JP2001289843A - Container for blood test - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a container for blood test by which a high blood coagulation performance can be displayed stably for a long period. SOLUTION: In the container for blood test, a blood coagulation accelerator is housed. In the container for blood test, the blood coagulation accelerator is prepared by drying an aqueous solution, at less than pH 7, wherein an enzyme (preferably, thrombin) by which the bond of arginine to an arbitrary amino acid residue and/or the bond of lysine to an arbitrary amino acid residure can be hydrolyzed in a peptide chain is derived and an aminocarboxylic acid (preferably, tranexamie acid) is dissolved.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a blood test container used for a clinical test such as a serum biochemical test, and more particularly to a blood test container containing a substance which promotes coagulation of a blood sample.

[0002]

2. Description of the Related Art In recent years, in order to collect blood for a serum test such as a serum biochemical test, a serum immunological test, and a blood cell test, it has become possible to shorten the coagulation time of blood and obtain serum quickly. Blood test containers are increasingly used. In such a blood test container, as a blood coagulation accelerator, an inorganic blood coagulation accelerator represented by silica or an enzyme-based blood coagulation promoter represented by thrombin is stored in advance. Blood clotting is promoted by the action of the drug.

[0003] Among them, an enzyme-based blood coagulation accelerator represented by thrombin has a blood coagulation activity several times higher than that of an inorganic blood coagulation accelerator, and exhibits extremely excellent blood coagulation activity. However, enzymatic blood coagulation promoters are extremely unstable and have a problem that blood coagulation activity decreases with time.

[0004] In order to solve this problem, a technique is also known in which an enzyme-based blood coagulation promoter such as thrombin is stabilized by freeze-drying. However, long-term stability is not always high only by freeze-drying.

On the other hand, as a technique for stabilizing thrombin or the like in an aqueous solution, a technique of adding a stabilizer to an aqueous solution as described in JP-A-64-40433,
As described in JP-A-62-106028, a technique for adjusting the pH of an aqueous solution is known. However, even these techniques have not been applicable to blood test containers that require particularly long-term storage stability.

[0006]

SUMMARY OF THE INVENTION An object of the present invention is to provide a blood test container capable of stably exhibiting high blood coagulation performance over a long period of time.

[0007]

According to the first aspect of the present invention,
In a blood test container containing a blood coagulation accelerator,
The blood coagulation promoter has an enzyme capable of hydrolyzing a bond between arginine and an arbitrary amino acid residue and / or a bond between lysine and an arbitrary amino acid residue in a peptide chain, and a pH 7 in which an aminocarboxylic acid is dissolved. A blood test container characterized by being prepared by drying less than an aqueous solution.

The invention according to claim 2 is the blood test container according to claim 1, wherein the aminocarboxylic acid is a substance having a cyclohexane ring.

The invention according to claim 3 is the blood test container according to claim 1, wherein the aminocarboxylic acid is at least one selected from the group consisting of tranexamic acid, glycine and alanine.

[0010] The invention according to claim 4 is characterized in that the aqueous solution has a pH
The blood test container according to any one of claims 1 to 3, wherein the number is 6 or more and less than 7.

Hereinafter, the present invention will be described in detail.

The blood test container of the present invention contains a blood coagulation accelerator. In the blood coagulation promoter, an enzyme capable of hydrolyzing a bond between arginine and an arbitrary amino acid residue and / or a bond between lysine and an arbitrary amino acid residue in a peptide chain and an aminocarboxylic acid are dissolved. It is prepared by drying an aqueous solution having a pH of less than 7.

An enzyme capable of hydrolyzing the bond between arginine and an arbitrary amino acid residue and / or the bond between lysine and an arbitrary amino acid residue in a peptide chain is generally a substance having an action of promoting blood coagulation alone.

Examples of the above-mentioned hydrolase include serine proteases such as trypsin, thrombin, and snake venom thrombin-like enzymes; thiol proteases such as cathepsin B and ficin; and metalloproteases such as kininase I. Thrombin is preferably used.

The method for preparing thrombin is not particularly limited, and for example, a method obtained by purifying thrombin from animal (human, bovine, etc.) plasma can be used.

