WO2014163170A1

WO2014163170A1 - Activity measurement reagent kit and activity measurement method for protein s

Info

Publication number: WO2014163170A1
Application number: PCT/JP2014/059905
Authority: WO
Inventors: 秀日金; 友秀津田
Original assignee: 株式会社シノテスト
Priority date: 2013-03-30
Filing date: 2014-03-28
Publication date: 2014-10-09
Also published as: JPWO2014163170A1; JP6634557B2

Abstract

Provided is an activity measurement reagent kit for protein S, comprising three constituent reagents that can be used in many general-purpose automatic biochemical analyzers. Also provided is an activity measurement method for protein S using three constituent reagents that can be used in many general-purpose automatic biochemical analyzers. An activity measurement reagent kit for protein S, characterized by comprising the following constituent reagents (i)-(iii). (i) Reagent containing activated protein C: containing at least activated protein C. (ii) Reagent containing activated blood coagulation factor V: containing at least activated blood coagulation factor V. (iii) Reagent containing prothrombin substrate: containing at least a substrate of prothrombin and thrombin. Additionally, an activity measurement method for protein S characterized by using an activity measurement reagent kit for protein S, comprising the above constituent reagents (i)-(iii).

Description

Protein S activity measuring reagent kit and activity measuring method

The present invention relates to an activity measuring reagent kit and an activity measuring method capable of accurately, simply and measuring the activity of protein S in a sample in a short time.
The present invention is particularly useful in the field of life science such as clinical examination, clinical pathology and medicine.

Protein S is a plasma protein that functions centrally in the control mechanism of the blood coagulation system in the living body.
This protein S is mainly present in blood and is a prosthetic factor (cofactor) of activated protein C, which can increase the activity of activated protein C, and is activated in blood. It is indispensable for the function of protein C.
In addition, activated protein C decomposes activated blood coagulation factor V (factor Va; FVA) and activated blood coagulation factor VIII (factor VIIIa; FVIIIa) that promote blood coagulation in humans. It is a factor that plays a role in suppressing the blood coagulation reaction.
Protein S specifically binds one-to-one with a C4b binding protein (complement factor 4b binding protein; C4bBP) to form a complex. That is, the C4b binding protein is a protein S ligand.
This complex formation reaction between protein S and C4b binding protein is as shown below, but this reaction is a reversible reaction.

In human blood, since the molar concentration of C4b binding protein is usually smaller than that of protein S and the dissociation constant is small, “protein S” and “protein S-C4b binding protein complex” are present in blood. Exists only. In other words, the equilibrium of the above reaction is completely to the right.
Usually, about 60% of protein S in the blood (plasma) of healthy individuals is “protein S-C4b binding protein complex” (ie, bound type), and about 40% of “protein S in free state”. S "(ie free).
Only free protein S (that is, free form) exhibits a coenzyme activity for activated protein C and can increase the activity of activated protein C.
In the present specification, the term “protein S” or “C4b binding protein” means that each of these substances, unless there is a description of complex (or binding type) or free (or free type). It shall mean the generic name of complex (or bound) and free (or free).
The schematic diagram of the control mechanism of the blood coagulation system by protein C and protein S is shown below.

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, and the presence of protein S increases the activity of activated protein C, and the activated blood coagulation factor V degradation reaction by activated protein C and The degradation reaction of activated blood coagulation factor VIII is promoted.
A decrease or abnormality in the activity of protein S, which has a function of suppressing the blood coagulation reaction, can cause thrombosis in vivo.
In fact, people with congenital anomalies of protein S often develop thrombosis such as deep vein thrombosis, superficial phlebitis or pulmonary infarction.
In addition, a decrease or abnormality in protein S activity is also observed in disseminated intravascular coagulation syndrome (DIC), vitamin K deficiency, or hepatic hypofunction.
That is, by measuring the activity of protein S in a sample such as plasma, it is possible to grasp the decrease or abnormality of protein S activity, thereby predicting the onset, early detection and treatment of diseases such as thrombosis. It plays an important role in determining effects.
As a method for measuring the activity of protein S in a sample, the sample is incubated with protein C-activator from snake venom under the formation of activated protein C, and blood coagulation factor XII, blood coagulation factor VII or blood A method of photometrically measuring the decrease in thrombin formation from prothrombin mediated by blood coagulation factor and its activator, using a chromogenic thrombin substrate, by adding an activator of coagulation factor II (prothrombin) Is disclosed (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).
Alternatively, 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 protein S activity and reagents for measuring the activity of protein S, at least one of the components (factors) of the blood coagulation reaction used in the measurement reaction is a component (factor) contained in the sample. They were used as they were. That is, it depends on the components contained in the sample.
Therefore, in the conventional method for measuring the activity of protein S and the reagent for measuring the activity of protein S using the components (factors) contained in the sample as they are, the components (factors) [for example, activated blood coagulation X When the factor] is deficient or decreased in the sample provider, at least one of the reaction components (factors) of the measurement reaction is not present in a sufficient amount, so the measurement reaction does not proceed sufficiently and the protein obtained In some cases, the S activity value has a problem that it is far from the original value and includes an error.
That is, there is a problem that the measured value is influenced by the amount (concentration) in the sample of the component (factor) related to the blood coagulation reaction or its variation.
Therefore, the present inventors are in a sample containing activated protein C, phospholipid, calcium ion, activated blood coagulation factor V, activated blood coagulation factor X, prothrombin and thrombin substrate that is not derived from the sample. A protein S activity measuring reagent, and activated protein C, phospholipid, calcium ion, activated blood coagulation factor V, activated blood coagulation factor X, prothrombin and thrombin substrate not derived from the sample Next, the amount of signal generated from the substrate of thrombin as a result of the reaction by each component described above is measured, and then the amount of signal whose generation is suppressed according to the activity of protein S contained in the sample is obtained. Invented and disclosed a method for measuring the activity of protein S in a sample to obtain the activity value of protein S contained in the sample ( Patent Document 4 and Non-Patent Document 1 see.).
In addition, the present inventors, for all protein S present in the sample [complex of protein S and C4b binding protein (binding type) and free protein S (free type)], that is, total protein S, Invented a measuring reagent and measuring method capable of measuring the activity of the total protein S (see Patent Document 5), and invented a measuring reagent and measuring method capable of measuring the protein amount of the total protein S In addition, a method for detecting protein S abnormality was invented based on the activity value and protein amount of the total protein S (see Patent Document 7).
By the way, the conventional protein S activity measurement method and activity measurement reagent use four or more reagents.
However, in general-purpose biochemical automatic analyzers that are frequently used in clinical laboratories of medical institutions such as hospitals and clinics, the number of reagents that can use four or more reagents is very limited. In the analyzer, four or more reagents cannot be used.
For this reason, in many facilities, the automatic analyzer cannot be used to measure the activity of protein S in a sample. Therefore, measurement must be carried out by a method, and therefore, four or more reagents are used. The complicated protein S activity measurement operation requires a considerable amount of manpower, is time consuming, and has problems in accuracy such as procedural problems.
Further, even in the limited automatic analyzer that can use four or more reagents, the measurement takes time.
For example, the Hitachi High-Technologies Corporation (Japan) 7170S type general-purpose automatic analyzer can use four reagents. Using this automatic analyzer, “diluent” (including surfactants), “ “First reagent” (including activated protein C, phospholipid, calcium chloride, and surfactant), “second reagent” (including activated blood coagulation factor V and the like), and “third reagent” ( When measuring protein S activity in a sample using a protein S activity measurement reagent consisting of four constituent reagents (including activated blood coagulation factor X, prothrombin, and thrombin substrate) (Plasma) 8 μL was added to and mixed with 112 μL of the “diluent”, then 2 μL of this mixture was dispensed, and then 98 μL of the “first reagent” was added and mixed at 37 ° C. For 1.4 minutes Then, 20 μL of the “second reagent” is added, mixed, and incubated at 37 ° C. for 8.3 minutes, and then 236 μL of the “third reagent” is added, mixed, and 12.3 minutes at 37 ° C. Absorbance (main wavelength: 405 nm, subwavelength: 505 nm) is measured at 47 and 53 measurement points after the incubation and addition of the “third reagent”, and the amount of change in absorbance between these points or its speed Further, the activity value of protein S contained in the sample is obtained.
In this case, it took about 22 minutes after the “first reagent” was added to the mixed solution of the sample and the “diluent” until the measurement was completed (see Patent Document 5).

JP-A-62-212569 Japanese National Patent Publication No. 4-506603 JP-T 6-504682 JP 2004-337143 A International Publication No. 2012/124798 JP 2012-193959 A JP 2012-191852 A

An object of the present invention is to provide a protein S activity measurement reagent kit comprising three constituent reagents that can be used in many general-purpose biochemical automatic analyzers. Furthermore, many general-purpose biochemical automatic analyzers are provided. It is to provide a method for measuring the activity of protein S using three constituent reagents that can be used in the above.
That is, it is an object to provide a protein S activity measuring reagent kit and an activity measuring method that are easy to automate the measurement and that can measure the activity of protein S contained in a sample accurately, simply, and in a short time.

As a result of repeated studies on the protein S activity measurement reagent kit and activity measurement method, the present inventors have found that the above problems can be solved when the combination of components contained in the constituent reagents is specific, and the present invention has been completed. It came to do.
The gist of the present invention is as follows.
(1) A protein S activity measurement reagent kit, which comprises the following components (i) to (iii):
(I) Activated protein C-containing reagent: Contains at least activated protein C.
(Ii) Activated blood coagulation factor V-containing reagent: Contains at least activated blood coagulation factor V.
(Iii) Prothrombin / substrate-containing reagent: Contains at least prothrombin and thrombin substrate.
(2) The protein S activity measurement reagent kit according to (1), wherein at least one reagent selected from an activated protein C-containing reagent and an activated blood coagulation factor V-containing reagent contains phospholipid.
(3) Reagent kit for measuring protein S activity according to (1) or (2), wherein at least one reagent selected from an activated protein C-containing reagent and an activated blood coagulation factor V-containing reagent contains calcium ions. .
(4) The aforementioned (1) to (1), wherein at least one reagent selected from an activated protein C-containing reagent, an activated blood coagulation factor V-containing reagent and a prothrombin / substrate-containing reagent contains activated blood coagulation factor X. 3. The reagent kit for measuring activity of protein S according to any one of 3).
(5) The activated protein C-containing reagent and the activated blood coagulation factor V-containing reagent contain neither prothrombin nor a thrombin substrate, according to any one of (1) to (4) above The reagent kit for measuring the activity of protein S as described.
(6) The reagent kit for measuring activity of protein S according to any one of (1) to (5), wherein protein S is total protein S.
(7) A method for measuring protein S activity, which comprises using the protein S activity measuring reagent kit according to any one of (1) to (6) above. Method.
(8) The method for measuring the activity of protein S according to (7), wherein the protein S is total protein S.

The protein S activity measurement reagent kit and activity measurement method of the present invention are easy to automate the measurement, and can measure the activity of protein S contained in a sample accurately, simply and in a short time.

It is the graph which showed the change by the time of the light absorbency (405 nm) when measuring 6 types of samples with known total protein S activity value by the protein S activity measuring reagent kit and activity measuring method of this invention. It is the graph which showed the change by the time of the light absorbency (405 nm) when measuring 6 types of samples with known total protein S activity value by the protein S activity measuring reagent kit and activity measuring method of this invention. It is the graph which showed the change with the time of the light absorbency (405 nm) when six types of samples with known protein S activity value are measured by the protein S activity measuring reagent kit and activity measuring method as a comparative example. It is the graph which showed the change with the time of the light absorbency (405 nm) when six types of samples with known protein S activity value are measured by the protein S activity measuring reagent kit and activity measuring method as a comparative example. It is the graph which showed the change with the time of the light absorbency (405 nm) when six types of samples with known protein S activity value are measured by the protein S activity measuring reagent kit and activity measuring method as a comparative example. It is the graph which showed the change with the time of the light absorbency (405 nm) when six types of samples with known protein S activity value are measured by the protein S activity measuring reagent kit and activity measuring method as a comparative example. It is the graph which showed the comparison of the measurement result of the protein S activity value by the protein S activity measurement reagent kit and the activity measurement method of this invention and each comparative example, and the measurement result of the protein amount of protein S. It is the graph which showed the change by the time of the light absorbency (405 nm) when measuring 6 types of samples with known total protein S activity value by the protein S activity measuring reagent kit and activity measuring method of this invention. It is the graph which showed the change by the time of the light absorbency (405 nm) when measuring 6 types of samples with known total protein S activity value by the protein S activity measuring reagent kit and activity measuring method of this invention. It is the graph which showed the change by the time of the light absorbency (405 nm) when measuring 6 types of samples with known total protein S activity value by the protein S activity measuring reagent kit and activity measuring method of this invention.

