JP6116231B2 - Tissue thromboplastin-containing blood coagulation ability measuring reagent and measuring method - Google Patents

Description

  The present invention relates to a reagent for measuring blood coagulation ability, particularly a reagent for measuring blood coagulation ability utilizing tissue thromboplastin.

Blood coagulation tests include prothrombin time (hereinafter abbreviated as PT) measurement, coagulation tests using chromogenic substrates, tests measuring individual components of the coagulation system such as endogenous thrombin generation ability (ETP), etc. Various inspection methods are known.
PT measurement is the most commonly used test in blood coagulation tests, and is mainly used for exogenous bleeding screening, monitoring of oral anticoagulants, and diagnosis of liver diseases. In general, PT measurement is performed by mixing a test plasma sample with a PT measurement reagent composed of a mixture of tissue thromboplastin and calcium ions, and the time from the moment of mixing until the formation of fibrin (coagulation) is observed (coagulation time). Is measured in seconds.
In a coagulation test where a chromogenic substrate is used, instead of clotting time, the time from the moment of mixing until a specific change in absorption is achieved is measured.
Tissue thromboplastin is also used in other test methods that do not measure clotting time but are useful for measuring individual components of the clotting system, such as, for example, endogenous thrombin generation capacity (ETP). (Patent Document 1).

  The main components of the PT measurement reagent are tissue thromboplastin and calcium ions. As the tissue thromboplastin, a naturally derived type generally extracted from a cerebrum such as a cow or a rabbit and a recombinant type in which a tissue factor prepared by genetic recombination is incorporated into a phospholipid are used.

  Many methods for obtaining tissue thromboplastin and producing PT measurement reagents are known, and many PT measurement reagents are commercially available. Natural tissue thromboplastin extracted from the cerebrum of animals contains impurities such as blood components, lipoproteins, and plasma proteins. When stored for a long period of time, insoluble substances are precipitated and the stability is poor. There was a problem. On the other hand, blood coagulation ability measuring reagents using genetically modified human tissue factor, bovine tissue factor, or rabbit tissue factor using Escherichia coli, yeast, insect cells or the like as a host are commercially available. However, even the genetically engineered tissue factor raw material has a problem that protein denaturation and biological activity decrease depending on the state of the storage solution, resulting in poor storage stability. Therefore, at present, most commercially available PT measurement reagents are sold in a lyophilized form. Therefore, before use, it must be dissolved in a reconstitution medium, such as distilled water or a buffer solution. The freeze-dried reagent requires a freeze-drying process that requires extra time and cost at the manufacturer, and the user needs to dissolve it using a solvent such as distilled water before use. In order to solve such problems, it is desired to provide a liquid and stable PT measurement reagent. If a liquid reagent can be achieved, these operations become unnecessary, and handling becomes easy, and the burden on the user can be reduced.

Regarding stabilization of tissue factor, tissue thromboplastin, and PT measurement reagent, for example, a method of adding albumin, sodium propionate, polyethylene glycol, or gluconic acid (Patent Document 2), a method of adding tartaric acid, gluconic acid, or lactic acid (Patent Document 3), a method of adding a nonionic surfactant and nickel ions (Patent Document 4), a method of adding a water-soluble polyphenol or catechol (Patent Document 5), a method of adding an antioxidant (Patent Document 3) Techniques such as 6) are disclosed.
However, these have problems such as insufficient stability and the need to add expensive substances. Further, although a PT measurement reagent that is stable in a lyophilized product by adding an amino acid or an amino acid derivative is disclosed (Patent Document 8), it is unclear whether it can be stabilized as a liquid reagent. As shown in FIG. 2, a stable effect was not obtained.

European Patent Application Publication No. EP420332A2 JP-A-6-201702 US Pat. No. 3,522,148 JP 2008-241621 A JP 2009-148261 A JP 11-316228 A JP-T 6-505562 JP 2002-303632 A

  The present invention has been made in view of these problems, and provides an inexpensive and stable blood coagulation ability measuring reagent and a measuring method containing tissue thromboplastin.

As a result of intensive studies in view of the above problems, the present inventors have stabilized a liquid prothrombin time (PT) measurement reagent containing tissue thromboplastin for a long period of time by containing an oligopeptide. Thus, the present inventors have found a blood coagulation ability measuring reagent and a measuring method which can be stably stored and can be measured with low cost and high accuracy.
The present invention has been accomplished based on this finding.