The above aminocarboxylic acid has an action of stabilizing an enzyme capable of hydrolyzing a bond between arginine and an arbitrary amino acid residue and / or a bond between lysine and an arbitrary amino acid residue in a peptide chain, in particular, stabilizing thrombin. Have.

The aminocarboxylic acid is a substance having an amino group and a carboxyl group in one molecule, and is not particularly limited in the present invention as long as it has a function of stabilizing the above-mentioned hydrolase.

Among the aminocarboxylic acids, it is preferable to use a substance having a cyclohexane ring in the molecular structure.
For example, tranexamic acid (4-aminomethylcyclohexanecarboxylic acid), 4-aminocyclohexanecarboxylic acid, and the like can be mentioned, and among them, tranexamic acid can be particularly preferably used.

Further, amino acids such as glycine and alanine can be used as the aminocarboxylic acid.

These aminocarboxylic acids may be used alone or in combination of two or more.

In preparing the blood coagulation promoter of the present invention, an enzyme capable of hydrolyzing a bond between arginine and an arbitrary amino acid residue and / or a bond between lysine and an arbitrary amino acid residue in the above peptide chain is first used. And an aqueous solution in which the above aminocarboxylic acid is dissolved.

The aqueous solution is prepared so as to have a pH of less than 7 (solution temperature of 20 ° C.) in a state in which the hydrolase and the aminocarboxylic acid are dissolved. This is the p
If H is 7 or more (particularly pH 7), the hydrolase will autolyze. Therefore, by setting the pH to less than 7, autolysis of the hydrolase is prevented.

The pH of the aqueous solution is preferably from 5 to less than 7, more preferably from 6 to less than 7. This is because when the pH of the aqueous solution is less than 5, the hydrolase is inactivated by the acid. The most preferable range of the pH of the aqueous solution is 6.0 to 6.7.

When preparing an aqueous solution having a pH of less than 7, it is not particularly limited, but it is convenient to dissolve the hydrolase and the aminocarboxylic acid in a buffer solution whose pH has been adjusted to less than 7 in advance. It is.

Examples of the buffer include phosphate buffer, citrate-disodium phosphate buffer, citrate-
Examples include a sodium citrate buffer, a sodium maleate-sodium hydroxide buffer, and the like.

The concentration of the enzyme capable of hydrolyzing the bond between arginine and any amino acid residue and / or the bond between lysine and any amino acid residue in the peptide chain in the aqueous solution is 5 to 10,000 IU / mL. Preferably, 10
5000 IU / mL is more preferred.

The concentration of aminocarboxylic acid in the above solution depends on the type and concentration of the hydrolase used in combination, but is preferably 0.001 to 30% (w / v). More preferably, it is set to be from 01 to 15% (w / v).

In particular, when thrombin is used as the hydrolase and tranexamic acid is used as the aminocarboxylic acid, the ratio of tranexamic acid is preferably adjusted to 0.001 to 0.1 mg per 1 IU of thrombin in the aqueous solution. Preferably, more preferably 0.01 to 0.0
Prepare so as to have a ratio of 5 mg.

The blood coagulation promoter in the present invention is prepared by preparing an aqueous solution in which the above-mentioned hydrolase and aminocarboxylic acid are dissolved, and then drying this aqueous solution. The reason for the necessity of the drying is that the hydrolytic enzyme has a property of being deactivated with time in water.

The drying method is not particularly limited, and the drying may be carried out naturally or using a hot air dryer or a dehumidifying dryer. However, when heating at the time of drying, it is preferable to dry at 50 ° C. or lower, and more preferably at 35 ° C. or lower, because if it is dried at high temperature, the hydrolase may be deactivated. .

In the manufacture of the blood test container of the present invention,
The step of drying the aqueous solution is not particularly limited. The aqueous solution may be dried after being stored in the container main body, or the blood coagulation promoter may be prepared by previously drying the aqueous solution outside the container to prepare the blood coagulation promoter. A coagulation accelerator may be stored in the container.

When the aqueous solution is dried after being stored in the container body, the storing method and storing form of the aqueous solution are not particularly limited. Examples of the storage method include a method in which the aqueous solution is spray-coated on the inner wall surface of the container, a method in which the aqueous solution is dropped on the bottom surface of the container, and the like.

The storage form may be held on the entire inner wall surface of the container, or may be held locally in the container.