[I] Protein S activity measurement reagent kit
1. General remarks
The reagent kit for measuring the activity of protein S of the present invention is a kit for measuring the activity of protein S and is characterized by comprising the following constituent reagents (i) to (iii).
(I) Activated protein C-containing reagent: Contains at least activated protein C.
(Ii) Activated blood coagulation factor V-containing reagent: Contains at least activated blood coagulation factor V.
(Iii) Prothrombin / substrate-containing reagent: Contains at least prothrombin and thrombin substrate.
In the reagent kit for measuring protein S activity of the present invention, at least one reagent selected from an activated protein C-containing reagent and an activated blood coagulation factor V-containing reagent contains phospholipid.
In the protein S activity measurement reagent kit of the present invention, at least one reagent selected from an activated protein C-containing reagent and an activated blood coagulation factor V-containing reagent contains calcium ions.
In the protein S activity measurement reagent kit of the present invention, at least one reagent selected from an activated protein C-containing reagent, an activated blood coagulation factor V-containing reagent and a prothrombin / substrate-containing reagent is used. Contains factor X.
In the protein S activity measurement reagent kit of the present invention, the activated protein C-containing reagent and the activated blood coagulation factor V-containing reagent contain neither prothrombin nor a thrombin substrate.
The reagent kit for measuring the activity of protein S of the present invention is suitable when protein S is total protein S.
2. Reagent containing activated protein C
In the present invention, the activated protein C-containing reagent contains at least activated protein C.
In the present invention, the activated protein C-containing reagent contains neither prothrombin nor a thrombin substrate.
(1) Activated protein C
In the present invention, the activated protein C contained in the activated protein C-containing reagent can be used without any 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.
Further, during the measurement reaction in the protein S activity measurement reagent kit and activity measurement method of the present invention, activated protein C is produced from protein C by a protein C activator or the like, and this is activated protein C in the present invention. It may be used as
In the present invention, when the activated protein C contained in the activated protein C-containing reagent contains protein S in the sample, the activated protein C is brought into contact with the protein S derived from the sample, so that phospholipids and calcium ions are brought into contact with the sample. In the presence of this, the activity of this activated protein C increases.
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.
In the present invention, the concentration at which the activated protein C contained in the activated protein C-containing reagent is used is brought into contact with activated blood coagulation factor V, and activated blood coagulation factor V in the presence of phospholipid and calcium ions. When decomposing a factor, it is usually preferably 1 pM to 200 nM.
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 20 nM, and more preferably 100 pM to 2 nM. Particularly preferred.
Further, in the protein S activity measuring reagent kit and the activity measuring method of the present invention, the activated protein C is adjusted so that the activated protein C has the aforementioned concentration during the degradation reaction of the activated blood coagulation factor V. It is preferable to make it contain in a contained reagent.
For example, in the activated protein C-containing reagent of the present invention, the activated protein C is preferably contained at 1 pM to 1000 nM, more preferably 10 pM to 100 nM, and particularly preferably 100 pM to 10 nM.
(2) Phospholipid
In the present invention, the activated protein C-containing reagent may contain a phospholipid.
In the present invention, this phospholipid must be contained in at least one of an activated protein C-containing reagent and an activated blood coagulation factor V-containing reagent.
A phospholipid may be contained in both the activated protein C-containing reagent and the activated blood coagulation factor V-containing reagent.
Further, phospholipids may be contained in both the activated protein C-containing reagent and the prothrombin / substrate-containing reagent.
Further, phospholipids may be contained in both the activated blood coagulation factor V-containing reagent and the prothrombin / substrate-containing reagent.
Further, phospholipids may be contained in three reagents: an activated protein C-containing reagent, an activated blood coagulation factor V-containing reagent, and a prothrombin / substrate-containing reagent.
Details of this phospholipid will be described later.
(3) Calcium ion
In the present invention, the activated protein C-containing reagent may contain calcium ions.
In the present invention, this calcium ion must be contained in at least one of an activated protein C-containing reagent and an activated blood coagulation factor V-containing reagent.
Note that calcium ions may be contained in both the activated protein C-containing reagent and the activated blood coagulation factor V-containing reagent.
Further, calcium ions may be contained in both the activated protein C-containing reagent and the prothrombin / substrate-containing reagent.
Further, calcium ions may be contained in both the activated blood coagulation factor V-containing reagent and the prothrombin / substrate-containing reagent.
In addition, calcium ions may be contained in two reagents: an activated protein C-containing reagent, an activated blood coagulation factor V-containing reagent, and a prothrombin / substrate-containing reagent.
Details of this calcium ion will be described later.
(4) Activated blood coagulation factor X
In the present invention, the activated protein C-containing reagent may contain activated blood coagulation factor X.
In the present invention, this activated blood coagulation factor X needs to be contained in at least one of an activated protein C-containing reagent, an activated blood coagulation factor V-containing reagent, or a prothrombin / substrate-containing reagent.
Note that activated blood coagulation factor X may be contained in both the activated protein C-containing reagent and the activated blood coagulation factor V-containing reagent.
In addition, activated blood coagulation factor X may be contained in both the activated protein C-containing reagent and the prothrombin / substrate-containing reagent.
In addition, activated blood coagulation factor X may be contained in both the activated blood coagulation factor V-containing reagent and the prothrombin / substrate-containing reagent.
In addition, activated blood coagulation factor X may be contained in three reagents: an activated protein C-containing reagent, an activated blood coagulation factor V-containing reagent, and a prothrombin / substrate-containing reagent.
Details of this activated blood coagulation factor X will be described later.
3. Activated blood coagulation factor V-containing reagent
In the present invention, the activated blood coagulation factor V-containing reagent contains at least activated blood coagulation factor V.
In the present invention, the activated blood coagulation factor V-containing reagent contains neither prothrombin nor a thrombin substrate.
(1) Activated blood coagulation factor V
In the present invention, the activated blood coagulation factor V (factor Va; FVa) contained in the activated blood coagulation factor V-containing reagent is not particularly limited and may be used without particular limitation. it can.
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 present invention, the activated blood coagulation factor V contained in the activated blood coagulation factor V-containing reagent is degraded by activated protein C in the presence of phospholipids and calcium ions.
When protein S is contained in the sample, the presence of protein S increases the activity of activated protein C and promotes the degradation reaction of this activated blood coagulation factor V.
Activated blood coagulation factor V promotes the reaction of generating thrombin from prothrombin catalyzed by activated blood coagulation factor X in the presence of phospholipids and calcium ions.
Therefore, if protein S is contained in the sample, the activity of activated protein C increases, the decomposition reaction of activated blood coagulation factor V is promoted, and the abundance of activated blood coagulation factor V ( (Concentration) decreases.
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.
In addition, activated blood coagulation factor V is brought into contact with activated protein C together with phospholipid and calcium ion, and incubated at least at least 1 minute, preferably at least 3 minutes, more preferably at least 5 minutes at room temperature or 37 ° C. After that, it is preferable to carry out a reaction for contacting with prothrombin or the like and incubating to produce thrombin from prothrombin.
When this activated blood coagulation factor V is used, the concentration of the activated blood coagulation factor V is contacted with activated protein C, and when the reaction for decomposing activated blood coagulation factor V in the presence of phospholipid and calcium ion is performed, Usually, it is preferably 0.5 pM to 25 nM.
However, when the concentration of this activated blood coagulation factor V is high, blood coagulation reaction proceeds due to components (factors) derived from the sample, and fibrin is precipitated or a large amount of color develops, resulting in accurate measurement. Therefore, the activated blood coagulation factor V concentration is more preferably 5 pM to 5 nM, and particularly preferably 20 pM to 1 nM.
In the reagent kit and activity measurement method for protein S of the present invention, the present invention is such that the activated blood coagulation factor V is at the above concentration during the degradation reaction of the activated blood coagulation factor V. It is preferable to contain in the activated blood coagulation factor V-containing reagent.
For example, activated blood coagulation factor V is preferably contained in the activated blood coagulation factor V-containing reagent of the present invention in an amount of 0.5 pM to 1.25 μM, more preferably 5 pM to 250 nM, and more preferably 20 pM to It is particularly preferable to contain 50 nM.
(2) Phospholipid
In the present invention, the activated blood coagulation factor V-containing reagent may contain a phospholipid.
In the present invention, this phospholipid must be contained in at least one of an activated protein C-containing reagent and an activated blood coagulation factor V-containing reagent.
A phospholipid may be contained in both the activated protein C-containing reagent and the activated blood coagulation factor V-containing reagent.
Further, phospholipids may be contained in both the activated protein C-containing reagent and the prothrombin / substrate-containing reagent.
Further, phospholipids may be contained in both the activated blood coagulation factor V-containing reagent and the prothrombin / substrate-containing reagent.
Further, phospholipids may be contained in three reagents: an activated protein C-containing reagent, an activated blood coagulation factor V-containing reagent, and a prothrombin / substrate-containing reagent.
Details of this phospholipid will be described later.
(3) Calcium ion
In the present invention, the activated blood coagulation factor V-containing reagent may contain calcium ions.
In order to stabilize activated blood coagulation factor V, the activated blood coagulation factor V-containing reagent preferably contains calcium ions.
In the present invention, this calcium ion must be contained in at least one of an activated protein C-containing reagent and an activated blood coagulation factor V-containing reagent.
Note that calcium ions may be contained in both the activated protein C-containing reagent and the activated blood coagulation factor V-containing reagent.
Further, calcium ions may be contained in both the activated protein C-containing reagent and the prothrombin / substrate-containing reagent.
Further, calcium ions may be contained in both the activated blood coagulation factor V-containing reagent and the prothrombin / substrate-containing reagent.
In addition, calcium ions may be contained in three reagents: an activated protein C-containing reagent, an activated blood coagulation factor V-containing reagent, and a prothrombin / substrate-containing reagent.
Details of this calcium ion will be described later.
(4) Activated blood coagulation factor X
In the present invention, the activated blood coagulation factor V-containing reagent may contain activated blood coagulation factor X.
In the present invention, this activated blood coagulation factor X needs to be contained in at least one of an activated protein C-containing reagent, an activated blood coagulation factor V-containing reagent, or a prothrombin / substrate-containing reagent.
Note that activated blood coagulation factor X may be contained in both the activated protein C-containing reagent and the activated blood coagulation factor V-containing reagent.
In addition, activated blood coagulation factor X may be contained in both the activated protein C-containing reagent and the prothrombin / substrate-containing reagent.
In addition, activated blood coagulation factor X may be contained in both the activated blood coagulation factor V-containing reagent and the prothrombin / substrate-containing reagent.
In addition, activated blood coagulation factor X may be contained in three reagents of an activated protein C-containing reagent, an activated blood coagulation factor V-containing reagent, and a prothrombin / substrate-containing reagent.
Details of this activated blood coagulation factor X will be described later.
4). Prothrombin / Substrate-containing reagent
In the present invention, the prothrombin / substrate-containing reagent contains at least prothrombin and a thrombin substrate.
(1) Prothrombin
In the present invention, the prothrombin contained in the prothrombin / substrate-containing reagent can be used without any 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 present invention, prothrombin contained in the prothrombin / substrate-containing reagent 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 protein S is contained in the sample, the activity of activated protein C increases, thereby promoting the degradation reaction of activated blood coagulation factor V, and thus activation. Since the promoting effect of activated blood coagulation factor V on the reaction of generating thrombin from prothrombin catalyzed by blood coagulation factor X is reduced, the reaction of generating thrombin from prothrombin is suppressed, and the amount of thrombin generated (Concentration) is reduced.
The concentration at which this prothrombin is used. Since this prothrombin is a substrate for activated blood coagulation factor X, it is preferable that the concentration be saturated in the catalytic reaction by activated blood coagulation factor X. The concentration of prothrombin is usually 1 nM to 100 μM when it is brought into contact with activated blood coagulation factor V and activated blood coagulation factor X to produce thrombin from prothrombin in the presence of phospholipids and calcium ions. It is preferably 10 nM to 10 μM, more preferably 100 nM to 1 μM.
Moreover, in the protein S activity measurement reagent kit and activity measurement method of the present invention, it is preferable that the prothrombin / substrate-containing reagent contains prothrombin so as to have the above concentration during the thrombin generation reaction.
For example, in the prothrombin / substrate-containing reagent in the present invention, prothrombin is preferably contained in the prothrombin / substrate-containing reagent in an amount of 1 nM to 1 mM, more preferably 10 nM to 100 μM, and particularly preferably 100 nM to 10 μM. preferable.
(2) Thrombin substrate
In the present invention, the thrombin substrate contained in the prothrombin / substrate-containing reagent is one that generates some signal upon receiving a catalytic action of thrombin as a protease (as a substrate of thrombin), or in addition to the catalytic reaction by thrombin. Any signal can be used as long as it produces a signal by continuing the reaction.
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.
Examples thereof include those in which the absorbance, transmittance, or fluorescence intensity changes due to the catalytic action of thrombin, in which the light absorption curve changes, or in which light is emitted.
Examples of this include peptides or proteins bound to compounds that change absorbance, transmittance or fluorescence intensity, or light absorption curves when released, or peptides bound to compounds that emit light when released or Examples thereof include proteins and the like, and substances that release the compound from the peptide or protein by the catalytic action of thrombin.
In such substances, thrombin is catalyzed, and when the compound is liberated, it can be detected as absorbance, transmittance or fluorescence intensity, light absorption curve change or light emission, etc. The enzyme activity can be determined by measuring the amount of the generated signal (absorbance, transmittance or fluorescence intensity, change in light absorption curve or light emission, etc.).
As such a substance, for example, “HD-phenylalanyl-L-pipecolyl-L-arginyl-p-nitroanilide dihydrochloride” [Test Team (registered trademark) chromogenic substrate S-2238] (manufacturer : Chromogenix-Instrumentation Laboratories [Italy], distributor: Sekisui Medical [Japan]], “HD-hexahydrotyrosyl-L-alanyl-L-arginyl-p-nitroanilide diacetate” [SPECTROZYME (registered trademark) TH] (AMERICANDIAGNOSTICA, Cosmo Bio), “Benzoyl-phenylalanyl-valinyl-arginyl-p-nitroanilide hydrochloride” [Thrombin Substrate I, Colorimetric] (CAL IOCHEM; Cosmo Bio), “Tosyl-glycyl-prolyl-arginyl-p-nitroanilide” [CHROMOZYME TH] (PENTAPHARM), “HD-phenylalanyl-prolyl-arginyl-3-carboxy-4- "Hydroxy-aniline" (Daiichi Sankyosha), "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, the activated blood coagulation factor X, prothrombin, and the like, and contacting the generated thrombin with the substrate of the thrombin are simultaneously performed.
In any case, the thrombin produced is brought into contact with the thrombin substrate and incubated at least at least 1 minute, preferably at least 3 minutes, more preferably at least 5 minutes at room temperature or 37 ° C. Generate a signal.
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.
In the protein S activity measurement reagent kit and the activity measurement method of the present invention, the thrombin substrate is contained in the prothrombin / substrate-containing reagent so that the thrombin substrate has the above-mentioned concentration when receiving the catalytic action of thrombin. Is preferred.
For example, the thrombin substrate is preferably contained in an amount of 5 μM to 200 mM, particularly preferably 50 μM to 20 mM.
(3) Phospholipid
In the present invention, the prothrombin / substrate-containing reagent may contain a phospholipid.
In the present invention, this phospholipid must be contained in at least one of an activated protein C-containing reagent and an activated blood coagulation factor V-containing reagent.
A phospholipid may be contained in both the activated protein C-containing reagent and the activated blood coagulation factor V-containing reagent.
Further, phospholipids may be contained in both the activated protein C-containing reagent and the prothrombin / substrate-containing reagent.
Further, phospholipids may be contained in both the activated blood coagulation factor V-containing reagent and the prothrombin / substrate-containing reagent.
Further, phospholipids may be contained in three reagents: an activated protein C-containing reagent, an activated blood coagulation factor V-containing reagent, and a prothrombin / substrate-containing reagent.
Details of this phospholipid will be described later.
(4) Calcium ion
In the present invention, the prothrombin / substrate-containing reagent may contain calcium ions.
In the present invention, this calcium ion must be contained in at least one of an activated protein C-containing reagent and an activated blood coagulation factor V-containing reagent.
Note that calcium ions may be contained in both the activated protein C-containing reagent and the activated blood coagulation factor V-containing reagent.
Further, calcium ions may be contained in both the activated protein C-containing reagent and the prothrombin / substrate-containing reagent.
Further, calcium ions may be contained in both the activated blood coagulation factor V-containing reagent and the prothrombin / substrate-containing reagent.
In addition, calcium ions may be contained in three reagents: an activated protein C-containing reagent, an activated blood coagulation factor V-containing reagent, and a prothrombin / substrate-containing reagent.
Details of this calcium ion will be described later.
(5) Activated blood coagulation factor X
In the present invention, the prothrombin / substrate-containing reagent may contain activated blood coagulation factor X.
In the present invention, this activated blood coagulation factor X needs to be contained in at least one of an activated protein C-containing reagent, an activated blood coagulation factor V-containing reagent, or a prothrombin / substrate-containing reagent.
Note that activated blood coagulation factor X may be contained in both the activated protein C-containing reagent and the activated blood coagulation factor V-containing reagent.
In addition, activated blood coagulation factor X may be contained in both the activated protein C-containing reagent and the prothrombin / substrate-containing reagent.
In addition, activated blood coagulation factor X may be contained in both the activated blood coagulation factor V-containing reagent and the prothrombin / substrate-containing reagent.
In addition, activated blood coagulation factor X may be contained in three reagents: an activated protein C-containing reagent, an activated blood coagulation factor V-containing reagent, and a prothrombin / substrate-containing reagent.
Details of this activated blood coagulation factor X will be described later.
5. Phospholipid
In the present invention, the activated protein C-containing reagent, activated blood coagulation factor V-containing reagent, or prothrombin / substrate-containing reagent may contain a phospholipid.
In the present invention, this phospholipid must be contained in at least one of an activated protein C-containing reagent and an activated blood coagulation factor V-containing reagent.
A phospholipid may be contained in both the activated protein C-containing reagent and the activated blood coagulation factor V-containing reagent.