The present invention relates to the following inventions:
[1] A tissue thromboplastin-containing reagent for measuring blood clotting ability, comprising an oligopeptide.
[2] The tissue thromboplastin-containing blood coagulation ability measurement reagent according to [1], wherein the oligopeptide consists of at least two amino acids.
[3] The tissue thromboplastin-containing blood coagulation ability measurement reagent according to [1] or [2], wherein the oligopeptide contains at least one amino acid selected from glycine, alanine, glutamic acid, and leucine.
[4] The oligopeptide is at least one oligopeptide selected from glycylglycine, glycylglycylglycine, alanylalanine, alanylglutamic acid , and glycylleucine [1] to [3] A reagent for measuring blood coagulation ability containing any tissue thromboplastin.
[5] The tissue thromboplastin-containing blood coagulation ability measurement reagent according to any one of [1] to [4], wherein the concentration of the oligopeptide is 0.1 mmol / L to 100 mmol / L.
[6] The tissue thromboplastin-containing blood coagulation ability measurement reagent according to any one of [1] to [5], comprising serum albumin.
[7] The tissue thromboplastin-containing blood coagulation ability measurement reagent according to [6], wherein the serum albumin concentration is 0.1 to 5 (W / V)%.
[8] The tissue thromboplastin-containing blood coagulation ability measurement reagent according to any one of [1] to [7], wherein the blood coagulation ability measurement reagent is a proton bin time measurement reagent.
[9] The tissue thromboplastin-containing blood coagulation according to any one of [1] to [7], wherein the blood coagulation ability measurement reagent is a complex factor measurement reagent for measuring coagulation factor II, coagulation factor VII, or coagulation factor X Measuring reagent.
[10] The tissue thromboplastin-containing blood coagulation ability measurement reagent according to any one of [1] to [7], wherein the blood coagulation ability measurement reagent is a specific factor activity measurement reagent.
[11] The tissue thromboplastin-containing blood coagulation ability measurement reagent according to any one of [1] to [10], wherein the blood coagulation ability measurement reagent is liquid.
[12] A method for producing a tissue thromboplastin-containing reagent for measuring blood clotting ability, comprising an oligopeptide.
[13] A method for measuring blood clotting ability containing tissue thromboplastin, comprising the step of contacting a test sample, tissue thromboplastin, calcium ion, and oligopeptide in a solution and measuring the time from the start of contact to coagulation. .
[14] A method for stabilizing a tissue thromboplastin-containing blood coagulation ability measuring reagent, characterized by causing an oligopeptide to coexist with a tissue thromboplastin-containing blood coagulation ability measurement reagent.

  According to the tissue thromboplastin-containing blood coagulation ability measuring reagent and measurement method of the present invention, blood coagulation ability can be measured with low cost and high accuracy, which can be stably stored. In particular, a tissue thromboplastin-containing reagent for measuring blood coagulation ability that can be stably stored for a long time not only as a lyophilized product but also as a liquid product can be produced.

It is a graph which shows the result of having calculated | required the extension degree of PT immediately after preparation by addition of oligopeptide, and PT after storage at 37 degreeC for 4 weeks.

Oligopeptides that can be used in the present invention are those in which at least two amino acids are peptide-bonded, and those composed of the same kind of amino acids or those composed of different kinds of amino acids can be appropriately selected and used. Preferably 2 to 10 amino acids, more preferably 2 to 5, more preferably 2 to 3 amino acids. The sequence of the oligonucleotide peptide is tissue thromboplastin-containing blood coagulation ability measuring reagent, and is not particularly limited as long as it particularly shows the stabilizing effect of the PT reagent. Examples include those containing glycine, alanine, glutamic acid, leucine and the like. Specific examples include glycylglycine, glycylglycylglycine, alanylalanine, alanylglutamic acid, and glycylleucine. These oligopeptides can be used alone or in combination of two or more.

  The term “oligopeptide” used in the present invention includes not only a narrowly defined oligopeptide (that is, an oligopeptide consisting of natural amino acids) but also derivatives thereof. Examples of such derivatives include peptide derivatives that have been subjected to various modifications that improve the stability of the peptide. Examples of the modification include D-formation of L-form amino acid (eg, D-formation of N-terminal amino acid, D-formation of C-terminal amino acid, D-formation of amino acids other than N-terminal and C-terminal), N-terminal amino acid Acetylation of the group, amidation of the C-terminal carboxyl group, substitution of natural amino acids with non-natural amino acids (similar in nature), or combinations thereof.

The oligopeptide may be added to a tissue thromboplastin-containing blood coagulation ability measurement reagent, preferably a PT measurement reagent.
The concentration of the oligopeptide to be added is preferably 0.1 mmol / L to 100 mmol / L in a liquid tissue thromboplastin-containing blood coagulation ability measurement reagent, preferably a PT measurement reagent, and 0.5 mmol / L. ˜100 mmol / L is more preferred, and 5˜30 mmol / L is particularly preferred.

Moreover, serum albumin can be contained as a preferred embodiment of the present invention. The presence of serum albumin is preferable because the stability of the reagent for measuring blood clotting ability containing tissue thromboplastin can be improved.
Serum albumin that can be used in the present invention is human or animal serum albumin, and natural or genetically modified ones can also be used.

The serum albumin may be added to a tissue thromboplastin-containing blood coagulation ability measurement reagent, preferably a PT measurement reagent.
The concentration of serum albumin to be added is preferably 0.1 to 5 (W / V)% in the liquid tissue thromboplastin-containing blood coagulation ability measurement reagent, preferably the concentration in the PT measurement reagent, and more preferably Is 0.5 to 2 (W / V)%.

