the sensitivity of the measurement is higher.

  From this table, the composition of phospholipids present during the catalytic reaction with activated protein C is “phosphatidylcholine 85%, phosphatidylserine 15%”, “phosphatidylcholine 80%, phosphatidylserine 20%”, and “phosphatidylcholine 75%, phosphatidylserine”. When the ratio is 25%, the ratio value is lower than that of other compositions, that is, the measurement reaction is promoted, and the measurement sensitivity is higher.

  From this, the composition of the phospholipids present during the further activation of the activated protein C by the protein S contained in the sample and during the degradation reaction of the activated blood coagulation factor V by the activated protein C It was confirmed that the reaction was promoted and the measurement sensitivity was increased by setting the composition ratio of phosphatidylcholine to 85% to 75% and the composition ratio of phosphatidylserine to 15% to 25%. .

[Example 4] (Examination of phospholipid used for catalytic reaction by activated blood coagulation factor X)
The composition of phospholipids used in the catalytic reaction to generate thrombin by activated blood coagulation factor X was examined.
In this study, since the purpose is to see the effect of phospholipid composition on the catalytic reaction of activated blood coagulation factor X, the reaction system starting from activated blood coagulation factor X was examined.

(1) Reagent for examining phospholipid composition (a) First reagent The following components were dissolved in pure water so as to have the respective concentrations described above to prepare a reagent having a pH of 7.5 (20 ° C).

Activated blood coagulation factor V 331pM
Phospholipid (phosphatidylcholine 80%, phosphatidylserine 20%) 10 μM
Calcium chloride 5 mM
Bovine serum albumin (BSA) 15 μM
Sodium chloride 150 mM
Tris (hydroxymethyl) aminomethane 50 mM

(B) Second reagent Four types of second reagents having different phospholipid compositions were prepared as follows.
The following components were dissolved in pure water so as to have the stated concentrations, and the pH was adjusted to pH 7.5 (20 ° C.).

Activated blood coagulation factor X 44pM
HD-Phenylalanyl-L-pipecholyl-L-arginyl-p-nitroanilide dihydrochloride (thrombin substrate) 750 μM
Prothrombin 695nM
Calcium chloride 5 mM
Bovine serum albumin (BSA) 15 μM
Sodium chloride 150 mM
Tris (hydroxymethyl) aminomethane 50 mM
Phospholipid 7.5μM
(A) Phosphatidylethanolamine 50%, phosphatidylserine 40%, phosphatidylcholine 10%
(B) Phosphatidylethanolamine 50%, phosphatidylserine 30%, phosphatidylcholine 20%
(C) Phosphatidylethanolamine 50%, phosphatidylserine 20%, phosphatidylcholine 30% or
(D) Phosphatidylethanolamine 50%, phosphatidylserine 10%, phosphatidylcholine 40%

(2) Measurement of effect by composition of phospholipid (a) 180 μL of the first reagent of (1) that had been incubated at 37 ° C. for 5 minutes and (1) that had been incubated at 37 ° C. for 5 minutes Were mixed with 360 μL of the second reagent and incubated at 37 ° C.

(B) After the addition and mixing in (a), the absorbance at a wavelength of 405 nm was measured.
A graph showing the change in absorbance is shown in FIG.
In this figure, the horizontal axis represents the time (minutes) after the addition and mixing in (a), and the vertical axis represents the absorbance (Abs.) At a wavelength of 405 nm.

  In this figure, “◯” indicates the absorbance when the phospholipid composition in the second reagent is “phosphatidylethanolamine 50%, phosphatidylserine 40%, phosphatidylcholine 10%”, and “◇” indicates that in the second reagent. Absorbance when the composition of phospholipid is “phosphatidylethanolamine 50%, phosphatidylserine 30%, phosphatidylcholine 20%”, “Δ” indicates the composition of phospholipid in the second reagent is “phosphatidylethanolamine 50%, phosphatidylserine” 20%, phosphatidylcholine 30% "and" □ "indicates the absorbance when the phospholipid composition in the second reagent is" phosphatidylethanolamine 50%, phosphatidylserine 10%, phosphatidylcholine 40% ". Show.

(3) Examination of effect by composition of phospholipid In this FIG. 4, as the rise in absorbance is faster, and the absorbance reaches a plateau within a shorter time, thrombin due to the catalytic action of activated blood coagulation factor X As a result, the sensitivity of the measurement becomes higher.