Further, phospholipids may be contained in both the activated protein C-containing reagent and the prothrombin / substrate-containing reagent.
Further, phospholipids may be contained in both the activated blood coagulation factor V-containing reagent and the prothrombin / substrate-containing reagent.
Further, phospholipids may be contained in three reagents: an activated protein C-containing reagent, an activated blood coagulation factor V-containing reagent, and a prothrombin / substrate-containing reagent.
In the reagent kit and activity measuring method for protein S of the present invention, the activated blood coagulation factor V is decomposed by activated protein C during the activity of increasing the activity of activated protein C by protein S contained in the sample. During the reaction and in the reaction of generating thrombin from prothrombin catalyzed by activated blood coagulation factor X in the presence of activated blood coagulation factor V, the activity of activated protein C is increased, activated blood coagulation factor V Phospholipid is present because it is necessary for the factor decomposition reaction and the reaction for generating thrombin from prothrombin.
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.
In the measurement reaction of protein S activity, the concentration at the time when this phospholipid is present is usually preferably 0.0005% (W / V) to 10% (W / V), and preferably 0.001 (W / V). V) to 5% (W / V) is more preferable, and 0.005 (W / V) to 3% (W / V) is particularly preferable.
In the protein S activity measurement reagent kit and the activity measurement method of the present invention, it is preferable that the constituent reagent contains a phospholipid so that the phospholipid has the above-mentioned concentration during the protein S activity measurement reaction. .
For example, in an activated protein C-containing reagent and / or an activated blood coagulation factor V-containing reagent (optionally in addition to a prothrombin / substrate-containing reagent), phospholipid is added in an amount of 0.0005% (W / V) to 20% ( W / V) is preferable, 0.001 (W / V) to 10% (W / V) is more preferable, and 0.005 (W / V) to 6% (W / V) is included. It is particularly preferred that
Examples of the phospholipid include glycerophospholipids such as phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol, and diphosphatidylglycerol, and sphingophospholipids such as sphingomyelin.
In addition, as this phospholipid, it is preferable for the protein S activity measurement to have phospholipid consisting of phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine at the time of the reaction for measuring protein S integrity.
Moreover, when the phospholipid consisting of this phosphatidylcholine, phosphatidylserine and phosphatidylethanolamine is present, the composition of these three types of phospholipids is a composition ratio of phosphatidylethanolamine of 10% (W / V) or more, and The composition ratio of phosphatidylserine is preferably 20% (W / V) or more.
In particular, the composition ratio of phosphatidylethanolamine is preferably 30% (W / V) or more, and the composition ratio of phosphatidylserine is preferably 30% (W / V) or more.
6). Calcium ion
In the present invention, the activated protein C-containing reagent, the activated blood coagulation factor V-containing reagent, or the prothrombin / substrate-containing reagent may contain calcium ions.
In order to stabilize activated blood coagulation factor V, the activated blood coagulation factor V-containing reagent preferably contains calcium ions.
In the present invention, this calcium ion must be contained in at least one of an activated protein C-containing reagent and an activated blood coagulation factor V-containing reagent.
Note that calcium ions may be contained in both the activated protein C-containing reagent and the activated blood coagulation factor V-containing reagent.
Further, calcium ions may be contained in both the activated protein C-containing reagent and the prothrombin / substrate-containing reagent.
Further, calcium ions may be contained in both the activated blood coagulation factor V-containing reagent and the prothrombin / substrate-containing reagent.
In addition, calcium ions may be contained in three reagents: an activated protein C-containing reagent, an activated blood coagulation factor V-containing reagent, and a prothrombin / substrate-containing reagent.
In the reagent kit and activity measuring method for protein S of the present invention, activated blood coagulation factor V by activated protein C is activated during the reaction of increasing the activity of activated protein C by protein S contained in the sample. Calcium ions must be present at the time of the decomposition reaction, and during the reaction of generating thrombin from prothrombin by activated blood coagulation factor X and activated blood coagulation factor V.
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 calcium ions used is that during activation of activated protein C by protein S contained in the sample, during degradation of activated blood coagulation factor V by activated protein C, and activity. In the reaction for producing thrombin from prothrombin by activated blood coagulation factor X and activated blood coagulation factor V, it is usually preferably 0.05 mM to 20 mM.
In the reagent kit and activity measuring method for protein S of the present invention, calcium ion is used during the activity-increasing reaction of activated protein C, during the decomposition reaction of activated blood coagulation factor V, and during the thrombin generation reaction. It is preferable to contain calcium ions in the constituent reagent so that the concentration becomes the above-mentioned concentration.
For example, calcium ion or a compound containing calcium ion is contained in an activated protein C-containing reagent and / or an activated blood coagulation factor V-containing reagent (in some cases, further in a prothrombin / substrate-containing reagent). It is preferable to make it.
7). Activated blood coagulation factor X
In the present invention, the activated protein C-containing reagent, the activated blood coagulation factor V-containing reagent, or the prothrombin / substrate-containing reagent may contain activated blood coagulation factor X.
In the present invention, this activated blood coagulation factor X needs to be contained in at least one of an activated protein C-containing reagent, an activated blood coagulation factor V-containing reagent, or a prothrombin / substrate-containing reagent.
Note that activated blood coagulation factor X may be contained in both the activated protein C-containing reagent and the activated blood coagulation factor V-containing reagent.
In addition, activated blood coagulation factor X may be contained in both the activated protein C-containing reagent and the prothrombin / substrate-containing reagent.
In addition, activated blood coagulation factor X may be contained in both the activated blood coagulation factor V-containing reagent and the prothrombin / substrate-containing reagent.
In addition, activated blood coagulation factor X may be contained in three reagents: an activated protein C-containing reagent, an activated blood coagulation factor V-containing reagent, and a prothrombin / substrate-containing reagent.
This activated blood coagulation factor X (factor Xa; FXa) can be used without any 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 protein S activity measurement reagent kit and activity measurement method of the present invention, activated blood coagulation factor X catalyzes a reaction for producing 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 protein S is contained in the sample, the activity of activated protein C is increased, thereby promoting the degradation reaction of activated blood coagulation factor V, and thus activation. Since the promoting effect of activated blood coagulation factor V on the reaction that generates thrombin catalyzed by blood coagulation factor X is reduced, 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 preferably 1 pM to 3 μM.
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 10 pM to 300 nM, particularly 100 pM to 30 nM. preferable.
In the protein S activity measurement reagent kit and activity measurement method of the present invention, the activated blood coagulation factor is added to the constituent reagent so that the activated blood coagulation factor X becomes the above concentration during the thrombin generation reaction. It is preferable to contain factor X.
For example, the activated blood coagulation factor X is preferably contained in an activated protein C-containing reagent, an activated blood coagulation factor V-containing reagent, and / or a prothrombin / substrate-containing reagent in an amount of 1 pM to 3 μM. It is more preferably contained, and particularly preferably 100 pM to 30 nM.
8). Surfactant
In the present invention, the activated protein C-containing reagent, the activated blood coagulation factor V-containing reagent, or the prothrombin / substrate-containing reagent may contain a surfactant.
Any one of these activated protein C-containing reagents, activated blood coagulation factor V-containing reagents, and prothrombin / substrate-containing reagents may contain a surfactant, or any two of them. One reagent may contain a surfactant, or any of these three reagents may contain a surfactant.
As the surfactant, one or more kinds of various surfactants such as a nonionic surfactant, an amphoteric surfactant, an anionic surfactant or a cationic surfactant are appropriately present. You can do it.
Examples of the surfactant include sorbitan fatty acid ester, glycerin fatty acid ester, decaglycerin fatty acid ester, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene glycerin fatty acid ester, polyethylene glycol fatty acid ester, polyoxyethylene alkyl ether, polyoxyethylene Nonionic surfactants such as phytosterol, phytostanol, polyoxyethylene polyoxypropylene alkyl ether, polyoxyethylene alkylphenyl ether, polyoxyethylene castor oil, hydrogenated castor oil or polyoxyethylene lanolin; amphoteric interfaces such as betaine acetate Activator; anion such as polyoxyethylene alkyl ether sulfate or polyoxyethylene alkyl ether acetate Sex surfactants; or, may be mentioned cationic surfactants such as amine salts or quaternary ammonium salts.
When this surfactant is a nonionic surfactant, its HLB (Hydrophile-Lipophile Balance) is preferably in the range of 10 to 20, more preferably in the range of 12 to 18, and 13 to 16 The thing of the range of is especially preferable.
As the nonionic surfactant, this surfactant is Triton X-100 [polyoxyethylene (n = 9,10) pt-octylphenyl ether, HLB: 13.5], Triton X-114 [ Polyoxyethylene (n = 7,8) pt-octylphenyl ether, HLB: 12.4], NP-10 [Polyoxyethylene (n = 10) nonylphenyl ether, HLB: 16.5], NP- 11 [polyoxyethylene (n = 11) nonylphenyl ether], NP-12 [polyoxyethylene (n = 12) nonylphenyl ether], NP-13 [polyoxyethylene (n = 13) nonylphenyl ether], NP -15 [polyoxyethylene (n = 15) nonylphenyl ether, HLB: 18.0], BT-9 [polyoxy Ethylene (n = 9) 2 alkyl ether, HLB: 13.5], or BT-12 [polyoxyethylene (n = 12) 2 alkyl ether, HLB: 14.5] and the like are preferable.
Moreover, as an amphoteric surfactant, AM-301 [lauryl dimethylamino acetic acid betaine aqueous solution] etc. are preferable.
And as anionic surfactant, sarcosinate LN [lauroyl sarcosine sodium] etc. are preferable.
Furthermore, as the cationic surfactant, CA-2350 [cetyltrimethylammonium chloride] and the like are preferable.
The concentration of the surfactant in the measurement reaction of protein S activity is not particularly limited, but is preferably 0.00001-5% (W / V), 0.0001-1% ( W / V) is more preferable, 0.001 to 0.1% (W / V) is further preferable, and 0.005 to 0.05% (W / V) is particularly preferable.
For nonionic surfactants, 0.001 to 0.01% (W / V) is very preferable, and for zwitterionic surfactants, 0.001 to 0.01% (W / V) is used. Highly preferred, 0.001 to 0.1% (W / V) is highly preferred for anionic surfactants, and 0.00001 to 0.001% (W / V) for cationic surfactants. Is highly preferred.
In the protein S activity measurement reagent kit and activity measurement method of the present invention, the constituent reagent may contain a surfactant so that the surfactant has the above-mentioned concentration during the protein S activity measurement reaction. preferable.
For example, it is preferable to contain 0.00001 to 10% (W / V) of a surfactant in an activated protein C-containing reagent, an activated blood coagulation factor V-containing reagent, and / or a prothrombin / substrate-containing reagent, It is more preferable to contain 0.0001 to 2% (W / V), still more preferably 0.001 to 0.2% (W / V), and 0.005 to 0.1% (W / V). It is particularly preferable to contain them.
For nonionic surfactants, 0.001 to 0.02% (W / V), for amphoteric surfactants, 0.001 to 0.02% (W / V), anionic interfaces It is very preferable that the activator is contained at a concentration of 0.001 to 0.2% (W / V), and the cationic surfactant is contained at a concentration of 0.00001 to 0.002% (W / V).
In the protein S activity measurement reagent kit and activity measurement method of the present invention, the activated blood coagulation caused by the protein C increased in activity and the activated protein C activated by protein S contained in the sample. The presence of a surfactant during the factor V decomposition reaction is preferred for measuring the activity of protein S contained in the sample.
9. protein
In the present invention, the activated protein C-containing reagent, activated blood coagulation factor V-containing reagent, or prothrombin / substrate-containing reagent is albumin such as human serum albumin (HSA), bovine serum albumin (BSA) or ovalbumin, or casein. Or a protein such as gelatin.
Any one of the activated protein C-containing reagent, the activated blood coagulation factor V-containing reagent, and the prothrombin / substrate-containing reagent may contain the protein, or any two of the two The reagent may contain the protein, or either of these two reagents may contain the protein.
The concentration of the protein is usually preferably 0.001% (w / v) to 10% (w / v), preferably 0.01% (w / v) when measuring the protein S activity. It is more preferably from 5% (w / v), particularly preferably from 0.1% (w / v) to 1% (w / v).
Moreover, in the protein S activity measurement reagent kit and activity measurement method of the present invention, it is preferable to contain the protein in the constituent reagents so that the protein has the aforementioned concentration when the protein S activity is measured.
For example, the protein is added to an activated protein C-containing reagent, an activated blood coagulation factor V-containing reagent, and / or a prothrombin / substrate-containing reagent in an amount of 0.001% (w / v) to 10% (w / v). It is preferably contained, more preferably 0.01% (w / v) to 5% (w / v), and 0.1% (w / v) to 1% (w / v). Is particularly preferred.
10. salt
In the present invention, the activated protein C-containing reagent, the activated blood coagulation factor V-containing reagent, or the prothrombin / substrate-containing reagent is a salt such as a halogen element and alkali metal salt or a halogen element and alkaline earth metal salt. It may be included.
Any one of the activated protein C-containing reagent, the activated blood coagulation factor V-containing reagent, and the prothrombin / substrate-containing reagent may contain the salt, or any two The reagent may contain this salt, or any of these three reagents may contain this salt.
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 of the salt is usually preferably 5 mM to 1 M, more preferably 50 mM to 250 mM, when measuring the protein S activity.
Further, in the protein S activity measurement reagent kit and activity measurement method of the present invention, it is preferable to contain the salt in the constituent reagent so that the salt has the above-mentioned concentration at the time of protein S activity measurement.
For example, the salt is preferably contained in an activated protein C-containing reagent, an activated blood coagulation factor V-containing reagent, and / or a prothrombin / substrate-containing reagent in an amount of 5 mM to 2 M, more preferably 50 mM to 500 mM. preferable.
11. pH
In the protein S activity measurement reagent kit and activity measurement method of the present invention, the protein S activity measurement reaction is preferably performed in the range of pH 6.0 to pH 10.0 (20 ° C.), and pH 6.5 to pH 8. It is particularly preferable to carry out within a range of 5 (20 ° C.).
In the protein S activity measurement reagent kit and activity measurement method of the present invention, it is preferable to set the pH of the constituent reagent so that the pH becomes the above-mentioned pH when protein S activity is measured.
In the protein S activity measurement reagent kit and activity measurement method of the present invention, a buffer having a buffering capacity in the pH range described above in order to keep the pH in the activity measurement reaction or the pH of the constituent reagent in the pH range. Is preferably present or contained as appropriate.
12 Other ingredients
In the protein S activity measurement reagent kit and activity measurement method of the present invention, components other than the above-described components such as preservatives, stabilizers, activators, or saccharides are appropriately added as necessary to the constituent reagents. Or can be present at the time of protein S activity measurement.
In the protein S activity measurement reagent kit and the activity measurement method of the present invention, components other than the above components can be included, if necessary, or can be present at the time of protein S activity measurement.
13. Specific example of protein S activity measurement reagent kit
The following (1) to (6) are shown as specific examples of the protein S activity measurement reagent kit of the present invention.
(1)
(I) Activated protein C-containing reagent: Contains activated protein C.
(Ii) Activated blood coagulation factor V-containing reagent: Contains activated blood coagulation factor V, activated blood coagulation factor X, phospholipid, and calcium salt.
(Iii) Prothrombin / substrate-containing reagent: Contains prothrombin and thrombin substrate.
(2)
(I) Reagent containing activated protein C: Contains activated protein C, phospholipid, and calcium salt.
(Ii) Activated blood coagulation factor V-containing reagent: Contains activated blood coagulation factor V and activated blood coagulation factor X.
(Iii) Prothrombin / substrate-containing reagent: Contains prothrombin and thrombin substrate.
(3)
(I) Activated protein C-containing reagent: Contains activated protein C.
(Ii) Activated blood coagulation factor V-containing reagent: comprises activated blood coagulation factor V, phospholipid, and calcium salt.
(Iii) Prothrombin / substrate-containing reagent: Contains prothrombin, a substrate for thrombin, and activated blood coagulation factor X.
(4)
(I) Reagent containing activated protein C: Contains activated protein C, phospholipid, and calcium salt.
(Ii) Activated blood coagulation factor V-containing reagent: Contains activated blood coagulation factor V.
(Iii) Prothrombin / substrate-containing reagent: Contains prothrombin, a substrate for thrombin, and activated blood coagulation factor X.
(5)
(I) Reagent containing activated protein C: Contains activated protein C and activated blood coagulation factor X.
(Ii) Activated blood coagulation factor V-containing reagent: comprises activated blood coagulation factor V, phospholipid, and calcium salt.
(Iii) Prothrombin / substrate-containing reagent: Contains prothrombin and thrombin substrate.
(6)
(I) Activated protein C-containing reagent: Contains activated protein C, activated blood coagulation factor X, phospholipid, and calcium salt.
(Ii) Activated blood coagulation factor V-containing reagent: Contains activated blood coagulation factor V.
(Iii) Prothrombin / substrate-containing reagent: Contains prothrombin and thrombin substrate.
[II] Method for measuring protein S activity
1. General remarks
The protein S activity measurement method of the present invention is a protein S activity measurement method, characterized by using the protein S activity measurement reagent kit.
The details of the protein S activity measurement reagent kit are as described in the above section “[I] Protein S activity measurement reagent kit”.
In addition, the method for measuring the activity of protein S of the present invention is suitable when protein S is total protein S.
2. Protein S activity measurement
(1) In the protein S activity measurement method of the present invention, an activated protein C-containing reagent, an activated blood coagulation factor V-containing reagent, and prothrombin, which are constituent reagents of the protein S activity measurement reagent kit to be used The measurement can be performed by mixing a substrate-containing reagent and a sample to be measured for protein S activity.
(2) More specifically, a sample is mixed with an activated protein C-containing reagent, an activated blood coagulation factor V-containing reagent, and a prothrombin / substrate-containing reagent, which are constituent reagents of a protein S activity measurement reagent kit Then, the amount of signal generated from the thrombin substrate by the reaction performed by the mixing is measured, and the activity value of protein S contained in the sample can be determined from the amount of this signal.
(3) For example, in the protein S activity measurement method of the present invention, using the protein S activity measurement reagent kit, the protein S activity contained in the sample is measured as follows. Can do.
(1-a) A sample and an activated protein C-containing reagent are mixed.
(1-b) All or a part of the mixture in (1-a) is mixed with an activated blood coagulation factor V-containing reagent.
(1-c) All or a part of the mixture in (1-b) is mixed with a prothrombin / substrate-containing reagent.
(1-d) The amount of signal generated from the thrombin substrate by the reaction in the step (1-c) is measured.
(1-e) The activity value of protein S contained in the sample is determined from the amount of signal measured in (1-d).
For example, the protein S activity measurement reagent kit may be used to measure the protein S activity contained in the sample as follows.
(2-a) A sample and an activated blood coagulation factor V-containing reagent are mixed.
(2-b) The whole or a part of the mixture in (2-a) is mixed with the activated protein C-containing reagent.
(2-c) The whole or a part of the mixture in (2-b) is mixed with the prothrombin / substrate-containing reagent.
(2-d) The amount of signal generated from the thrombin substrate by the reaction in the step (2-c) is measured.
(2-e) The activity value of protein S contained in the sample is determined from the amount of signal measured in (2-d).
(4) More specifically, in the protein S activity measurement method of the present invention, the above-described protein S activity measurement reagent kit is used, for example, as shown in [A] to [F] below. The activity of protein S contained in the sample can be measured (see the following reaction formulas).