Even when the blood coagulation accelerator obtained by drying the aqueous solution is stored in a container, the storage method and storage form are not particularly limited.

As a storage method, the above blood coagulation promoter is
It may be tableted and stored in a container, or may be stored in powder form. Further, the blood coagulation promoter may be stored directly in the container, may be stored in the container via a carrier, or may be stored so as to be held on the wall surface of the container.

The shape of the container body constituting the blood test container of the present invention is not particularly limited as long as it can store blood and react with a drug.
Preferable examples include a bottomed tubular container constituting a general blood collection tube.

The content of the container body is not particularly limited, and is about 1 to 20 mL, more preferably 2 to 1 mL.
A container body having a content of about 0 mL can be used.

[0038] The amount of the above-mentioned hydrolase stored in the container body is adjusted according to the amount of blood to be collected. If the amount is too small, the coagulation time may be prolonged or coagulation may be incomplete. If the amount is too large, the test value may be adversely affected. Therefore, the amount is preferably 1 to 20 IU per 1 ml of collected blood, more preferably 3 to 15 IU.

Examples of the material of the container body include thermoplastic resins such as polyethylene, polypropylene, polystyrene, polyethylene terephthalate, polymethyl methacrylate, and polyacrylonitrile; thermosetting resins such as unsaturated polyester resins, epoxy resins, and epoxy-acrylate resins. Resins; modified natural resins such as cellulose acetate, cellulose propionate, ethyl cellulose, ethyl chitin; silicate glass such as soda-lime glass, phosphosilicate glass, borosilicate glass; glass such as quartz glass;
And those containing these as main components.

The opening of the container body may be sealed with a sealing member. The blood test container of the present invention can be used as a so-called vacuum blood collection tube by sealing the opening and reducing the pressure inside the container. The use of a vacuum blood collection tube has the advantage that blood collection is simplified and the stability of the blood coagulation accelerator is further enhanced.

The member for sealing the opening of the container body may be a sheet-shaped sealing member in addition to a rubber stopper formed to fit into the opening. Examples of the material forming the plug include materials having excellent sealing properties, such as butyl rubber, chlorinated butyl rubber, brominated butyl rubber, and elastomer, and examples of the material forming the sealing member include metal foil such as aluminum.

In the blood test container of the present invention, a serum separating agent may be stored at the bottom. The serum separating agent moves between the clot and the serum by centrifugation after blood collection, and separates the serum by forming a septum.

The serum separating agent is a gel having thixotropic properties. For example, additives such as a thixotropic agent, a specific gravity adjusting agent, and a viscosity adjusting agent are added to a synthetic resin having fluidity at room temperature. And obtained by mixing.

Examples of the synthetic resin include oligomers of dicyclopentadiene and the like. Examples of the thixotropic agent include condensates of sorbitol and aromatic aldehyde and polyoxyethylene polyoxyalkylene block copolymer. As the specific gravity adjuster, for example, silica or the like, and as the viscosity adjuster, for example,
And phthalic acid esters.

The blood test container of the present invention may further contain a blood clot adhesion preventing component. This can prevent blood clot components from adhering to the inner surface of the container after coagulation of blood, and can effectively separate blood clots and serum without restricting the movement of blood clots during centrifugation.

As the blood clot adhesion preventing component, a hydrophilic substance which is hardly soluble or insoluble in water can be used, for example, an aliphatic modified silicone oil (eg, dimethylpolysiloxane), an aromatic modified silicone oil (E.g., methylphenylpolysiloxane), partially saponified polyvinyl alcohol, and vinylpyrrolidone-vinyl acetate copolymer. When modified silicone oils are used, it is preferable to further add a water-soluble substance in order to prevent a decrease in blood coagulation accelerating performance caused by covering the surface of the blood coagulation accelerating agent. Examples of the water-soluble substance include polyvinyl alcohol and polyvinyl pyrrolidone.

By adding the above-mentioned clot adhesion preventing component to an aqueous solution in which the above-mentioned hydrolase and aminocarboxylic acid are dissolved, it can be held on the inner wall surface of the container simultaneously with the blood coagulation promoter.