  From this figure, the composition of phospholipids present during the catalytic reaction by activated blood coagulation factor X is “phosphatidylethanolamine 50%, phosphatidylserine 40%, phosphatidylcholine 10%” and “phosphatidylethanolamine 50%, phosphatidylserine”. In the case of “30%, phosphatidylcholine 20%”, the rise in absorbance increases earlier than in other compositions, and the absorbance reaches a plateau within a shorter time. That is, it can be seen that the thrombin generation reaction by activated blood coagulation factor X is promoted, and the sensitivity of the measurement is higher.

  From this, the composition of phospholipids to be present during the reaction for generating thrombin from prothrombin catalyzed by activated blood coagulation factor X is 50% or more of the composition ratio of phosphatidylethanolamine, and the composition ratio of phosphatidylserine is It was confirmed that the reaction was promoted and the measurement sensitivity was increased by setting it to 30% or more.

Claims (7)

A method for measuring an activity value of a blood coagulation reaction-suppressing plasma protein in a sample, comprising:
(A) Contacting a sample with activated protein C or a protein C activator, phospholipid, calcium ion, activated blood coagulation factor V, activated blood coagulation factor X, prothrombin and thrombin substrate not derived from the sample Let
(B) measuring the amount of signal generated from the substrate of thrombin as a result of the reaction by each component of (a),
(C) determining the amount of signal whose production is suppressed according to the activity of the blood coagulation reaction suppression plasma protein contained in the sample;
The blood coagulation reaction suppression plasma protein activity measuring method in the sample which obtains the activity value of the blood coagulation reaction suppression plasma protein contained in the sample. A reaction system composed of activated protein C or protein C activator, phospholipid, calcium ion, activated blood coagulation factor V, activated blood coagulation factor X, prothrombin and thrombin substrate not derived from the sample A method for measuring an activity value of a blood coagulation reaction-suppressing plasma protein in a sample, comprising:
(A) contacting the sample with at least activated protein C or protein C activator;
(B) When the sample contains blood coagulation reaction-suppressed plasma protein, the activated blood coagulation factor V degradation reaction catalyzed by the activated protein C produced or further activated in the presence of phospholipids and calcium ions Promoted,
(C) The reaction to generate thrombin from prothrombin catalyzed by activated blood coagulation factor X in the presence of phospholipids and calcium ions, promoted by activated blood coagulation factor V, is activated in (b) above. Suppressed by promoting the degradation reaction of blood coagulation factor V, reducing the production of thrombin,
(D) By reducing the generation of thrombin in (c), the reaction that generates a signal from the thrombin substrate catalyzed by thrombin is suppressed,
(E) measuring the amount of signal whose production is suppressed by the suppression of the reaction in (d),
The activity measurement method of the blood coagulation reaction suppression plasma protein in the sample according to claim 1, wherein the activity value of the blood coagulation reaction suppression plasma protein contained in the sample is obtained.   The method for measuring the activity of a blood coagulation reaction-suppressed plasma protein in a sample according to claim 1 or 2, wherein the signal amount is measured by measuring absorbance, transmittance, or fluorescence intensity.   The numerical value of the amount of change in the measured signal amount per unit time, the numerical value calculated by first-derivative of the measured signal amount value with respect to time, or the numerical value calculated by second-order differentiation, and in the sample The activity value of the blood coagulation reaction-suppressing plasma protein contained in the sample is obtained using a calibration curve representing the relationship with the activity value of the blood coagulation reaction-suppressing plasma protein. The method for measuring the activity of blood coagulation reaction-suppressed plasma protein in the sample according to 1.   The amount of change per minute in the absorbance obtained by the measurement, the rate of change in absorbance obtained by first derivative of the absorbance obtained by the measurement with respect to time, or the absorbance obtained by the measurement with respect to time. Using a calibration curve representing the relationship between the acceleration of absorbance change obtained by second-order differentiation and the activity value of blood coagulation reaction-inhibited plasma protein in the sample, the activity value of blood coagulation reaction-inhibiting plasma protein contained in the sample The method for measuring the activity of a blood coagulation reaction-suppressed plasma protein in a sample according to any one of claims 1 to 4, wherein:   Plasma protein inhibiting blood coagulation reaction in a sample containing activated protein C or protein C activator, phospholipid, calcium ion, activated blood coagulation factor V, activated blood coagulation factor X, prothrombin and thrombin substrate Activity measuring reagent.   6. An activated protein C or protein C activator, phospholipid, calcium ion, activated blood coagulation factor V, activated blood coagulation factor X, prothrombin and thrombin substrate, A reagent for measuring the activity of a blood coagulation reaction-suppressing plasma protein in a sample, which is used in the method for measuring the activity of a blood coagulation reaction-suppressing plasma protein in a sample according to any one of the above items.

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