[A]
◎ Reagent kit for protein S activity measurement
(I) Activated protein C-containing reagent: Contains activated protein C.
(Ii) Activated blood coagulation factor V-containing reagent: Contains activated blood coagulation factor V, activated blood coagulation factor X, phospholipid, and calcium salt.
(Iii) Prothrombin / substrate-containing reagent: Contains prothrombin and thrombin substrate.
◎ Measurement
(A-1) The sample and the activated protein C-containing reagent are mixed.
(A-2) By mixing all or part of the mixture of (A-1) with an activated blood coagulation factor V-containing reagent and bringing each component into contact with each other, in the coexistence of phospholipids and calcium ions, When the sample contains protein S, the presence of protein S increases the activity of activated protein C.
In the presence of phospholipids and calcium ions, a decomposition reaction of activated blood coagulation factor V catalyzed by activated protein C is performed.
In addition, the decomposition reaction of the activated blood coagulation factor V is promoted by the increased activity of the activated protein C.
(A-3) Activated blood coagulation in the coexistence of phospholipids and calcium ions by mixing all or part of the mixture of (A-2) with a prothrombin / substrate-containing reagent and bringing the components into contact with each other. A reaction is carried out to generate thrombin from prothrombin catalyzed by factor X.
Then, a reaction for generating a signal from a substrate of thrombin catalyzed by the generated thrombin is performed.
Since the thrombin generation reaction is promoted by activated blood coagulation factor V, the activation of activated protein C in the step (A-2) increases the activity of (A-2). By promoting the decomposition reaction of activated blood coagulation factor V in the process, the thrombin generation reaction is suppressed, and the thrombin generation is reduced.
The reduction in the generation of thrombin suppresses the reaction that generates a signal from the thrombin substrate catalyzed by thrombin.
(A-4) The amount of signal generated from the thrombin substrate by the reaction catalyzed by thrombin generated in the step (A-3) is measured.
(A-5) The activity value of protein S contained in the sample is determined from the amount of signal measured in the step (A-4).
In addition, the amount of the signal measured in the step (A-4) is the amount of the signal whose generation is suppressed by the suppression of the reaction that generates the signal from the thrombin substrate in the step (A-3). Therefore, the activity value of protein S contained in the sample can be determined from the amount of the signal in which this generation is suppressed.
[B]
◎ Reagent kit for protein S activity measurement
(I) Reagent containing activated protein C: Contains activated protein C, phospholipid, and calcium salt.
(Ii) Activated blood coagulation factor V-containing reagent: Contains activated blood coagulation factor V and activated blood coagulation factor X.
(Iii) Prothrombin / substrate-containing reagent: Contains prothrombin and thrombin substrate.
◎ Measurement
(B-1) The presence of protein S in the coexistence of phospholipids and calcium ions when the sample is mixed with the reagent containing activated protein C and brought into contact with each component so that the sample contains protein S Increases the activity of activated protein C.
(B-2) By mixing all or part of the mixture of (B-1) with an activated blood coagulation factor V-containing reagent and bringing the components into contact with each other, in the coexistence of phospholipids and calcium ions, A decomposition reaction of activated blood coagulation factor V catalyzed by activated protein C is performed.
In addition, the decomposition reaction of the activated blood coagulation factor V is promoted by the increased activity of activated protein C in the step (B-1).
(B-3) Activated blood coagulation in the presence of phospholipids and calcium ions by mixing all or part of the mixture of (B-2) with a prothrombin / substrate-containing reagent and bringing the components into contact with each other. A reaction is carried out to generate thrombin from prothrombin catalyzed by factor X.
Then, a reaction for generating a signal from a substrate of thrombin catalyzed by the generated thrombin is performed.
Since the thrombin generation reaction is promoted by activated blood coagulation factor V, the activation of activated protein C in the step (B-1) increases the activity of (B-2). By promoting the decomposition reaction of activated blood coagulation factor V in the process, the thrombin generation reaction is suppressed, and the thrombin generation is reduced.
The reduction in the generation of thrombin suppresses the reaction that generates a signal from the thrombin substrate catalyzed by thrombin.
(B-4) The amount of signal generated from the thrombin substrate by the reaction catalyzed by the thrombin generated in the step (B-3) is measured.
(B-5) The activity value of protein S contained in the sample is determined from the amount of signal measured in the step (B-4).
The amount of the signal measured in the step (B-4) is the amount of the signal whose production was suppressed by the suppression of the reaction that generates the signal from the thrombin substrate in the step (B-3). Therefore, the activity value of protein S contained in the sample can be determined from the amount of the signal in which this generation is suppressed.
[C]
◎ Reagent kit for protein S activity measurement
(I) Activated protein C-containing reagent: Contains activated protein C.
(Ii) Activated blood coagulation factor V-containing reagent: comprises activated blood coagulation factor V, phospholipid, and calcium salt.
(Iii) Prothrombin / substrate-containing reagent: Contains prothrombin, a substrate for thrombin, and activated blood coagulation factor X.
◎ Measurement
(C-1) A sample and an activated protein C-containing reagent are mixed.
(C-2) By mixing all or part of the mixture of (C-1) with an activated blood coagulation factor V-containing reagent and bringing the components into contact with each other, in the coexistence of phospholipids and calcium ions, When the sample contains protein S, the presence of protein S increases the activity of activated protein C.
In the presence of phospholipids and calcium ions, a decomposition reaction of activated blood coagulation factor V catalyzed by activated protein C is performed.
In addition, the decomposition reaction of the activated blood coagulation factor V is promoted by the increased activity of the activated protein C.
(C-3) Activated blood coagulation in the presence of phospholipids and calcium ions by mixing all or part of the mixture of (C-2) with a prothrombin / substrate-containing reagent and bringing the components into contact with each other. A reaction is carried out to generate thrombin from prothrombin catalyzed by factor X.
Then, a reaction for generating a signal from a substrate of thrombin catalyzed by the generated thrombin is performed.
Since the thrombin generation reaction is promoted by activated blood coagulation factor V, the activation of activated protein C in the step (C-2) increases the activity of (C-2). By promoting the decomposition reaction of activated blood coagulation factor V in the process, the thrombin generation reaction is suppressed, and the thrombin generation is reduced.
The reduction in the generation of thrombin suppresses the reaction that generates a signal from the thrombin substrate catalyzed by thrombin.
(C-4) The amount of signal produced from the thrombin substrate by the reaction catalyzed by the thrombin produced in the step (C-3) is measured.
(C-5) The activity value of protein S contained in the sample is determined from the amount of signal measured in the step (C-4).
In addition, the amount of the signal measured in the step (C-4) is the amount of the signal whose generation is suppressed by the suppression of the reaction that generates the signal from the thrombin substrate in the step (C-3). Therefore, the activity value of protein S contained in the sample can be determined from the amount of the signal in which this generation is suppressed.
[D]
◎ Reagent kit for protein S activity measurement
(I) Reagent containing activated protein C: Contains activated protein C, phospholipid, and calcium salt.
(Ii) Activated blood coagulation factor V-containing reagent: Contains activated blood coagulation factor V.
(Iii) Prothrombin / substrate-containing reagent: Contains prothrombin, a substrate for thrombin, and activated blood coagulation factor X.
◎ Measurement
(D-1) The presence of protein S in the coexistence of phospholipids and calcium ions when the sample is mixed with the reagent containing activated protein C and brought into contact with each component so that the sample contains protein S Increases the activity of activated protein C.
(D-2) By mixing all or part of the mixture of (D-1) with an activated blood coagulation factor V-containing reagent and bringing the components into contact with each other, in the coexistence of phospholipids and calcium ions, A decomposition reaction of activated blood coagulation factor V catalyzed by activated protein C is performed.
In addition, the decomposition reaction of the activated blood coagulation factor V is promoted by the increased activity of activated protein C in the step (D-1).
(D-3) Activated blood coagulation in the presence of phospholipids and calcium ions by mixing all or part of the mixture of (D-2) with a prothrombin / substrate-containing reagent and bringing the components into contact with each other. A reaction is carried out to generate thrombin from prothrombin catalyzed by factor X.
Then, a reaction for generating a signal from a substrate of thrombin catalyzed by the generated thrombin is performed.
Since the thrombin generation reaction is promoted by activated blood coagulation factor V, the activation of activated protein C in the step (D-1) increases the activity of (D-2). By promoting the decomposition reaction of activated blood coagulation factor V in the process, the thrombin generation reaction is suppressed, and the thrombin generation is reduced.
The reduction in the generation of thrombin suppresses the reaction that generates a signal from the thrombin substrate catalyzed by thrombin.
(D-4) The amount of signal generated from the thrombin substrate by the reaction catalyzed by the thrombin generated in the step (D-3) is measured.
(D-5) The activity value of protein S contained in the sample is determined from the amount of signal measured in the step (D-4).
In addition, the amount of the signal measured in the step (D-4) is the amount of the signal whose generation is suppressed by suppressing the reaction that generates the signal from the thrombin substrate in the step (D-3). Therefore, the activity value of protein S contained in the sample can be determined from the amount of the signal in which this generation is suppressed.
[E]
◎ Reagent kit for protein S activity measurement
(I) Reagent containing activated protein C: Contains activated protein C and activated blood coagulation factor X.
(Ii) Activated blood coagulation factor V-containing reagent: comprises activated blood coagulation factor V, phospholipid, and calcium salt.
(Iii) Prothrombin / substrate-containing reagent: Contains prothrombin and thrombin substrate.
◎ Measurement
(E-1) The sample and the activated protein C-containing reagent are mixed.
(E-2) By mixing all or part of the mixture of (E-1) with an activated blood coagulation factor V-containing reagent and bringing the components into contact with each other, in the presence of phospholipids and calcium ions, When the sample contains protein S, the presence of protein S increases the activity of activated protein C.
In the presence of phospholipids and calcium ions, a decomposition reaction of activated blood coagulation factor V catalyzed by activated protein C is performed.
In addition, the decomposition reaction of the activated blood coagulation factor V is promoted by the increased activity of the activated protein C.
(E-3) Activated blood coagulation in the presence of phospholipids and calcium ions by mixing all or part of the mixture of (E-2) with a prothrombin / substrate-containing reagent and bringing each component into contact with each other. A reaction is carried out to generate thrombin from prothrombin catalyzed by factor X.
Then, a reaction for generating a signal from a substrate of thrombin catalyzed by the generated thrombin is performed.
Since the thrombin generation reaction is promoted by activated blood coagulation factor V, the activation of activated protein C in the step (E-2) increases the activity of (E-2). By promoting the decomposition reaction of activated blood coagulation factor V in the process, the thrombin generation reaction is suppressed, and the thrombin generation is reduced.
The reduction in the generation of thrombin suppresses the reaction that generates a signal from the thrombin substrate catalyzed by thrombin.
(E-4) The amount of signal generated from the thrombin substrate by the reaction catalyzed by thrombin generated in the step (E-3) is measured.
(E-5) The activity value of protein S contained in the sample is determined from the amount of signal measured in the step (E-4).
In addition, the amount of the signal measured in the step (E-4) is the amount of the signal whose production is suppressed by the suppression of the reaction that generates the signal from the thrombin substrate in the step (E-3). Therefore, the activity value of protein S contained in the sample can be determined from the amount of the signal in which this generation is suppressed.
[F]
◎ Reagent kit for protein S activity measurement
(I) Activated protein C-containing reagent: Contains activated protein C, activated blood coagulation factor X, phospholipid, and calcium salt.
(Ii) Activated blood coagulation factor V-containing reagent: Contains activated blood coagulation factor V.
(Iii) Prothrombin / substrate-containing reagent: Contains prothrombin and thrombin substrate.
◎ Measurement
(F-1) The presence of protein S in the coexistence of phospholipid and calcium ion when the sample and protein S-containing reagent are mixed and the components are brought into contact with each other so that the sample contains protein S. Increases the activity of activated protein C.
(F-2) By mixing all or part of the mixture of (F-1) with an activated blood coagulation factor V-containing reagent and bringing each component into contact with each other, in the coexistence of phospholipids and calcium ions, A decomposition reaction of activated blood coagulation factor V catalyzed by activated protein C is performed.
In addition, the decomposition reaction of the activated blood coagulation factor V is promoted by the increased activity of activated protein C in the step (F-1).
(F-3) Activated blood coagulation in the presence of phospholipids and calcium ions by mixing all or part of the mixture of (F-2) with a prothrombin / substrate-containing reagent and bringing them into contact with each other. A reaction is carried out to generate thrombin from prothrombin catalyzed by factor X.
Then, a reaction for generating a signal from a substrate of thrombin catalyzed by the generated thrombin is performed.
Since the thrombin generation reaction is promoted by activated blood coagulation factor V, the increase in activated protein C activity in the step (F-1) results in the above (F-2). By promoting the decomposition reaction of activated blood coagulation factor V in the process, the thrombin generation reaction is suppressed, and the thrombin generation is reduced.
The reduction in the generation of thrombin suppresses the reaction that generates a signal from the thrombin substrate catalyzed by thrombin.
(F-4) The amount of signal generated from the thrombin substrate by the reaction catalyzed by thrombin generated in the step (F-3) is measured.
(F-5) The activity value of protein S contained in the sample is determined from the amount of signal measured in the step (F-4).
In addition, the amount of the signal measured in the step (F-4) is the amount of the signal whose generation is suppressed by suppressing the reaction that generates the signal from the thrombin substrate in the step (F-3). Therefore, the activity value of protein S contained in the sample can be determined from the amount of the signal in which this generation is suppressed.
(5) Moreover, in the protein S activity measurement method of the present invention, the above-described protein S activity measurement reagent kit is used, for example, as shown in [G] and [H] below. It is also possible to measure the activity of the protein S that has been stored (see the above reaction formula [Chemical Formula 3], respectively).
[G]
◎ Reagent kit for protein S activity measurement
(I) Reagent containing activated protein C: Contains activated protein C, phospholipid, and calcium salt.
(Ii) Activated blood coagulation factor V-containing reagent: Contains activated blood coagulation factor V and activated blood coagulation factor X.
(Iii) Prothrombin / substrate-containing reagent: Contains prothrombin and thrombin substrate.
◎ Measurement
(G-1) A sample and an activated blood coagulation factor V-containing reagent are mixed and each component is brought into contact.
(G-2) When protein S is contained in the sample by mixing all or part of the mixture of (G-1) with an activated protein C-containing reagent and bringing each component into contact, In the presence of lipid and calcium ions, the presence of protein S increases the activity of activated protein C.
In the presence of phospholipids and calcium ions, a decomposition reaction of activated blood coagulation factor V catalyzed by activated protein C is performed.
Note that, due to the increased activity of activated protein C due to the presence of protein S, the degradation reaction of this activated blood coagulation factor V is promoted.
(G-3) Activated blood coagulation in the presence of phospholipids and calcium ions by mixing all or part of the mixture of (G-2) with a prothrombin / substrate-containing reagent and bringing them into contact with each other. A reaction is carried out to generate thrombin from prothrombin catalyzed by factor X.
Then, a reaction for generating a signal from a substrate of thrombin catalyzed by the generated thrombin is performed.
Since the thrombin generation reaction is promoted by activated blood coagulation factor V, activated blood coagulation factor V due to increased activity of activated protein C in the step (G-2). By promoting the decomposition reaction, thrombin generation reaction is suppressed, and thrombin generation is reduced.
The reduction in the generation of thrombin suppresses the reaction that generates a signal from the thrombin substrate catalyzed by thrombin.
(G-4) The amount of signal generated from the thrombin substrate by the reaction catalyzed by thrombin generated in the step (G-3) is measured.
(G-5) The activity value of protein S contained in the sample is determined from the amount of signal measured in the step (G-4).
In addition, the amount of the signal measured in the step (G-4) is the amount of the signal whose generation is suppressed by suppressing the reaction that generates the signal from the thrombin substrate in the step (G-3). Therefore, the activity value of protein S contained in the sample can be determined from the amount of the signal in which the generation is suppressed.
[H]
◎ Reagent kit for protein S activity measurement
(I) Activated protein C-containing reagent: Contains activated protein C, activated blood coagulation factor X, phospholipid, and calcium salt.
(Ii) Activated blood coagulation factor V-containing reagent: Contains activated blood coagulation factor V.
(Iii) Prothrombin / substrate-containing reagent: Contains prothrombin and thrombin substrate.
◎ Measurement
(H-1) A sample and an activated blood coagulation factor V-containing reagent are mixed and each component is brought into contact.
(H-2) When all or part of the mixture of (H-1) is mixed with an activated protein C-containing reagent and brought into contact with each component, the protein S is contained in the sample. In the presence of lipid and calcium ions, the presence of protein S increases the activity of activated protein C.
In the presence of phospholipids and calcium ions, a decomposition reaction of activated blood coagulation factor V catalyzed by activated protein C is performed.
Note that, due to the increased activity of activated protein C due to the presence of protein S, the degradation reaction of this activated blood coagulation factor V is promoted.
(H-3) Activated blood coagulation in the presence of phospholipids and calcium ions by mixing all or part of the mixture of (H-2) with a prothrombin / substrate-containing reagent and bringing the components into contact with each other. A reaction is carried out to generate thrombin from prothrombin catalyzed by factor X.
Then, a reaction for generating a signal from a substrate of thrombin catalyzed by the generated thrombin is performed.
Since the thrombin generation reaction is promoted by activated blood coagulation factor V, activated blood coagulation factor V due to increased activity of activated protein C in the step (H-2). By promoting the decomposition reaction, thrombin generation reaction is suppressed, and thrombin generation is reduced.
The reduction in the generation of thrombin suppresses the reaction that generates a signal from the thrombin substrate catalyzed by thrombin.
(H-4) The amount of signal produced from the thrombin substrate by the reaction catalyzed by the thrombin produced in the step (H-3) is measured.
(H-5) The activity value of protein S contained in the sample is determined from the amount of signal measured in the step (H-4).
In addition, the amount of the signal measured in the step (H-4) is the amount of the signal whose generation is suppressed by the suppression of the reaction that generates the signal from the thrombin substrate in the step (H-3). Therefore, the activity value of protein S contained in the sample can be determined from the amount of the signal in which this generation is suppressed.
3. sample
In the reagent kit and activity measurement method for protein S of the present invention, the sample is a substance that may contain protein S and is a substance for which the activity of protein S is to be measured.
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.
4). Measurement of signal amount
The thrombin substrate in the protein S activity measurement reagent kit and activity measurement method of the present invention generates a signal by being catalyzed by thrombin generated from prothrombin.
In the protein S activity measurement reagent kit and activity measurement method of the present invention, the amount of signal generated from the thrombin substrate is measured.
In addition, when protein S 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 protein S contained 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, fluorescence intensity, light absorption curve or light emission, the absorbance, transmittance, fluorescence intensity, emission intensity, or the like is measured.
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 may be measured by an end point method (end point method) or a reaction rate method (rate method) for obtaining an amount of change in absorbance per unit time (that is, a rate of change in absorbance).
5. Activity value of protein S contained in the sample
In the protein S activity measurement reagent kit and activity measurement method of the present invention, if the sample contains protein S, the activity of activated protein C increases, and the degradation reaction of activated blood coagulation factor V occurs. Promoted, the abundance (concentration) of activated blood coagulation factor V decreases.
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 protein S contained in the sample.
Therefore, in the protein S activity measurement reagent kit and activity measurement method of the present invention, the amount of signal generated by contacting the sample with activated protein C or the like and carrying out the reaction as described above is measured, that is, By measuring the amount of signal whose production is suppressed, the activity value of protein S contained in the sample is obtained.
Obtaining the activity value of protein S contained in the sample after measuring the amount of signal in which this generation is suppressed may be appropriately performed. For example, it can be performed as follows.
For at least one sample whose protein S activity value is known and for a sample whose protein S activity value is known to be “zero” (such as physiological saline or pure water), perform the measurement operation as described above. The amount of signal generated (the amount of signal with suppressed generation) is determined.
Then, a calibration curve is created by expressing the relationship between the generated signal amount (signal amount in which generation is suppressed) and the activity value of protein S contained in the sample in a mathematical expression or a graph.
This calibration curve represents the relationship between the degree of inhibition of signal generation and the activity value of protein S contained in the sample.
Next, a measurement operation is performed in the same manner as described above for a sample whose protein S activity value is unknown, and the amount of signal generated (the amount of signal with suppressed generation) is determined.
This signal amount is applied to the calibration curve, and the corresponding protein S activity value is determined.
Thereby, the activity value of protein S of the sample can be obtained from the amount of signal generated in the sample whose protein S activity value is unknown (the amount of signal whose generation is suppressed).
The calibration curve represents the relationship between the amount of signal (the amount of signal with suppressed production), that is, the degree of inhibition of signal production, and the activity value of protein S contained in the sample. The amount of signal in (the amount of signal in which production is suppressed) 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 measured signal amount and the activity value of protein S 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 in absorbance per unit time (for example, 1 minute) obtained by measurement, or the rate of change in absorbance obtained by first-order differentiation of the absorbance obtained by measurement with respect to time, or obtained by measurement The acceleration of the change in absorbance obtained by second-order differentiation of the obtained absorbance with respect to time can be exemplified.
6). Sample dilution
In the reagent kit and activity measuring method for protein S of the present invention, it is preferable for measuring the activity of protein S that the sample is diluted in measuring the activity of protein S contained in the sample.
This sample is diluted. For example, the sample and the reagent of the reagent kit for measuring the activity of protein S of the present invention (reactive protein C-containing reagent, activated blood coagulation factor V-containing reagent, and / or prothrombin) This sample can be diluted by mixing with (substrate-containing reagent).
In the protein S activity measurement reagent kit and activity measurement method of the present invention, a sample, an activated protein C-containing reagent, and an activated blood coagulation factor V-containing reagent were mixed to form a mixed solution thereof. Preferably, the sample is diluted in stages.
Although it is a dilution ratio when the sample is diluted by mixing this sample with the constituent reagent of the protein S activity measurement reagent kit, etc., there is no particular limitation, but the measurement of the activity of protein S contained in the sample is not limited. Therefore, it is preferably 2 times to 50000 times, more preferably 50 times to 40000 times, and particularly preferably 500 times to 20000 times.
Therefore, in the protein S activity measurement reagent kit and activity measurement method of the present invention, when the sample and the component reagent of the protein S activity measurement reagent kit are mixed, the dilution ratio of the sample after the mixing is It is preferable to select the mixing ratio of the sample and the constituent reagents (ratio of the respective addition amounts) so that the magnification becomes.
In the protein S activity measurement reagent kit and activity measurement method of the present invention, when measuring the activity of protein S contained in a sample using an automatic analyzer or the like, there is a function of diluting the sample in the device. When equipped, use any of the constituent reagents of the protein S activity measurement reagent kit of the present invention as a diluent in the apparatus, mix the sample and the constituent reagent (dilute the sample), and then You may measure in the said apparatus by mixing all or one part of a liquid mixture with another component reagent.
7). Measurement method
In the protein S activity measurement reagent kit and activity measurement method of the present invention, protein S activity may be measured by a method or using an apparatus such as an automatic analyzer. It is preferable to perform measurement using an automatic analyzer or the like because it can be measured easily and in a short time.
8). Specific examples of protein S activity measurement in samples
Specific examples of the protein S activity measurement contained in the sample in the protein S activity measurement reagent kit and activity measurement method of the present invention are described below.
[A] Specific example-1
(1) Reagent kit for protein S activity measurement
(I) Reactive protein C-containing reagent
The following reagent components were dissolved in pure water so as to have the respective concentrations described above, and the pH was adjusted to 7.0 (20 ° C.) to prepare an activated protein C-containing reagent.
Activated Protein C (Purified Human Activated Protein C; Enzyme Research Laboratories, Inc. [USA]) 893 pM
Sodium chloride <Salt> 0.1M
Bovine serum albumin (BSA) <Protein> 0.1% (W / V)
Sodium azide <Preservative> 0.05% (W / V)
3-morpholinopropanesulfonic acid (MOPS) <Buffer> 50 mM
(Ii) Activated blood coagulation factor V-containing reagent
The following reagent components were dissolved in pure water so as to have the respective concentrations described above, and the pH was adjusted to 7.0 (20 ° C.) to prepare an activated blood coagulation factor V-containing reagent.
Activated blood coagulation factor V (purified human activated blood coagulation factor V; Haematologic Technologies, Inc. [USA]) 300 pM
Activated blood coagulation factor X (purified bovine activated blood coagulation factor X; New England Biolabs, Inc. [USA]) 157 pM
Phospholipid (The composition ratio of phosphatidylserine [PS], phosphatidylcholine [PC] and phosphatidylethanolamine [PE] is 1: 1: 1 [33.33% (W / V): 33.33% (W / V): 33.33% (W / V)]; NOF Corporation [Japan]) 0.05% (W / V)
Calcium chloride 5 mM
Sodium chloride <Salt> 0.1M
Bovine serum albumin (BSA) <Protein> 0.1% (W / V)
Sodium azide <Preservative> 0.05% (W / V)
3-morpholinopropanesulfonic acid (MOPS) <Buffer> 50 mM
(Iii) Prothrombin / substrate-containing reagent
The following reagent components were dissolved in pure water so that each concentration was as described, and the pH was adjusted to 7.0 (20 ° C.) to prepare a prothrombin / substrate-containing reagent.
Prothrombin (purified human prothrombin; Enzyme Research Laboratories, Inc. [USA]) 694 nM
Test Team (registered trademark) Chromogenic Substrate S-2238 (Thrombin Substrate [Thrombin Chromogenic Substrate]; Manufacturer: Chromogenix-Instrumentation Laboratories [Italy], Distributor: Sekisui Medical Inc. [Japan]) 1.5 mM
Sodium chloride <Salt> 0.1M
Bovine serum albumin (BSA) <Protein> 0.1% (W / V)
Sodium azide <Preservative> 0.05% (W / V)
3-morpholinopropanesulfonic acid (MOPS) <Buffer> 50 mM
(2) Sample
Human plasma was used as a sample.
(3) Measurement
(I) First stage
The sample of (2) and the activated protein C-containing reagent of (i) of (1) are mixed to prepare a mixed solution (first mixed solution) of the sample and the activated protein C-containing reagent, whereby the sample Protein S and activated protein C contained in are contacted. (Note that this dilutes the sample.)
Note that the mixing ratio of the sample and the activated protein C-containing reagent is determined based on the dilution ratio when the sample is diluted by mixing with the activated protein C-containing reagent or the like.
The preferable range of the dilution ratio is as described above.
(Ii) Second stage
All or part of the first mixed solution is mixed with the activated blood coagulation factor V-containing reagent (ii) of (1) to prepare a mixed solution (second mixed solution).
The amount of the first mixed solution to be mixed and the amount of the activated blood coagulation factor V-containing reagent are respectively the amount of the prothrombin / substrate-containing reagent, the activity value of protein S contained in the sample, and the reaction of this activity measurement. What is necessary is just to determine suitably according to a measurement wavelength, the specification of the analyzer to be used, etc., other conditions, etc., such as the extinction coefficient of the substance which measures the activity value or density | concentration of each component, and a light absorbency.
In general, for example, the amount of the first mixed solution mixed with the activated blood coagulation factor V-containing reagent is 20 to 500 μL, and the amount of the activated blood coagulation factor V-containing reagent is A range of 50 to 300 μL is preferred.
After the preparation of the second mixed solution, incubation is performed.
The incubation time is not particularly limited, but is usually preferably 1 minute or longer, more preferably 3 minutes or longer, and particularly preferably 5 minutes or longer.
Moreover, the temperature at the time of incubation should just be the temperature above the temperature which freezes the said 2nd liquid mixture.
In general, the higher the temperature during the measurement reaction, the higher the reaction rate.
However, if the temperature is too high, the protein S and the components involved in this activity measurement reaction may be denatured, inactivated, denatured or decomposed. It is necessary that the temperature be lower than the temperature at which denaturation, deactivation, alteration or decomposition occurs.
The incubation temperature is usually 2 to 70 ° C., preferably 20 to 37 ° C., more preferably 30 to 37 ° C.
In addition, if the component related to the activity measurement reaction is a heat-resistant material, it may be at a higher temperature.
When the sample contains protein S in the coexistence of phospholipid and calcium ions by the preparation and incubation of the second mixed solution, the activity of the activated protein C is increased by the presence of the protein S.
In the presence of phospholipids and calcium ions, a decomposition reaction of activated blood coagulation factor V catalyzed by activated protein C is performed.
In addition, the decomposition reaction of the activated blood coagulation factor V is promoted by the increased activity of the activated protein C.
(Iii) Third stage
The prothrombin / substrate-containing reagent (iii) of (1) is mixed with all or part of the second mixed solution prepared in the second step to prepare a mixed solution (third mixed solution). This is the final reaction solution.
The amount of the second mixed solution and the amount of the prothrombin / substrate-containing reagent to be mixed are respectively the amount of the first mixed solution, the amount of activated blood coagulation factor V-containing reagent, and the activity of protein S contained in the sample. Value, the activity value or concentration of each component involved in the reaction of this activity measurement, the extinction coefficient of the substance to be measured, the measurement wavelength, the specifications of the analyzer to be used, and other conditions, etc. That's fine.
In general, for example, the amount of the prothrombin / substrate-containing reagent is preferably in the range of 10 to 500 μL.
After the preparation of the third mixed solution (final reaction solution), incubation is performed.
The incubation time is not particularly limited, but is usually preferably 1 minute or longer, more preferably 3 minutes or longer, and particularly preferably 5 minutes or longer.
Moreover, the temperature at the time of incubation should just be temperature higher than the temperature which freezes the said 3rd liquid mixture (final reaction liquid).
In general, the higher the temperature during the measurement reaction, the higher the reaction rate.
However, if the temperature is too high, the protein S and the components involved in this activity measurement reaction may be denatured, inactivated, denatured or decomposed. It is necessary that the temperature be lower than the temperature at which denaturation, deactivation, alteration or decomposition occurs.
The incubation temperature is usually 2 to 70 ° C., preferably 20 to 37 ° C., more preferably 30 to 37 ° C.
In addition, if the component related to the activity measurement reaction is a heat-resistant material, it may be at a higher temperature.
By preparing and incubating the third mixed solution (final reaction solution), the measurement reaction in the third step is started following the reaction in the second step, and activated blood coagulation is performed in the presence of phospholipids and calcium ions. A reaction is carried out to generate thrombin from prothrombin catalyzed by factor X.
Then, a reaction for generating a signal from a substrate of thrombin catalyzed by the generated thrombin is performed.
Specifically, “Test Team (registered trademark) chromogenic substrate S-2238”, which is a substrate of thrombin, is hydrolyzed by thrombin, and a reaction in which p-nitroaniline is generated is performed.
Since the thrombin generation reaction is promoted by activated blood coagulation factor V, the activated blood coagulation factor V in the second stage due to the increased activity of activated protein C in the second stage. By promoting the factor decomposition reaction, the thrombin generation reaction is suppressed, and thrombin generation is reduced.
The reduction in the generation of thrombin suppresses the reaction that generates a signal from the thrombin substrate catalyzed by thrombin.
Specifically, the reaction of “Test Team (registered trademark) chromogenic substrate S-2238”, which is a substrate of thrombin, is hydrolyzed by thrombin to produce p-nitroaniline.
Next, the color of the generated p-nitroaniline, which is a signal generated from the thrombin substrate, is measured by measuring the absorbance (or transmittance) of the third mixed solution (final reaction solution).
The absorbance (or transmittance) may be measured by a one-wavelength method performed at only one wavelength or a two-wavelength method performed at two wavelengths, respectively, and may be selected as appropriate.
Further, the wavelength at which the absorbance (or transmittance) is measured may be appropriately selected according to the color absorption wavelength of the dye (p-nitroaniline or the like).
Since p-nitroaniline has an absorption maximum in the vicinity of 400 nm, it is preferable to measure the absorbance (or transmittance) at, for example, 405 nm.
The absorbance (or transmittance) may be measured by appropriately selecting a method such as an end point method (end point method) or a reaction rate method (rate method).
It is a signal generated from the thrombin substrate that the activity value of protein S contained in the sample is calculated from the measured absorbance (or transmittance) or the amount of change in the measured absorbance (or transmittance). A method of calculating from the absorbance (or transmittance) measured based on the molar extinction coefficient of coloring of the dye, or a protein S-containing substance (standard substance or standard solution, etc.) whose protein S activity value (or titer) is known A method such as a method of calculating in comparison with the absorbance (or transmittance) of) may be selected as appropriate.
In the present invention, the activity value of protein S contained in the sample is obtained by subtracting the reagent blind (reagent blank) from the absorbance (or transmittance) obtained by measuring the activity of protein S contained in the sample. Is preferably calculated.
The operation of measuring the activity of protein S contained in the sample in the present invention may be performed by the measurer himself or herself, using an apparatus such as an automatic analyzer, or the method used. And a method using an apparatus such as an automatic analyzer may be combined.
For example, the first stage of (i) may be performed using a technique, and the second stage of (ii) and the third stage of (iii) may be performed using an apparatus such as an automatic analyzer.
In addition, for example, the first stage of (i), the second stage of (ii), and the third stage of (iii) are all fully automated using a device such as an automatic analyzer. You may go on.
In addition, since measurement can be performed accurately, simply, and in a short time, it is preferable to perform measurement completely fully automatically using an apparatus such as an automatic analyzer as described above.
[B] Specific example-2
(1) Reagent kit for protein S activity measurement
(I) Reactive protein C-containing reagent
The following reagent components were dissolved in pure water so as to have the respective concentrations described above, and the pH was adjusted to 7.0 (20 ° C.) to prepare an activated protein C-containing reagent.
Activated Protein C (Purified Human Activated Protein C; Enzyme Research Laboratories, Inc. [USA]) 893 pM
Sodium chloride <Salt> 0.1M
Bovine serum albumin (BSA) <Protein> 0.1% (W / V)
Sodium azide <Preservative> 0.05% (W / V)
3-morpholinopropanesulfonic acid (MOPS) <Buffer> 50 mM
(Ii) Activated blood coagulation factor V-containing reagent
The following reagent components were dissolved in pure water so as to have the respective concentrations described above, and the pH was adjusted to 7.0 (20 ° C.) to prepare an activated blood coagulation factor V-containing reagent.
Activated blood coagulation factor V (purified human activated blood coagulation factor V; Haematologic Technologies, Inc. [USA]) 300 pM
Phospholipid (The composition ratio of phosphatidylserine [PS], phosphatidylcholine [PC] and phosphatidylethanolamine [PE] is 1: 1: 1 [33.33% (W / V): 33.33% (W / V): 33.33% (W / V)]; NOF Corporation [Japan]) 0.05% (W / V)
Calcium chloride 5 mM
Sodium chloride <Salt> 0.1M
Bovine serum albumin (BSA) <Protein> 0.1% (W / V)
Sodium azide <Preservative> 0.05% (W / V)
3-morpholinopropanesulfonic acid (MOPS) <Buffer> 50 mM
(Iii) Prothrombin / substrate-containing reagent
The following reagent components were dissolved in pure water so that each concentration was as described, and the pH was adjusted to 7.0 (20 ° C.) to prepare a prothrombin / substrate-containing reagent.
Prothrombin (purified human prothrombin; Enzyme Research Laboratories, Inc. [USA]) 694 nM
Test Team (registered trademark) Chromogenic Substrate S-2238 (Thrombin Substrate [Thrombin Chromogenic Substrate]; Manufacturer: Chromogenix-Instrumentation Laboratories [Italy], Distributor: Sekisui Medical Inc. [Japan]) 1.5 mM
Activated blood coagulation factor X (purified bovine activated blood coagulation factor X; New England Biolabs, Inc. [USA]) 470 pM
Sodium chloride <Salt> 0.1M
Bovine serum albumin (BSA) <Protein> 0.1% (W / V)
Sodium azide <Preservative> 0.05% (W / V)
3-morpholinopropanesulfonic acid (MOPS) <Buffer> 50 mM
(2) Sample
Human plasma was used as a sample.
(3) Measurement
(1) (i) an activated protein C-containing reagent, (ii) an activated blood coagulation factor V-containing reagent, and (iii) a prothrombin / substrate-containing reagent of (2) By measuring the sample as described in (3) of [A] above, the activity value of protein S contained in the sample can be determined.