[Action] In the blood test container of the present invention, the blood coagulation promoter hydrolyzes the bond between arginine and any amino acid residue and / or the bond between lysine and any amino acid residue in the peptide chain. Aminocarboxylic acid is incorporated as a possible enzyme stabilizer. In addition, the blood coagulation accelerator is used to suppress autolysis of enzymes.
H is prepared by drying the aqueous solution in a predetermined range.
Therefore, the blood test container of the present invention can exhibit stable blood coagulation performance over a long period of time.

[0049]

Next, an embodiment of the present invention will be described. However, the present invention is not limited to only these examples.

In the examples and comparative examples, the following were used as reagents and containers.・ Thrombin (trade name: Thrombin Mochida, manufactured by Mochida Pharmaceutical Co., Ltd.) ・ Tranexamic acid (manufactured by Aldrich) ・ β-alanine (manufactured by Wako Pure Chemical Industries, Ltd., special grade reagent) ・ Test container: polyethylene terephthalate Inner volume: 10 ml (12φ × 100mm) (Sekisui Chemical Co., Ltd.)-Sealing member Butyl rubber stopper-Purified water (water for injection (Japanese Pharmacopoeia))

Example 1 250,000 IU of thrombin
And 3 g of tranexamic acid in 20 mM phosphate buffer (pH
(6.5, 20 ° C.). 10 μL of this aqueous solution (pH 6.5, 20 ° C.) was spray-coated on the inner surface of a 10 mL polyethylene terephthalate tube, and dried at 23 ° C. for 24 hours using a dehumidifying dryer.
The opening of the ethylene terephthalate tube was sealed with a plug made of butyl rubber to obtain a blood test container of the present invention.

Example 2 250,000 IU of thrombin
And 1.5 g of alanine in 20 mM phosphate buffer (pH 6.
(5, 20 ° C.) in 100 mL. This aqueous solution (p
(H6.5, 20 ° C.) 10 μL was spray-coated on the inner surface of a polyethylene terephthalate tube having an internal volume of 10 mL, and dried at 23 ° C. for 24 hours using a dehumidifying dryer. The opening of the ethylene terephthalate tube was sealed with a plug made of butyl rubber to obtain a blood test container of the present invention.

[Comparative Example 1] 250,000 IU of thrombin
Was dissolved in 100 mL of purified water. 10 μL of this aqueous solution
Is spray-coated on the inner surface of a polyethylene terephthalate tube having a content of 10 mL, and is dried at 23 ° C. using a dehumidifying dryer.
Let dry for hours. The opening of the ethylene terephthalate tube was sealed with a plug made of butyl rubber to obtain a blood test container of a comparative example.

[Evaluation of Stability] The blood test containers of the above Examples and Comparative Examples were stored at 35 ° C. and 75% RH before storage.
, The amount of thrombin after storage for one week was measured according to the thrombin quantification method of the thirteenth revised Japanese Pharmacopoeia, and the residual ratio of thrombin was determined. The results are shown in Table 1.

[0055]

[Table 1]

As is clear from Table 1, the Example of the present invention has a thrombin residual ratio about twice as high as that of the Comparative Example.
It can be seen that the blood coagulation activity has excellent stability over time.

[0057]

The blood test container of the present invention is as described above, wherein the blood coagulation accelerator in the container contains aminocarboxylic acid, and the pH of the aqueous solution of the coagulation accelerator before drying is less than 7. Therefore, stable blood coagulation performance can be exhibited for a long period of time.

 ──────────────────────────────────────────────────続 き Continued on the front page F term (reference) 2G045 AA01 BB38 BB48 BB52 CA25 FB01 HA02 HA06 4C038 TA10 UA02

Claims (4)

[Claims] 1. A blood test container containing a blood coagulation promoter, wherein the blood coagulation promoter comprises a bond between arginine and any amino acid residue and / or a lysine and any amino acid residue in a peptide chain. A blood test container, which is prepared by drying an aqueous solution having a pH of less than 7 in which an enzyme capable of hydrolyzing a bond of the above and an aminocarboxylic acid are dissolved. 2. The blood test container according to claim 1, wherein the aminocarboxylic acid is a substance having a cyclohexane ring. 3. The method of claim 1, wherein the aminocarboxylic acid is tranexamic acid,
The blood test container according to claim 1, wherein the container is at least one selected from the group consisting of glycine and alanine. 4. The blood test container according to claim 1, wherein the aqueous solution has a pH of 6 or more and less than 7.