EXAMPLES Hereinafter, although an Example demonstrates this invention more specifically in detail, this invention is not limited by these Examples.
[Example 1] (Protein S activity measurement reagent kit and activity measurement method of the present invention)
The activity of protein S contained in the sample was measured using the protein S activity measurement reagent kit and the activity measurement method of the present invention and the comparative example, respectively.
1. Reagent kit for protein S activity measurement
The protein S activity measurement reagent kit of the present invention was prepared as follows.
In addition, a reagent kit for measuring protein S activity as a comparative example for the present invention was also prepared as follows.
The main components contained in each component reagent of the protein S activity measurement reagent kit are shown in Table 1.

(1) Reagent / method of the present invention-1
“Reagent / method-1 of the present invention” which is a reagent kit for measuring the activity of protein S of the present invention was prepared as follows.
(I) Reactive protein C-containing reagent
The following reagent components were dissolved in pure water so as to have the respective concentrations described above, and the pH was adjusted to 7.0 (20 ° C.) to prepare an activated protein C-containing reagent.
Activated Protein C (Purified Human Activated Protein C; Enzyme Research Laboratories, Inc. [USA]) 893 pM
Sodium chloride <Salt> 0.1M
Bovine serum albumin (BSA; Sigma-Aldrich [USA]) <Protein> 0.1% (W / V)
Sodium azide <Preservative> 0.05% (W / V)
3-morpholinopropanesulfonic acid (MOPS) <Buffer> 50 mM
(Ii) Activated blood coagulation factor V-containing reagent
Prepare the activated blood coagulation factor V-containing reagent (Invention-1) by dissolving the following reagent components in pure water so as to have the respective concentrations described above, and adjusting the pH to pH 7.0 (20 ° C.). did.
Activated blood coagulation factor V (purified human activated blood coagulation factor V; Haematologic Technologies, Inc. [USA]) 300 pM
Activated blood coagulation factor X (purified bovine activated blood coagulation factor X; New England Biolabs, Inc. [USA]) 157 pM
Phospholipid (phosphatidylserine [PS], phosphatidylcholine [PC] and phosphatidylethanolamine [PE] having a composition ratio of 1: 1: 1; NOF Corporation [Japan]) 0.05% (W / V)
Calcium chloride 5 mM
Sodium chloride <Salt> 0.1M
Bovine serum albumin (BSA; Sigma-Aldrich [USA]) <Protein> 0.1% (W / V)
Sodium azide <Preservative> 0.05% (W / V)
3-morpholinopropanesulfonic acid (MOPS) <Buffer> 50 mM
(Iii) Prothrombin / substrate-containing reagent
The following reagent components were dissolved in pure water to the respective concentrations described above, and the pH was adjusted to 7.0 (20 ° C.) to prepare a prothrombin / substrate-containing reagent (present invention-1).
Prothrombin (purified human prothrombin; Enzyme Research Laboratories, Inc. [USA]) 694 nM
Test Team (registered trademark) Chromogenic Substrate S-2238 (Thrombin Substrate [Thrombin Chromogenic Substrate]; Manufacturer: Chromogenix-Instrumentation Laboratories [Italy], Distributor: Sekisui Medical Inc. [Japan]) 1.5 mM
Sodium chloride <Salt> 0.1M
Bovine serum albumin (BSA; Sigma-Aldrich [USA]) <Protein> 0.1% (W / V)
Sodium azide <Preservative> 0.05% (W / V)
3-morpholinopropanesulfonic acid (MOPS) <Buffer> 50 mM
(2) Reagent / method-2 of the present invention
“Reagent / method-2 of the present invention” which is a reagent kit for measuring the activity of protein S of the present invention was prepared as follows.
(I) Reactive protein C-containing reagent
Prepared as described in (i) (i) above, this was used as an activated protein C-containing reagent.
(Ii) Activated blood coagulation factor V-containing reagent
Prepare the activated blood coagulation factor V-containing reagent (Invention-2) by dissolving the following reagent components in pure water so that each concentration is as described, and adjusting the pH to pH 7.0 (20 ° C.). did.
Activated blood coagulation factor V (purified human activated blood coagulation factor V; Haematologic Technologies, Inc. [USA]) 300 pM
Phospholipid (The composition ratio of phosphatidylserine [PS], phosphatidylcholine [PC] and phosphatidylethanolamine [PE] is 1: 1: 1 [33.33% (W / V): 33.33% (W / V): 33.33% (W / V)]; NOF Corporation [Japan]) 0.05% (W / V)
Calcium chloride 5 mM
Sodium chloride <Salt> 0.1M
Bovine serum albumin (BSA; Sigma-Aldrich [USA]) <Protein> 0.1% (W / V)
Sodium azide <Preservative> 0.05% (W / V)
3-morpholinopropanesulfonic acid (MOPS) <Buffer> 50 mM
(Iii) Prothrombin / substrate-containing reagent
The following reagent components were dissolved in pure water to the respective concentrations described above, and the pH was adjusted to 7.0 (20 ° C.) to prepare a prothrombin / substrate-containing reagent (present invention-2).
Prothrombin (purified human prothrombin; Enzyme Research Laboratories, Inc. [USA]) 694 nM
Test Team (registered trademark) Chromogenic Substrate S-2238 (Thrombin Substrate [Thrombin Chromogenic Substrate]; Manufacturer: Chromogenix-Instrumentation Laboratories [Italy], Distributor: Sekisui Medical Inc. [Japan]) 1.5 mM
Activated blood coagulation factor X (purified bovine activated blood coagulation factor X; New England Biolabs, Inc. [USA]) 470 pM
Sodium chloride <Salt> 0.1M
Bovine serum albumin (BSA; Sigma-Aldrich [USA]) <Protein> 0.1% (W / V)
Sodium azide <Preservative> 0.05% (W / V)
3-morpholinopropanesulfonic acid (MOPS) <Buffer> 50 mM
(3) Comparative Reagent / Method-1
“Comparative Reagent / Method-1” which is a reagent kit for measuring protein S activity as a comparative example was prepared as follows.
(I) Reactive protein C-containing reagent
Prepared as described in (i) (i) above, this was used as an activated protein C-containing reagent.
(Ii) Activated blood coagulation factor V-containing reagent
Each of the following reagent components was dissolved in pure water so as to have the indicated concentrations, and the pH was adjusted to 7.0 (20 ° C.) to prepare an activated blood coagulation factor V-containing reagent (Comparative-1). .
Activated blood coagulation factor V (purified human activated blood coagulation factor V; Haematologic Technologies, Inc. [USA]) 300 pM
Test Team (registered trademark) Chromogenic Substrate S-2238 (Thrombin Substrate [Thrombin Chromogenic Substrate]; Manufacturer: Chromogenix-Instrumentation Laboratories [Italy], Distributor: Sekisui Medical (Japan)) 0.5 mM
Phospholipid (The composition ratio of phosphatidylserine [PS], phosphatidylcholine [PC] and phosphatidylethanolamine [PE] is 1: 1: 1 [33.33% (W / V): 33.33% (W / V): 33.33% (W / V)]; NOF Corporation [Japan]) 0.05% (W / V)
Calcium chloride 5 mM
Sodium chloride <Salt> 0.1M
Bovine serum albumin (BSA; Sigma-Aldrich [USA]) <Protein> 0.1% (W / V)
Sodium azide <Preservative> 0.05% (W / V)
3-morpholinopropanesulfonic acid (MOPS) <Buffer> 50 mM
(Iii) Prothrombin / substrate-containing reagent
The following reagent components were dissolved in pure water to the respective concentrations described above, and the pH was adjusted to 7.0 (20 ° C.) to prepare a prothrombin / substrate-containing reagent (Comparative-1).
Prothrombin (purified human prothrombin; Enzyme Research Laboratories, Inc. [USA]) 694 nM
Activated blood coagulation factor X (purified bovine activated blood coagulation factor X; New England Biolabs, Inc. [USA]) 470 pM
Sodium chloride <Salt> 0.1M
Bovine serum albumin (BSA; Sigma-Aldrich [USA]) <Protein> 0.1% (W / V)
Sodium azide <Preservative> 0.05% (W / V)
3-morpholinopropanesulfonic acid (MOPS) <Buffer> 50 mM
The "prothrombin / substrate-containing reagent (Comparative-1)" does not contain a thrombin substrate, but for comparison with the "prothrombin / substrate-containing reagent" in the present invention, It will be referred to as “containing reagent (Comparison-1)”.
(4) Comparative reagent / method-2
“Comparative Reagent / Method-2” which is a reagent kit for measuring protein S activity as a comparative example was prepared as follows.
(I) Reactive protein C-containing reagent
It was prepared as described in (i) of (1) above, and this was used as an activated protein C-containing reagent.
(Ii) Activated blood coagulation factor V-containing reagent
The following reagent components were dissolved in pure water so that each concentration was as described, and the pH was adjusted to 7.0 (20 ° C.) to prepare an activated blood coagulation factor V-containing reagent (Comparative-2). .
Activated blood coagulation factor V (purified human activated blood coagulation factor V; Haematologic Technologies, Inc. [USA]) 300 pM
Prothrombin (purified human prothrombin; Enzyme Research Laboratories, Inc. [USA]) 231 nM
Phospholipid (The composition ratio of phosphatidylserine [PS], phosphatidylcholine [PC] and phosphatidylethanolamine [PE] is 1: 1: 1 [33.33% (W / V): 33.33% (W / V): 33.33% (W / V)]; NOF Corporation [Japan]) 0.05% (W / V)
Calcium chloride 5 mM
Sodium chloride <Salt> 0.1M
Bovine serum albumin (BSA; Sigma-Aldrich [USA]) <Protein> 0.1% (W / V)
Sodium azide <Preservative> 0.05% (W / V)
3-morpholinopropanesulfonic acid (MOPS) <Buffer> 50 mM
(Iii) Prothrombin / substrate-containing reagent
The following reagent components were dissolved in pure water to the respective concentrations described above, and the pH was adjusted to 7.0 (20 ° C.) to prepare a prothrombin / substrate-containing reagent (Comparative-2).
Test Team (registered trademark) Chromogenic Substrate S-2238 (Thrombin Substrate [Thrombin Chromogenic Substrate]; Manufacturer: Chromogenix-Instrumentation Laboratories [Italy], Distributor: Sekisui Medical Inc. [Japan]) 1.5 mM
Activated blood coagulation factor X (purified bovine activated blood coagulation factor X; New England Biolabs, Inc. [USA]) 470 pM
Sodium chloride <Salt> 0.1M
Bovine serum albumin (BSA; Sigma-Aldrich [USA]) <Protein> 0.1% (W / V)
Sodium azide <Preservative> 0.05% (W / V)
3-morpholinopropanesulfonic acid (MOPS) <Buffer> 50 mM
The "prothrombin / substrate-containing reagent (Comparison-2)" does not contain prothrombin, but for the sake of convenience, for comparison with the "prothrombin / substrate-containing reagent" in the present invention, (Comparison-2) ".
(5) Comparative reagent / method-3
“Comparative Reagent / Method-3” which is a reagent kit for measuring protein S activity as a comparative example was prepared as follows.
(I) Reactive protein C-containing reagent
It was prepared as described in (i) of (1) above, and this was used as an activated protein C-containing reagent.
(Ii) Activated blood coagulation factor V-containing reagent
The following reagent components were dissolved in pure water so that each concentration was as described, and the pH was adjusted to 7.0 (20 ° C.) to prepare an activated blood coagulation factor V-containing reagent (Comparative-3). .
Activated blood coagulation factor V (purified human activated blood coagulation factor V; Haematologic Technologies, Inc. [USA]) 300 pM
Prothrombin (purified human prothrombin; Enzyme Research Laboratories, Inc. [USA]) 231 nM
Test Team (registered trademark) Chromogenic Substrate S-2238 (Thrombin Substrate [Thrombin Chromogenic Substrate]; Manufacturer: Chromogenix-Instrumentation Laboratories [Italy], Distributor: Sekisui Medical [Japan]] 0.5 mM
Phospholipid (The composition ratio of phosphatidylserine [PS], phosphatidylcholine [PC] and phosphatidylethanolamine [PE] is 1: 1: 1 [33.33% (W / V): 33.33% (W / V): 33.33% (W / V)]; NOF Corporation [Japan]) 0.05% (W / V)
Calcium chloride 5 mM
Sodium chloride <Salt> 0.1M
Bovine serum albumin (BSA; Sigma-Aldrich [USA]) <Protein> 0.1% (W / V)
Sodium azide <Preservative> 0.05% (W / V)
3-morpholinopropanesulfonic acid (MOPS) <Buffer> 50 mM
(Iii) Prothrombin / substrate-containing reagent
The following reagent components were dissolved in pure water to the respective concentrations described above, and the pH was adjusted to 7.0 (20 ° C.) to prepare a prothrombin / substrate-containing reagent (Comparative-3).
Activated blood coagulation factor X (purified bovine activated blood coagulation factor X; New England Biolabs, Inc. [USA]) 470 pM
Sodium chloride <Salt> 0.1M
Bovine serum albumin (BSA; Sigma-Aldrich [USA]) <Protein> 0.1% (W / V)
Sodium azide <Preservative> 0.05% (W / V)
3-morpholinopropanesulfonic acid (MOPS) <Buffer> 50 mM
This “prothrombin / substrate-containing reagent (Comparative-3)” contains neither prothrombin nor a thrombin substrate. For the sake of convenience, the “prothrombin / substrate-containing reagent” is used for comparison with the “prothrombin / substrate-containing reagent” in the present invention. -Substrate containing reagent (Comparison-3) ".
(6) Comparative reagent / method-4
“Comparative Reagent / Method-4” which is a reagent kit for measuring protein S activity as a comparative example was prepared as follows.
(I) Reactive protein C-containing reagent
Prepared as described in (i) (i) above, this was used as an activated protein C-containing reagent.
(Ii) Activated blood coagulation factor V-containing reagent
Each of the following reagent components was dissolved in pure water so as to have the indicated concentrations, and the pH was adjusted to 7.0 (20 ° C.) to prepare an activated blood coagulation factor V-containing reagent (Comparative-4). .
Activated blood coagulation factor V (purified human activated blood coagulation factor V; Haematologic Technologies, Inc. [USA]) 300 pM
Activated blood coagulation factor X (purified bovine activated blood coagulation factor X; New England Biolabs, Inc. [USA]) 157 pM
Test Team (registered trademark) Chromogenic Substrate S-2238 (Thrombin Substrate [Thrombin Chromogenic Substrate]; Manufacturer: Chromogenix-Instrumentation Laboratories [Italy], Distributor: Sekisui Medical [Japan]] 0.5 mM
Phospholipid (The composition ratio of phosphatidylserine [PS], phosphatidylcholine [PC] and phosphatidylethanolamine [PE] is 1: 1: 1 [33.33% (W / V): 33.33% (W / V): 33.33% (W / V)]; NOF Corporation [Japan]) 0.05% (W / V)
Calcium chloride 5 mM
Sodium chloride <Salt> 0.1M
Bovine serum albumin (BSA; Sigma-Aldrich [USA]) <Protein> 0.1% (W / V)
Sodium azide <Preservative> 0.05% (W / V)
3-morpholinopropanesulfonic acid (MOPS) <Buffer> 50 mM
(Iii) Prothrombin / substrate-containing reagent
The following reagent components were dissolved in pure water to the respective concentrations described above, and the pH was adjusted to 7.0 (20 ° C.) to prepare a prothrombin / substrate-containing reagent (Comparative-4).
Prothrombin (purified human prothrombin; Enzyme Research Laboratories, Inc. [USA]) 694 nM
Sodium chloride <Salt> 0.1M
Bovine serum albumin (BSA; Sigma-Aldrich [USA]) <Protein> 0.1% (W / V)
Sodium azide <Preservative> 0.05% (W / V)
3-morpholinopropanesulfonic acid (MOPS) <Buffer> 50 mM
The "prothrombin / substrate-containing reagent (Comparison-4)" does not contain a thrombin substrate, but for comparison with the "prothrombin / substrate-containing reagent" in the present invention, It will be referred to as “containing reagent (Comparison-4)”.
2. sample
Human plasma is divided, and one of these is passed through a column (affinity column) in which an anti-human protein S antibody is bound to a carrier, thereby immobilizing protein S contained in the human plasma in this column, The protein S-deficient plasma not containing protein S was obtained.
The following five types of human plasma samples were prepared by mixing the protein S-deficient plasma and the previously divided human plasma (containing protein S) at various ratios.
In addition, the numerical value shown for each sample represents the total protein S activity value (μg / mL equivalent) of each sample.
The protein S-deficient plasma was used as a sample having a total protein S activity value of 0 μg / mL equivalent.
The total protein S activity value of the sample is, as described above, all protein S existing in the sample [complex of protein S and C4b binding protein (binding type) and free protein S (free Type)].
In the specification and drawings of this patent application, the protein S activity value of 1 μg / mL of purified human protein S (Enzyme Research Laboratories, Inc. [USA]) is defined as the protein S activity value of 1 μg / mL equivalent. The total protein S activity value of 1 μg / mL of the purified human protein S was defined as the total protein S activity value of 1 μg / mL equivalent.
Therefore, in the specification and drawings of this patent application, the activity of protein S and the activity of total protein S are respectively expressed using “μg / mL equivalent”.
(1) Sample a (total protein S activity value: 0 μg / mL equivalent)
(2) Sample b (Total protein S activity value: 6.2 μg / mL equivalent)
(3) Sample c (Total protein S activity value: 12.3 μg / mL equivalent)
(4) Sample d (total protein S activity value: 18.5 μg / mL equivalent)
(5) Sample e (Total protein S activity value: 24.6 μg / mL equivalent)
(6) Sample f (total protein S activity value: 31.5 μg / mL equivalent)
3. Measurement
The protein S activity of each sample was measured as follows using a general-purpose automatic analyzer TBA-120FR (Toshiba Medical Systems [Japan]).
(1) With the sample dilution function of the general-purpose automatic analyzer TBA-120FR, activation of 2 μL and (1) (i) of each of (2) and (1) of each of the samples (1) to (6) above 270 μL of protein C-containing reagent was mixed.
By mixing the sample with the activated protein C-containing reagent, this sample was diluted at a dilution ratio of 136 times.
(2) Next, 3 μL of the mixed solution of (1) is sampled and discharged to the cuvette, and the activated blood coagulation factor V-containing reagent of (ii) of (ii) above (this invention- 150 μL of 1) was added and mixed. [Measurement points: 0 points]
And it incubated at 37 degreeC.
(3) Next, the measurement point was changed from 16 points (4 minutes 40.46 seconds after addition of the activated blood coagulation factor V-containing reagent (present invention-1)) to 17 points (4 minutes 50 after addition of the reagent). (49)), 50 μL of the (iii) prothrombin / substrate-containing reagent (Invention-1) of (1) above was added to the mixture of (2) and mixed.
And it incubated at 37 degreeC until the measurement point 33 points [9 minutes and 47 seconds after adding the said activated blood coagulation factor V containing reagent (this invention-1)].
(4) The change in absorbance (wavelength: 405 nm) of the mixed solution in the cuvette was measured at 1 point [9.93 seconds after adding the above-mentioned activated blood coagulation factor V-containing reagent (present invention-1). ] To 33 measurement points [9 minutes 47 seconds after addition of the same reagent] and recorded.
That is, the change in absorbance (wavelength: 405 nm) when the protein S activity of each sample was measured by the reagent / method-1 of the present invention (1) was measured and recorded.
(5) In (2), instead of the activated blood coagulation factor V-containing reagent (present invention-1), the activated blood coagulation factor V-containing reagent (this) in (ii) in (ii) above (this book) The invention (2) is used, and the prothrombin / substrate-containing reagent (Invention-2) in (1) (2) is used instead of the prothrombin / substrate-containing reagent (Invention-1) in (3). Except for the use, the procedure of (1) to (4) was followed, and the absorbance (when protein S activity of each sample was measured by the reagent / method-2 of the present invention of (1) above (2) ( Wavelength: 405 nm) change was measured and recorded.
(6) The activated blood coagulation factor V-containing reagent of (ii) in (ii) above (Comparison) in place of the activated blood coagulation factor V-containing reagent (Invention-1) in (2) -1), and in (3) above, (iii) (iii) prothrombin / substrate containing reagent (Comparative -1) in 1 above is used instead of the prothrombin / substrate containing reagent (present invention-1). Except for the above, the procedure of (1) to (4) was followed, and the absorbance (wavelength: 405 nm) when the protein S activity of each sample was measured by the comparative reagent / method-1 of (1) above. ) Was measured and recorded.
(7) In addition, the activated blood coagulation factor V-containing reagent of (ii) in (1) above (in comparison with the activated blood coagulation factor V-containing reagent (present invention-1) in (2) (comparison) -2) and (iii) using the prothrombin / substrate-containing reagent (Comparison-2) of (4) in (1) instead of the prothrombin / substrate-containing reagent (Invention-1) in (3) Except for the above, the procedure of (1) to (4) was followed, and the absorbance (wavelength: 405 nm) when the protein S activity of each sample was measured by the comparative reagent / method-2 of (1) above. ) Was measured and recorded.
(8) In addition, instead of the activated blood coagulation factor V-containing reagent (present invention-1) in (2), the activated blood coagulation factor V-containing reagent of (ii) in (1) above (comparison) -3), and in (3), instead of the prothrombin / substrate-containing reagent (present invention-1), (iii) (iii) prothrombin / substrate-containing reagent (Comparison-3) is used. Except for the above, the procedure of (1) to (4) was followed, and the absorbance (wavelength: 405 nm) when the protein S activity of each sample was measured by the comparative reagent / method-3 of (1) above. ) Was measured and recorded.
(9) The activated blood coagulation factor V-containing reagent of (ii) in (ii) above (in comparison with the activated blood coagulation factor V-containing reagent (present invention-1) in (2) (comparison) -4), and in (3) above, instead of the prothrombin / substrate-containing reagent (present invention-1), (iii) (iii) prothrombin / substrate-containing reagent (Comparative -4) in 1 above is used. Except for the above, the procedure of (1) to (4) was followed, and the absorbance (wavelength: 405 nm) when the protein S activity of each sample was measured by the comparative reagent / method-4 of (1) above. ) Was measured and recorded.
4). Measurement result
(1) The measurement results in 3 above, that is, the absorbance (wavelength: when measuring the activity of protein S contained in the sample by the protein S activity measurement reagent kit and the activity measurement method as the present invention and the comparative example, respectively. 405 nm) are shown in FIGS. 1 to 6 and Tables 2 to 7.
The results of measurement using the reagent / method-1 of the present invention (1) are shown in FIG. 1 and Table 2, and the results of measurement using the reagent / method-2 of the present invention (2) are shown in FIG. 3 and Table 3, the results of the measurement by the comparative reagent / method-1 of the above (3) are shown in FIG. 3 and Table 4, and the results of the measurement by the comparative reagent / method-2 of the above (4) are shown. FIG. 4 and Table 5 show the results of measurement using the comparative reagent / method-3 of (1) in (1) above, and FIG. 5 and Table 6 show the results of the comparison reagent / method-4 of (1) in (1) above. The measurement results are shown in FIG.
(2) In these FIGS. 1 to 6, which protein S activity measurement reagent kit and activity measurement method were used was shown above the figure.
In these figures, the horizontal axis indicates the measurement points in the general-purpose automatic analyzer TBA-120FR used for the measurement, and the vertical axis indicates the absorbance (wavelength: 405 nm) at each measurement point.
Further, in these figures, “□” indicates a measured value (the absorbance described above) when measured for the sample a (2) (1) (total protein S activity value: 0 μg / mL equivalent). Indicates the measured value (the absorbance described above) when measured for the sample b (2) (total protein S activity value: 6.2 μg / mL equivalent) in 2 above, and “Δ” indicates that in 2 (3) above. The measurement value (the absorbance described above) when measured with respect to the sample c (total protein S activity value: 12.3 μg / mL equivalent), and “◯” indicates the sample d (total protein S activity value) in (2) above. : 18.5 μg / mL equivalent) measured value (absorbance described above), “+” indicates the sample e in 2 (5) above (total protein S activity value: 24.6 μg / mL equivalent) The measured value (absorbance described above) when measured for "-" Indicates a measured value (the absorbance described above) when measured for the sample f (2) (6) (total protein S activity value: 31.5 μg / mL equivalent).
(3) In Tables 2 to 7, in the upper left part of the table, which protein S activity measurement reagent kit and activity measurement method was used is shown.
In these tables, in order from the top, the measured value (the absorbance described above) when measuring the sample a (2) (1) (total protein S activity value: 0 μg / mL equivalent), 2) Sample b (total protein S activity value: 6.2 μg / mL equivalent), measured value (absorbance described above), sample c of 2 (3) above (total protein S activity value: 12. Measured value when measured for 3 μg / mL equivalent) (absorbance described above), Measured value when measured for sample d of 2 above (4) (total protein S activity value: 18.5 μg / mL equivalent) ), (2) (5) sample e (total protein S activity value: 24.6 μg / mL equivalent) measured value (absorbance), (2) sample (6) f Total protein S activity: 31.5 μg mL showing the measurement value when measured equiv) (the absorbance).
In these tables, in order from the left side, the measured value when the measurement point is 1 point (absorbance described above), the measured value when the measurement point is 2 points (absorbance described above), and the measured point is 3 The measured value (absorbance described above) when it is a point,... The measured value (absorbance described above) when the measured point is 33 points are shown.

5). Summary
(1) From FIG. 1 to FIG. 6 and Tables 2 to 7 which are measurement results in 3 above, the following can be understood.
That is, in the measurement using the reagent / method-1 of the present invention (1) and the measurement using the reagent / method-2 of the present invention (2), the samples a to (6) of (2) are used. It can be seen that the absorbance after addition and mixing of the prothrombin / substrate-containing reagent in (3) above differs from sample to sample in each sample f of (3).
In both the measurement by the reagent / method-1 of the present invention and the measurement by the reagent / method-2 of the present invention, the absorbance increases in order as the activity value of protein S contained in the sample decreases from a low value to a high value. It turns out that it is low in inverse proportion.
That is, in the measurement by the reagent / method-1 of the present invention and the measurement by the reagent / method-2 of the present invention, there is a reaction that generates a signal from the thrombin substrate in proportion to the activity of protein S contained in the sample. From this, it can be seen that the activity of protein S contained in the sample can be quantitatively determined by measuring the amount of the signal in which the production is suppressed.
Namely, an activated protein C-containing reagent containing at least activated protein C, an activated blood coagulation factor V-containing reagent containing at least activated blood coagulation factor V, and a prothrombin / substrate-containing reagent containing at least prothrombin and a thrombin substrate The protein S activity contained in the sample in each of the above-described measurement using the reagent / method-1 of the present invention and the measurement using the reagent / method-2 of the present invention using the reagent kit for measuring the protein S activity comprising It can be seen that can be measured quantitatively.
(2) On the other hand, the measurement by the comparative reagent / method-1 of (1) in (1) above, the measurement by the comparative reagent / method-2 of (1) in (1) above, In the measurement by the method-3 and the measurement by the comparative reagent / method-4 in the above (6), each of the above-mentioned 3 (3) in each of the samples a to (6) in the above (2) (1) The absorbance after addition and mixing of the prothrombin / substrate-containing reagent in FIG. 3 is almost the same for each sample, and it can be seen that in any sample measurement, if the measurement point is the same, the absorbance is almost the same.
That is, in the measurement using the above-described comparative reagent / method-1 to comparative reagent / method-4, the reaction that generates a signal from the substrate of thrombin is not suppressed according to the activity of protein S contained in the sample. Therefore, it is understood that the activity of protein S contained in the sample cannot be quantitatively determined by measuring the amount of the signal.
Namely, an activated protein C-containing reagent containing at least activated protein C, an activated blood coagulation factor V-containing reagent containing at least activated blood coagulation factor V, and a prothrombin / substrate-containing reagent containing at least prothrombin and a thrombin substrate Using the reagent kit for measuring the activity of protein S, which is different from the kit for measuring the activity of protein S comprising the above-mentioned constituent reagents, the measurement by the comparative reagent / method-1, the measurement by the comparative reagent / method-2, and the comparison It can be seen that the measurement by the reagent / method-3 and the measurement by the comparative reagent / method-4 cannot quantitatively measure the protein S activity contained in the sample in any case.
(3) Therefore, from the results of the study in this Example, an activated protein C-containing reagent containing at least activated protein C, an activated blood coagulation factor V-containing reagent containing at least activated blood coagulation factor V, and at least prothrombin And a protein S activity measurement reagent kit according to the present invention, and a protein S activity measurement reagent kit according to the present invention. The method for measuring the activity of protein S of the present invention can quantitatively measure the activity of protein S contained in a sample, can be easily automated, and the activity of protein S contained in a sample. It was confirmed that it can be measured accurately, simply and in a short time.
[Example 2] (Comparison with conventional measuring reagents and measuring methods)
The protein S activity measurement reagent kit and activity measurement method of the present invention, and the conventional protein S activity measurement reagent kit and activity measurement method, respectively. The amount of protein was compared.
I. Protein S activity measurement reagent kit and activity measurement method of the present invention
1. Reagent kit for protein S activity measurement
◎ Reagent and method of the present invention-1
(I) Reactive protein C-containing reagent
It was prepared as described in 1 (1) (i) of Example 1 and used as an activated protein C-containing reagent.
(Ii) Activated blood coagulation factor V-containing reagent
It was prepared as described in (ii) of (1) of Example 1 above and used as an activated blood coagulation factor V-containing reagent (Invention-1).
(Iii) Prothrombin / substrate-containing reagent
Prepared as described in (iii) of (1) in Example 1 above, this was used as a prothrombin / substrate-containing reagent (Invention-1).
2. sample
The following (1) and (2) were used as samples.
(1) Plasma of 32 healthy people
(2) One plasma known to be a PS-K155E heterozygote (protein S Tokushima, which is one of the abnormalities of protein S), which is a genetic mutation of protein S
3. Measurement
The protein S activity of each sample was measured using a 7170S type general-purpose automatic analyzer (Hitachi High-Technologies Corporation [Japan]) as follows.
(1) With the sample dilution function of the 7170S general-purpose automatic analyzer, 2 μL of each of the samples of (1) and (2) above and the activated protein C-containing reagent of (i) above 270 μL was mixed.
By mixing the sample with the activated protein C-containing reagent, the sample was diluted at a dilution ratio of 136 times.
(2) Next, 3 μL of the mixed solution of (1) is sampled and discharged to a cuvette, and 150 μL of the activated blood coagulation factor V-containing reagent (1) of (ii) is added thereto. Was added and mixed. [Measurement point: 0th point]
And this liquid mixture was incubated at 37 degreeC.
(3) Next, the measurement point was changed from 16 points (4 minutes 30.093 seconds after addition of the above-mentioned activated blood coagulation factor V-containing reagent (present invention-1)) to 17 points (4 minutes 46 after addition of the reagent). (977 seconds), 50 μL of the above-mentioned (iii) prothrombin / substrate-containing reagent (Invention-1) was added to the mixed solution of (2) and mixed.
And this liquid mixture was incubated at 37 degreeC.
(4) Next, with respect to the liquid mixture in the cuvette, 28 points from 25 measurement points [7 minutes 9.732 seconds after adding the above-mentioned activated blood coagulation factor V-containing reagent (present invention-1)] The amount of change in absorbance was measured at a wavelength of 405 nm over 8 minutes and 0.36 seconds after adding the above-mentioned activated blood coagulation factor V-containing reagent (present invention-1).
Then, the amount of change in absorbance per minute was determined from the measured amount of change in absorbance.
(5) Next, a sample with a known activity value of total protein S was measured as described in (1) to (4) above, and the amount of change in absorbance per minute was determined.
(6) Next, the amount of change in absorbance per minute in the sample obtained in (4) above, and the amount of change in absorbance per minute in samples whose activity value of total protein S obtained in (5) is known And the activity value (μg / mL equivalent) of total protein S contained in the sample was calculated.
In this way, the total protein S activity value of each sample of the above 2 was obtained.
The results obtained by measuring the activity of total protein S for each of the above two samples are shown in FIG. 7 and Table 8.
II. Measurement using conventional protein S · activity measurement reagent kit and activity measurement method
1. Reagent kit for protein S activity measurement
(I) Diluent
The following components were dissolved in pure water so as to have the stated concentrations, and the pH was adjusted to 8.0 (20 ° C.) to prepare a diluted solution.
Triton X-100 (Wako Pure Chemical Industries, Ltd. [Japan]) <Surfactant> 0.06% (W / V)
Bovine serum albumin (BSA; Sigma-Aldrich [USA]) <Protein> 0.1% (W / V)
Sodium chloride <Salt> 0.1M
Trisodium citrate 10.6 mM
Tris (hydroxymethyl) aminomethane <Buffer> 50 mM
(Ii) First reagent
The following components were dissolved in pure water so that each concentration was as described, and the pH was adjusted to pH 8.0 (20 ° C.) to prepare the first reagent.
Activated protein C (purified human activated protein C; Enzyme Research Laboratories, Inc. [USA]) 397 pM
Triton X-100 (Wako Pure Chemical Industries, Ltd. [Japan]) <Surfactant> 0.006% (W / V)
Phospholipid (The composition ratio of phosphatidylserine [PS], phosphatidylcholine [PC] and phosphatidylethanolamine [PE] is 1: 1: 1 [33.33% (W / V): 33.33% (W / V): 33.33% (W / V)]; NOF Corporation [Japan]) 0.05% (W / V)
Calcium chloride 2.5 mM
Bovine serum albumin (BSA; Sigma-Aldrich [USA]) <Protein> 0.1% (W / V)
Sodium chloride <Salt> 0.1M
Tris (hydroxymethyl) aminomethane <Buffer> 50 mM
(Iii) Second reagent
The following components were dissolved in pure water so as to have the respective concentrations described above, and the pH was adjusted to 8.0 (20 ° C.) to prepare a second reagent.
Activated blood coagulation factor V (purified human activated blood coagulation factor V; Haematologic Technologies, Inc. [USA]) 357 pM
Triton X-100 (Wako Pure Chemical Industries, Ltd. [Japan]) <Surfactant> 0.006% (W / V)
Phospholipid (The composition ratio of phosphatidylserine [PS], phosphatidylcholine [PC] and phosphatidylethanolamine [PE] is 1: 1: 1 [33.33% (W / V): 33.33% (W / V): 33.33% (W / V)]; NOF Corporation [Japan]) 0.05% (W / V)
Calcium chloride 2.5 mM
Bovine serum albumin (BSA; Sigma-Aldrich [USA]) <Protein> 0.1% (W / V)
Sodium chloride <Salt> 0.1M
Tris (hydroxymethyl) aminomethane <Buffer> 50 mM
(Iv) Third reagent
The following components were dissolved in pure water so as to have the respective concentrations described above, and the pH was adjusted to pH 7.5 (20 ° C.) to prepare a third reagent.
Activated blood coagulation factor X (purified bovine activated blood coagulation factor X; New England Biolabs, Inc. [USA]) 50 pM
Prothrombin (purified human prothrombin; Enzyme Research Laboratories, Inc. [USA]) 738 nM
Test Team (registered trademark) chromogenic substrate S-2238 (thrombin substrate [thrombin chromogenic substrate]; manufacturer: Chromogenix-Instrumentation Laboratories [Italy], distributor: Sekisui Medical [Japan]) 750 μM
Phospholipid (having a composition ratio of phosphatidylserine [PS], phosphatidylcholine [PC] and phosphatidylethanolamine [PE] of 3: 2: 5) 7.5 μM
Calcium chloride 5.0 mM
Bovine serum albumin (BSA; Sigma-Aldrich [USA]) <Protein> 0.1% (W / V)
Sodium chloride <Salt> 0.15M
Tris (hydroxymethyl) aminomethane <Buffer> 50 mM
The above-mentioned phospholipids were phosphatidylserine (porcine brain phosphatidylserine; PS; DOOSSAN Serial Research Laboratories [Korea]) 0.6 mg, phosphatidylcholine (porcine liver phosphatidylcholine; PC; DOOSAN Secondary Research Laboratories [Korea]). 4 mg and 1.0 mg of phosphatidylethanolamine (porcine liver phosphatidylethanolamine; PE; DOOSAN Serial Research Laboratories [Korea]) were each collected in a test tube, and after evaporation of chloroform as a solvent with an evaporator, distilled water was used. Was added, and the mixture was vigorously stirred for 1 minute. Phospholipids having a composition ratio of di-serine, phosphatidylcholine and phosphatidylethanolamine of 3: 2: 5 (ie, 30% (W / V): 20% (W / V): 50% (W / V)) It is contained so as to have a concentration.
2. sample
Each of the samples I (2) (1) and (2) was used as a sample.
3. Measurement
The protein S activity of each sample was measured using a 7170S type general-purpose automatic analyzer (Hitachi High-Technologies Corporation [Japan]) as follows.
(1) With the sample dilution function of the 7170S type general-purpose automatic analyzer, 8 μL of each of the samples of (2) and (1) above was mixed with 112 μL of the diluted solution of (1) above. .
By mixing this sample with the diluent, this sample was diluted at a dilution ratio of 15 times.
(2) Next, 2 μL of the mixed solution of (1) was sampled and discharged to the cuvette, and 98 μL of the first reagent of (ii) was added thereto and mixed. [Measurement point: 0th point]
And this liquid mixture was incubated at 37 degreeC.
(3) Next, when the measurement point is between 5 points (1 minute 18.654 seconds after addition of the first reagent) and 6 points (1 minute 34.593 seconds after addition of the same reagent), (2) 20 μL of the above-mentioned first (iii) second reagent was added and mixed.
And this liquid mixture was incubated at 37 degreeC.
(4) Next, when the measurement point is between 33 points (9 minutes 29.582 seconds after addition of the first reagent) and 34 points (9 minutes 47.426 seconds after addition of the same reagent), (3) 236 μL of the above (iv) third reagent was added to the mixed solution and mixed.
And this liquid mixture was incubated at 37 degreeC to the measurement point 73 points [21 minutes 47.426 seconds after the same reagent addition].
(5) The absorbance of the liquid mixture in the cuvette from 39 measurement points (11 minutes 49.432 seconds after addition of the first reagent) to 52 points (15 minutes 38.504 seconds after addition of the same reagent). The amount of change was measured at a main wavelength of 405 nm and a sub wavelength of 505 nm.
Then, the amount of change in absorbance per minute was determined from the measured amount of change in absorbance.
(6) Next, a sample with a known total protein S activity value was measured as described in (1) to (5) above, and the amount of change in absorbance per minute was determined.
(7) Next, the amount of change in absorbance per minute in the sample determined in (5) above, and the amount of change in absorbance per minute in samples whose activity value of total protein S determined in (6) is known And the activity value (μg / mL equivalent) of total protein S contained in the sample was calculated.
In this way, the total protein S activity value of each sample of the above 2 was obtained.
The results obtained by measuring the activity of total protein S for each of the above two samples are shown in FIG. 7 and Table 8.
III. Determination of protein amount of protein S by latex turbidimetry with reagent kit and measurement method
1. Reagent kit for measuring protein S protein activity by latex turbidimetry
(I) First reagent
C4b binding protein was obtained from B. The method of Dahlbeack et al. [Biochem. J. et al. 209, 847-856, 1983].
Next, a 50 mM MES-hydrochloric acid buffer solution (pH 6.2 (20 ° C.)) containing 0.1% (W / V) BSA, 0.3 M sodium chloride and 0.05% sodium azide was prepared.
Here, the C4b binding protein prepared as described above was added to a concentration of 25 μg / mL, and used as the first reagent.
(Ii) Second reagent
(A) Preparation of anti-protein S antibody
The present inventors prepared anti-protein S antibody according to a conventional method using purified free protein S as an immunizing antigen.
A hybridoma producing a monoclonal antibody that specifically binds to a site other than the binding site of protein S to the C4b binding protein was screened to obtain a mouse / mouse hybridoma (strain 9H6).
5 mg of mouse anti-protein S monoclonal antibody produced from this hybridoma (9H6 strain) was dissolved in 0.5 mL of sodium phosphate buffer (pH 7.5).
To this, 0.01 mL of N, N-dimethylformamide in which 0.6 mg of S-acetylmercaptosuccinic anhydride was dissolved was added and incubated at room temperature for 30 minutes.
Next, 0.02 mL of 0.1 M EDTA aqueous solution, 0.1 mL of 1 M tris (hydroxymethyl) aminomethane buffer (pH 7.0), and 0 of 1 M hydroxylamine hydrochloride buffer (pH 7.0) were added thereto. Each 1 mL was added and incubated at 30 ° C. for 30 minutes.
Thereafter, this was subjected to gel filtration with a Sephadex G-25 column, which had been equilibrated with 0.1 M sodium phosphate buffer (pH 6.0) containing 5 mM EDTA, and mercapto-succinylated mouse anti-protein S. A monoclonal antibody was obtained.
(B) Preparation of anti-protein S antibody-immobilized latex particle suspension
1.0 mL of a 10% suspension of latex particles having an average particle size of 0.192 μm and 1.0 mL of 50 mM MES-hydrochloric acid buffer (pH 6.0 (20 ° C.)) are mixed, and further 320 mM carbodiimide (Dojindo Laboratories) Product number: 348-03631) 32 μL of an aqueous solution was added and mixed, and allowed to stand on ice for 10 minutes.
To 1.2 mL of the latex particle suspension that was allowed to stand on ice for 10 minutes, the mercapto-succinylated mouse anti-protein S monoclonal antibody prepared in (a) was added at a concentration of 0.083 g / dL in 50 mM MES-HCl buffer. The liquid [pH 6.0 (20 degreeC)] 1.8mL mixed liquid was added, and it stirred at 4 degreeC overnight.
Next, after removing the supernatant by centrifugation, the precipitate was suspended in 50 mM Tris-HCl buffer (pH 8.0 (20 ° C.)) containing 0.8% BSA and left at 37 ° C. for 3 hours. The blocking process was performed.
Next, after collecting the precipitate by centrifugation, it was redispersed with a 0.05% sodium azide aqueous solution containing 0.1% BSA and suspended so that the absorbance at a wavelength of 700 nm was 15.0 OD. .
This was designated as an anti-protein S antibody-immobilized latex particle suspension.
(C) Preparation of second reagent
The anti-protein S antibody-immobilized latex particle suspension prepared in (b) above was diluted 10-fold with a 0.05% aqueous sodium azide solution containing 0.1% BSA to obtain 0.1% “anti-protein S”. A suspension containing “antibody-immobilized latex particles” was prepared.
This was used as the second reagent.
2. sample
Each of the above samples I (2) (1) and (2) was used as a sample.
3. Measurement
(A) Measurement was performed using a general-purpose automatic analyzer TBA-120FR (Toshiba Medical Systems [Japan]).
First, 3 μL of the sample 2 was added to a measurement cell (cuvette).
Next, 100 μL of the first reagent (1) was added to these measurement cells (cuvettes) and mixed.
These measurement cells (cuvettes) were allowed to stand at 37 ° C.
Thereby, the complex of the free protein S contained in the sample and the C4b binding protein in the first reagent was formed.
That is, all the protein S contained in the sample became a “complex of protein S and C4b binding protein (binding type)”.
(B) At 4 minutes and 40 seconds after the addition of the first reagent (measurement point: 16 points), the liquid mixture in these measurement cells (cuvettes) is further added to (c) of (ii) above. 100 μL of the second reagent of) was added and mixed.
(C) At 5 minutes and 35 seconds after the addition of the first reagent (measurement point: 19 points), the absorbance (wavelength 700 nm) of the mixed solution in these measurement cells (cuvettes) was measured as a sample blind test. .
These measurement cells (cuvettes) were allowed to stand at 37 ° C. for reaction.
As a result, an antigen-antibody reaction between the anti-protein S antibody immobilized on the latex particles and the protein S contained in the sample was performed to generate latex particle aggregates.
(D) At 9 minutes and 47 seconds (measurement point: 33 points) after the addition of the first reagent, the absorbance (wavelength 700 nm) of the reaction mixture in the measurement cell (cuvette) is the measured value of the sample. As measured.
(E) The absorbance (sample blind) measured in (c) was subtracted from the absorbance (measured value) measured in (d) to obtain an absorbance difference.
This difference in absorbance is proportional to the total protein S protein amount (concentration) contained in the sample.
A calibration curve is prepared from the absorbance difference of a sample having a known protein concentration of total protein S, and the absorbance difference obtained by measuring each of the above two samples by the same method is applied to the calibration curve to obtain the total amount contained in the sample. The protein concentration of protein S was determined.
Thus, the protein amount of the total protein S of each sample of 2 was obtained.
FIG. 7 and Table 8 show the results obtained by measuring the protein amount of the total protein S for each of the two samples.
IV. Measurement result
Measurement results of the protein S activity measurement reagent kit and activity measurement method of the present invention in I, measurement results of the conventional protein S activity measurement reagent kit and activity measurement method in II, and latex turbidimetry in III FIG. 7 shows a diagram comparing the measurement results obtained by the reagent kit for measuring the protein amount of protein S and the measurement method by Table 1, and Table 8 shows the table for comparison.
1. Description of figure
In FIG. 7, the horizontal axis indicates the measured value (unit: μg / mL) of the total protein S contained in the sample by the latex nephelometry in III.
In FIG. 7, the vertical axis indicates the measured value of the activity of total protein S contained in the sample according to the present invention in I and the conventional protein S activity measuring reagent kit and activity measuring method in II (unit: μg / mL equivalent).
In FIG. 7, “◯” indicates the measurement results when the activity of the total protein S contained in the sample was measured by the protein S activity measurement reagent kit and activity measurement method of the present invention in I above, “Δ” indicates the measurement result when the activity of the total protein S contained in the sample was measured by the conventional protein S activity measuring reagent kit and activity measuring method in II.
2. Table description
In Table 8, in order from the left side, the measurement value (unit: μg / mL) of the protein amount of the total protein S contained in the sample by the latex turbidimetry method in III, the reagent for measuring the activity of protein S of the present invention in I above Measured value of total protein S activity (unit: μg / mL equivalent) contained in sample by kit and activity measurement method, and contained in sample by conventional protein S activity measurement reagent kit and activity measurement method in II above The measured values (unit: μg / mL equivalent) of the activity of total protein S are shown respectively.

V. Summary
(1) From FIG. 7 and Table 8, which are measurement results in the IV, the following can be understood.
That is, the activity measurement result (measurement value) of the protein S activity measurement reagent kit and activity measurement method of the present invention in I above and the activity measurement of the conventional protein S activity measurement reagent kit and activity measurement method in II above When comparing the results (measured values) of the present invention, the activity of the present invention in the above-mentioned I in both of the plasma samples of 32 healthy subjects and the sample of one plasma subject to protein S abnormality (protein S Tokushima) It can be seen that most of the measurement values obtained by the measurement reagent kit and activity measurement method and the measurement values obtained by the conventional activity measurement reagent kit and activity measurement method in II are the same.
Thus, the protein S activity measurement reagent kit and activity measurement method of the present invention can be operated by reducing the constituent reagents and measurement steps (measurement steps) compared to the conventional activity measurement reagent kit and activity measurement method. The measurement results are the same as those of conventional activity measurement reagent kits and activity measurement methods with many constituent reagents and measurement steps (measurement steps), despite the fact that the time required for measurement is greatly reduced. I understand that.
(2) In addition, the result of measurement of activity by the reagent kit and activity measurement method for protein S of the present invention in I above (measurement value) and the result of measurement of protein amount by latex turbidimetry in III above (measurement value) ) In most samples, the specific activity obtained by dividing the activity value of total protein S by the protein amount of total protein S is almost y = x. You can see that it is on the line.
Therefore, also from this, it can be seen that the measured values by the protein S activity measuring reagent kit and the activity measuring method of the present invention are accurate.
In FIG. 7, the measured value of the activity of the total protein S is clearly lower than the measured value of the protein amount of the total protein S, and is far below the y = x line 3 Of the two samples, one sample is one sample that has been found to have protein S abnormality (Protein S Tokushima), but the other two samples also have protein S type II abnormality. It is estimated that.
(3) Therefore, also from the examination results in this example, an activated protein C-containing reagent containing at least activated protein C, an activated blood coagulation factor V-containing reagent containing at least activated blood coagulation factor V, and at least A protein S activity measurement reagent kit according to the present invention, and a protein S activity measurement reagent kit according to the present invention, each of which comprises prothrombin and a component reagent of a prothrombin / substrate-containing reagent containing a thrombin substrate. The method for measuring the activity of protein S of the present invention reduces the number of constituent reagents and measurement steps (measurement steps) without sacrificing the accuracy of the measurement, thereby saving labor in the measurement and shortening the time required for the measurement. Measures the protein S activity contained in the sample accurately, simply and in a short time. It was confirmed that it was possible.
[Example 3] (Protein S activity measurement reagent kit and activity measurement method-2 of the present invention)
The activity of protein S contained in the sample was measured with the reagent kit and activity measurement method for protein S of the present invention.
1. Reagent kit for protein S activity measurement
The protein S activity measurement reagent kit of the present invention was prepared as follows.
The main components contained in each component reagent of the protein S activity measurement reagent kit are shown in Table 9.

(1) Reagent / method-3 of the present invention
“Reagent / method-3 of the present invention” which is a reagent kit for measuring the activity of protein S of the present invention was prepared as follows.
(I) Reactive protein C-containing reagent
The following reagent components were dissolved in pure water to the respective concentrations described above, and the pH was adjusted to 7.0 (20 ° C.) to prepare an activated protein C-containing reagent (present invention-3).
Activated protein C (purified human activated protein C; Enzyme Research Laboratories, Inc. [USA]) 3.6 nM
Activated blood coagulation factor X (purified bovine activated blood coagulation factor X; New England Biolabs, Inc. [USA]) 5.9 nM
Sodium chloride <Salt> 0.1M
Bovine serum albumin (BSA; Sigma-Aldrich [USA]) <Protein> 0.1% (W / V)
Sodium azide <Preservative> 0.05% (W / V)
3-morpholinopropanesulfonic acid (MOPS) <Buffer> 50 mM
(Ii) Activated blood coagulation factor V-containing reagent
Prepare the activated blood coagulation factor V-containing reagent (Invention-3) by dissolving the following reagent components in pure water so as to have the concentrations indicated, and adjusting the pH to pH 7.0 (20 ° C.). did.
Activated blood coagulation factor V (Purified human activated blood coagulation factor V; Haematologic Technologies, Inc. [USA]) 150 pM
Phospholipid (The composition ratio of phosphatidylserine [PS], phosphatidylcholine [PC] and phosphatidylethanolamine [PE] is 1: 1: 1 [33.33% (W / V): 33.33% (W / V): 33.33% (W / V)]; NOF Corporation [Japan]) 0.05% (W / V)
Calcium chloride 5 mM
Sodium chloride <Salt> 0.1M
Bovine serum albumin (BSA; Sigma-Aldrich [USA]) <Protein> 0.1% (W / V)
Sodium azide <Preservative> 0.05% (W / V)
3-morpholinopropanesulfonic acid (MOPS) <Buffer> 50 mM
(Iii) Prothrombin / substrate-containing reagent
The following reagent components were dissolved in pure water to the respective concentrations described above, and the pH was adjusted to 7.0 (20 ° C.) to prepare a prothrombin / substrate-containing reagent (present invention-3).
Prothrombin (purified human prothrombin; Enzyme Research Laboratories, Inc. [USA]) 694 nM
Test Team (registered trademark) Chromogenic Substrate S-2238 (Thrombin Substrate [Thrombin Chromogenic Substrate]; Manufacturer: Chromogenix-Instrumentation Laboratories [Italy], Distributor: Sekisui Medical Inc. [Japan]) 1.5 mM
Sodium chloride <Salt> 0.1M
Bovine serum albumin (BSA; Sigma-Aldrich [USA]) <Protein> 0.1% (W / V)
Sodium azide <Preservative> 0.05% (W / V)
3-morpholinopropanesulfonic acid (MOPS) <Buffer> 50 mM
(2) Reagent / method of the present invention-4
“Reagent / method-4 of the present invention” which is a reagent kit for measuring the activity of protein S of the present invention was prepared as follows.
(I) Reactive protein C-containing reagent
The following reagent components were dissolved in pure water to the respective concentrations described above, and the pH was adjusted to 7.0 (20 ° C.) to prepare an activated protein C-containing reagent (present invention-4).
Activated protein C (Purified human activated protein C; Enzyme Research Laboratories, Inc. [USA]) 72 pM
Phospholipid (The composition ratio of phosphatidylserine [PS], phosphatidylcholine [PC] and phosphatidylethanolamine [PE] is 1: 1: 1 [33.33% (W / V): 33.33% (W / V): 33.33% (W / V)]; NOF Corporation [Japan]) 0.05% (W / V)
Calcium chloride 5 mM
Sodium chloride <Salt> 0.1M
Bovine serum albumin (BSA; Sigma-Aldrich [USA]) <Protein> 0.1% (W / V)
Sodium azide <Preservative> 0.05% (W / V)
3-morpholinopropanesulfonic acid (MOPS) <Buffer> 50 mM
(Ii) Activated blood coagulation factor V-containing reagent
Prepare the activated blood coagulation factor V-containing reagent (Invention-4) by dissolving the following reagent components in pure water so as to have the concentrations indicated, and adjusting the pH to 7.0 (20 ° C.). did.
Activated blood coagulation factor V (purified human activated blood coagulation factor V; Haematologic Technologies, Inc. [USA]) 7.5 nM
Activated blood coagulation factor X (purified bovine activated blood coagulation factor X; New England Biolabs, Inc. [USA]) 5.9 nM
Calcium chloride 5 mM
Sodium chloride <Salt> 0.1M
Bovine serum albumin (BSA; Sigma-Aldrich [USA]) <Protein> 0.1% (W / V)
Sodium azide <Preservative> 0.05% (W / V)
3-morpholinopropanesulfonic acid (MOPS) <Buffer> 50 mM
(Iii) Prothrombin / substrate-containing reagent
The following reagent components were dissolved in pure water to the respective concentrations described above, and the pH was adjusted to 7.0 (20 ° C.) to prepare a prothrombin / substrate-containing reagent (present invention-4).
Prothrombin (purified human prothrombin; Enzyme Research Laboratories, Inc. [USA]) 694 nM
Test Team (registered trademark) Chromogenic Substrate S-2238 (Thrombin Substrate [Thrombin Chromogenic Substrate]; Manufacturer: Chromogenix-Instrumentation Laboratories [Italy], Distributor: Sekisui Medical Inc. [Japan]) 1.5 mM
Sodium chloride <Salt> 0.1M
Bovine serum albumin (BSA; Sigma-Aldrich [USA]) <Protein> 0.1% (W / V)
Sodium azide <Preservative> 0.05% (W / V)
3-morpholinopropanesulfonic acid (MOPS) <Buffer> 50 mM
(3) Reagent / method of the present invention-5
“Reagent / method-5 of the present invention” which is a reagent kit for measuring the activity of protein S of the present invention was prepared as follows.
(I) Reactive protein C-containing reagent
The following reagent components were dissolved in pure water to the respective concentrations described above, and the pH was adjusted to 7.0 (20 ° C.) to prepare an activated protein C-containing reagent (present invention-5).
Activated protein C (Purified human activated protein C; Enzyme Research Laboratories, Inc. [USA]) 72 pM
Activated blood coagulation factor X (purified bovine activated blood coagulation factor X; New England Biolabs, Inc. [USA]) 117 pM
Phospholipid (The composition ratio of phosphatidylserine [PS], phosphatidylcholine [PC] and phosphatidylethanolamine [PE] is 1: 1: 1 [33.33% (W / V): 33.33% (W / V): 33.33% (W / V)]; NOF Corporation [Japan]) 0.05% (W / V)
Calcium chloride 5 mM
Sodium chloride <Salt> 0.1M
Bovine serum albumin (BSA; Sigma-Aldrich [USA]) <Protein> 0.1% (W / V)
Sodium azide <Preservative> 0.05% (W / V)
3-morpholinopropanesulfonic acid (MOPS) <Buffer> 50 mM
(Ii) Activated blood coagulation factor V-containing reagent
Prepare the activated blood coagulation factor V-containing reagent (Invention-5) by dissolving the following reagent components in pure water to the indicated concentrations and adjusting the pH to 7.0 (20 ° C). did.
Activated blood coagulation factor V (purified human activated blood coagulation factor V; Haematologic Technologies, Inc. [USA]) 7.5 nM
Calcium chloride 5 mM
Sodium chloride <Salt> 0.1M
Bovine serum albumin (BSA; Sigma-Aldrich [USA]) <Protein> 0.1% (W / V)
Sodium azide <Preservative> 0.05% (W / V)
3-morpholinopropanesulfonic acid (MOPS) <Buffer> 50 mM
(Iii) Prothrombin / substrate-containing reagent
The following reagent components were dissolved in pure water so as to have the respective concentrations described above, and the pH was adjusted to 7.0 (20 ° C.) to prepare a prothrombin / substrate-containing reagent (present invention-5).
Prothrombin (purified human prothrombin; Enzyme Research Laboratories, Inc. [USA]) 694 nM
Test Team (registered trademark) Chromogenic Substrate S-2238 (Thrombin Substrate [Thrombin Chromogenic Substrate]; Manufacturer: Chromogenix-Instrumentation Laboratories [Italy], Distributor: Sekisui Medical Inc. [Japan]) 1.5 mM
Sodium chloride <Salt> 0.1M
Bovine serum albumin (BSA; Sigma-Aldrich [USA]) <Protein> 0.1% (W / V)
Sodium azide <Preservative> 0.05% (W / V)
3-morpholinopropanesulfonic acid (MOPS) <Buffer> 50 mM
2. sample
Human plasma is divided into two, and one of these is passed through a column (affinity column) in which an anti-human protein S antibody is bound to a carrier, thereby immobilizing protein S contained in the human plasma in this column, The flow-through fraction was fractionated to obtain protein S-deficient plasma not containing protein S.
The following five types of human plasma samples were prepared by mixing this protein S-deficient plasma and the other human plasma (including protein S) divided at various ratios.
In addition, the numerical value shown for each sample represents the total protein S activity value (μg / mL equivalent) of each sample.
The protein S-deficient plasma was used as a sample having a total protein S activity value of 0 μg / mL equivalent.
(1) Sample a (total protein S activity value: 0 μg / mL equivalent)
(2) Sample b (Total protein S activity value: 6.2 μg / mL equivalent)
(3) Sample c (Total protein S activity value: 12.3 μg / mL equivalent)
(4) Sample d (total protein S activity value: 18.5 μg / mL equivalent)
(5) Sample e (Total protein S activity value: 24.6 μg / mL equivalent)
(6) Sample f (total protein S activity value: 31.5 μg / mL equivalent)
3. Measurement
The protein S activity of each sample was measured using a 7170S type general-purpose automatic analyzer (Hitachi High-Technologies Corporation [Japan]) as follows.
(1) With the sample dilution function provided in the 7170S general-purpose automatic analyzer, 2 μL of each of the samples (1) to (6) above and the activated protein (i) of (1) above 270 μL of C-containing reagent (Invention-3) was mixed.
By mixing this sample with the activated protein C-containing reagent (present invention-3), this sample was diluted at a dilution ratio of 136 times.
(2) Next, 3 μL of the mixed solution of (1) is sampled and discharged to a cuvette, and the activated blood coagulation factor V-containing reagent of (ii) of (ii) above (this invention- 150 μL of 3) was added and mixed. [Measurement points: 0 points]
And it incubated at 37 degreeC.
(3) Next, the measurement point was changed from 16 points (4 minutes 30.093 seconds after addition of the above-mentioned activated blood coagulation factor V-containing reagent (present invention-3)) to 17 points (4 minutes 46 after addition of the reagent). (977 seconds), 50 μL of (iii) prothrombin / substrate-containing reagent (Invention-3) of (1) above was added to the mixture of (2) and mixed.
And it incubated at 37 degreeC until the measurement point 34 point [The said activated blood coagulation factor V containing reagent (invention-3) was added and 9 minutes 47.426 seconds later].
(4) Change in absorbance (wavelength: 405 nm) of the mixed solution in the cuvette was measured at 1 point [7267 seconds after adding the above-mentioned activated blood coagulation factor V-containing reagent (present invention-3) To 34 measurement points [9 minutes 47.426 seconds after addition of the same reagent] and recorded.
That is, the change in absorbance (wavelength: 405 nm) when the protein S activity of each sample was measured by the reagent / method-3 of the present invention (1) was measured and recorded.
(5) The activated blood coagulation factor V-containing reagent of (ii) of (ii) above (Invention-4) instead of the activated protein C-containing reagent (Invention-3) in (1) ), And in (2), instead of the activated blood coagulation factor V-containing reagent (present invention-3), the activated protein C-containing reagent (present invention-4) of (1) in (1) above ), And (iii) using the prothrombin / substrate-containing reagent (Invention-4) in (2) above instead of the prothrombin / substrate-containing reagent (Invention-3) in (3) Is the absorbance (wavelength: 405 nm) when the protein S activity of each sample was measured by the reagent / method-4 of the present invention (1) of (1) above. ) Was measured and recorded.
(6) The activated blood coagulation factor V-containing reagent of (ii) in (ii) above (Invention-5) instead of the activated protein C-containing reagent (Invention-3) in (1) ), (1) the activated protein C-containing reagent of the above (3) (i) (the present invention-5) instead of the activated blood coagulation factor V-containing reagent (the present invention-3) in the above (2). ) And (iii) (iii) (iii) using a prothrombin / substrate-containing reagent (this invention-5) instead of the prothrombin / substrate-containing reagent (this invention-3) in the above (3). Is the absorbance (wavelength: 405 nm) when the procedure of (1) to (4) is performed and the activity of protein S of each sample is measured by the reagent / method-5 of the present invention (1). ) Was measured and recorded.
4). Measurement result
(1) The measurement result in 3 above, that is, the change in absorbance (wavelength: 405 nm) when the activity of protein S contained in a sample is measured by the protein S activity measurement reagent kit and activity measurement method of the present invention. 8 to FIG. 10 and Tables 10 to 12.
The results of measurement using the reagent / method-3 of the present invention 1 (1) are shown in FIG. 8 and Table 10, and the results of measurement using the reagent / method-4 of the present invention 1 (2) are shown in FIG. 10 and Table 12 show the results of the measurement by the reagent / method-5 of the present invention (1) in (1) above.
(2) In these FIGS. 8 to 10, which protein S activity measurement reagent kit and activity measurement method were used was shown above the figure.
In these drawings, the horizontal axis represents the measurement points in the 7170S general-purpose automatic analyzer used for the measurement, and the vertical axis represents the absorbance (wavelength: 405 nm) at each measurement point.
Further, in these figures, “□” indicates a measured value (the absorbance described above) when measured for the sample a (2) (1) (total protein S activity value: 0 μg / mL equivalent). Indicates the measured value (the absorbance described above) when measured for the sample b (2) (total protein S activity value: 6.2 μg / mL equivalent) in 2 above, and “Δ” indicates that in 2 (3) above. The measurement value (the absorbance described above) when measured with respect to the sample c (total protein S activity value: 12.3 μg / mL equivalent), and “◯” indicates the sample d (total protein S activity value) in (2) above. : 18.5 μg / mL equivalent) measured value (absorbance described above), “+” indicates the sample e in 2 (5) above (total protein S activity value: 24.6 μg / mL equivalent) The measured value (absorbance described above) when measured for "-" Indicates a measured value (the absorbance described above) when measured for the sample f (2) (6) (total protein S activity value: 31.5 μg / mL equivalent).
(3) In Tables 10 to 12, the upper left of the table indicates which protein S activity measurement reagent kit and activity measurement method was used.
In these tables, in order from the top, the measured value (the absorbance described above) when measuring the sample a (2) (1) (total protein S activity value: 0 μg / mL equivalent), 2) Sample b (total protein S activity value: 6.2 μg / mL equivalent), measured value (absorbance described above), sample c of 2 (3) above (total protein S activity value: 12. Measured value when measured for 3 μg / mL equivalent) (absorbance described above), Measured value when measured for sample d of 2 above (4) (total protein S activity value: 18.5 μg / mL equivalent) ), (2) (5) sample e (total protein S activity value: 24.6 μg / mL equivalent) measured value (absorbance), (2) sample (6) f Total protein S activity: 31.5 μg mL showing the measurement value when measured equiv) (the absorbance).
In these tables, in order from the left side, the measured value when the measurement point is 1 point (absorbance described above), the measured value when the measurement point is 2 points (absorbance described above), and the measured point is 3 The measured value when the point is (the absorbance described above),... The measured value when the measurement point is 34 points (the absorbance described above) are shown.

5. Summary
(1) From the measurement results in 3 above, FIGS.
That is, the measurement according to (1) of the present reagent / method-3 of (1), the measurement of (1) of the present invention / method-4 of (1), and the present (1) of the present reagent / method of (3). In the measurement according to 5, the absorbance after addition of each prothrombin / substrate-containing reagent in 3 and mixing in each of the samples a to 6 in (2) (1) to (6) is different for each sample. I understand.
Then, the measurement according to (1) of the present reagent / method-3 of (1), the measurement of (1) of the present invention / method-4 of (1), and the present reagent / method of (1) of (3) In any of the measurements according to 5, it can be seen that the absorbance gradually decreases as the activity value of protein S contained in the sample increases from a low value to a high value.
That is, the measurement according to 1 (1) of the reagent / method-3 of the present invention, the measurement according to (1) of the present invention / method-4 of 1), and the reagent / method of the present invention of (1) above- In the measurement according to 5, as the activity of protein S contained in the sample increases, the reaction that generates a signal from the thrombin substrate is suppressed. From this, the amount of the signal whose generation is suppressed is reduced. By measuring, it turns out that the activity of the protein S contained in the sample can be obtained quantitatively.
Namely, an activated protein C-containing reagent containing at least activated protein C, an activated blood coagulation factor V-containing reagent containing at least activated blood coagulation factor V, and a prothrombin / substrate-containing reagent containing at least prothrombin and a thrombin substrate (1) Measurement by the reagent / method-3 of the present invention (1) using a protein S activity measurement reagent kit comprising the constituent reagents of (1), Measurement by the reagent / method-4 of the present invention (1), It can also be seen that the activity of protein S contained in the sample can be quantitatively measured in the measurement by the reagent / method-5 of the present invention (3) in 1 above.
(3) Therefore, also from the examination results in this example, an activated protein C-containing reagent containing at least activated protein C, an activated blood coagulation factor V-containing reagent containing at least activated blood coagulation factor V, and at least A protein S activity measurement reagent kit according to the present invention, and a protein S activity measurement reagent kit according to the present invention, each of which comprises prothrombin and a component reagent of a prothrombin / substrate-containing reagent containing a thrombin substrate. The method for measuring protein S activity of the present invention is capable of quantitatively measuring the activity of protein S contained in a sample, is easy to automate the measurement, and is capable of measuring protein S contained in a sample. It was confirmed that the activity can be measured accurately, simply and in a short time.

Claims

A protein S activity measurement reagent kit, comprising the following constituent reagents (i) to (iii):
(I) Activated protein C-containing reagent: Contains at least activated protein C.
(Ii) Activated blood coagulation factor V-containing reagent: Contains at least activated blood coagulation factor V.
(Iii) Prothrombin / substrate-containing reagent: Contains at least prothrombin and thrombin substrate.
The protein S activity measurement reagent kit according to claim 1, wherein at least one reagent selected from an activated protein C-containing reagent and an activated blood coagulation factor V-containing reagent contains phospholipid.
The protein S activity measurement reagent kit according to claim 1 or 2, wherein at least one reagent selected from an activated protein C-containing reagent and an activated blood coagulation factor V-containing reagent contains calcium ions.
4. The method according to claim 1, wherein at least one reagent selected from an activated protein C-containing reagent, an activated blood coagulation factor V-containing reagent, and a prothrombin / substrate-containing reagent contains activated blood coagulation factor X. A reagent kit for measuring the activity of protein S according to claim 1.
The protein S according to any one of claims 1 to 4, wherein the activated protein C-containing reagent and the activated blood coagulation factor V-containing reagent contain neither prothrombin nor a thrombin substrate. Activity measurement reagent kit.
6. The protein S activity measurement reagent kit according to claim 1, wherein the protein S is total protein S.
A method for measuring protein S activity, which comprises using the protein S activity measuring reagent kit according to any one of claims 1 to 6.
The method for measuring the activity of protein S according to claim 7, wherein the protein S is total protein S.