  Generally, tissue thromboplastin is a lipoprotein composed of a complex of tissue factor and phospholipid. Natural tissue thromboplastin is extracted from human or animal organs. Produced from genetically engineered E. coli, yeast, or animal cells that have artificially incorporated a gene containing the cDNA sequence of tissue factor such as human, rabbit, bovine, etc., or from cultured cells of tissue factor-expressing animal cells It can also be prepared by artificially binding phospholipids to the tissue factor. Many reports have already been made on methods for preparing complexes of tissue factor and phospholipid vesicles of appropriate composition (for example, Patent Document 7). In the present invention, both natural and genetically engineered tissue thromboplastins can be used.

  The tissue thromboplastin-containing blood coagulation ability measurement reagent and measurement method of the present invention are characterized in that the known tissue thromboplastin-containing blood coagulation ability measurement reagent contains an oligopeptide that can be used in the present invention. The oligopeptide may be added at the time of measurement or may be added at the time of reagent production, but is preferably added at the time of reagent production. The oligopeptide stabilizes the tissue thromboplastin-containing blood coagulation ability measurement reagent, so that long-term storage stability during use (measurement) after preparation of the reagent can be desired.

  In addition, as an embodiment of the tissue thromboplastin-containing blood coagulation ability measurement reagent and measurement method of the present invention, a liquid product or a frozen product may be used. Even if it is a reagent, since it can be stabilized for a long period of time, it is particularly preferable to apply it to a liquid product.

  Moreover, as an embodiment of the tissue thromboplastin-containing blood coagulation ability measuring reagent and measuring method of the present invention, a preparation comprising a plurality of reagents such as one reagent system or two reagent system may be used. Specifically, when the PT measurement reagent is provided as a liquid reagent, a one-reagent preparation in which tissue thromboplastin (coagulation factor III), which is a reaction main component of the reagent, and calcium ion, which is a coagulation factor IV, are mixed. As a preparation having longer-term storage stability, it can also be provided as a two-reagent preparation prepared by separating an aqueous solution of tissue thromboplastin and a calcium compound, or a lyophilized preparation.

  As a first embodiment of the tissue thromboplastin-containing blood coagulation ability measurement reagent and measurement method of the present invention, it is constituted as a PT measurement reagent, reacted with a specimen, and among the blood coagulation factors, Factor II, Factor V, It can be used as a reagent for examining the overall activity of Factor VII and Factor X. Specifically, the PT measurement reagent is obtained by adding calcium ions at a concentration capable of expressing blood coagulation activity to the natural tissue thromboplastin described above or tissue thromboplastin prepared from genetically modified cells or cultured cells. Provided. Preservatives, pH buffering agents, additives for avoiding the influence of anticoagulant substances that affect PT measurement, and the like can be added to the reagent.

As a second embodiment of the tissue thromboplastin-containing blood coagulation ability measurement reagent and measurement method of the present invention, the plasma is composed in the same manner as the PT measurement reagent, and the factor V is excluded from the coagulation factors (deficient plasma: That is, as a coagulation factor, it is mixed with Factor II, Factor VII, and Factor X), and can also be used as a complex factor measurement reagent for examining the activity of Factor II, Factor VII, and Factor X Use.
The second tissue thromboplastin-containing blood coagulation ability measurement reagent can be configured as a two-reagent system in which the PT measurement reagent and the deficient plasma are separately prepared, or prepared by mixing the PT measurement reagent and the deficient plasma. It can also be configured as a single reagent system. As an example of the two-reagent system, the first reagent is PT measurement reagent, the second reagent is deficient plasma, the sample is reacted with the first reagent, and then reacted with the deficient plasma for measurement. As an example of the one-reagent system, a reagent in which a PT measurement reagent and deficient plasma are mixed is formed, and a sample is reacted to perform measurement. A person skilled in the art can carry out by appropriately selecting which reagent configuration to take.

As a third embodiment of the tissue thromboplastin-containing blood coagulation ability measurement reagent and measurement method of the present invention, a factor deficiency comprising the same constitution as the PT measurement reagent and lacking any one of the coagulation factors In combination with plasma, it can be used as a reagent for measuring a specific factor activity for examining a specific factor activity. For example, the factor VII activity of a test sample is measured by combining a factor VII-deficient plasma (that is, coagulation factors include factor II, factor V, and factor X) and a PT measurement reagent. be able to. Further, factor VII activity in a test sample is measured by combining factor II-deficient plasma (that is, coagulation factors include factor V, factor VII, and factor X) and a PT measurement reagent. be able to. In addition, the factor VII activity of a test sample is measured by combining a factor X-deficient plasma (that is, coagulation factors include factor II, factor V, and factor VII) and a PT measurement reagent. be able to.
The third tissue thromboplastin-containing blood coagulation ability measuring reagent can be configured as a two-reagent system in which the PT measuring reagent and the deficient plasma are separately prepared, or prepared by mixing the PT measuring reagent and the deficient plasma. It can also be configured as a single reagent system. As an example of the two-reagent system, the first reagent is deficient plasma, the second reagent is PT measuring reagent, the sample is reacted with the first reagent, and then reacted with the deficient plasma for measurement. As an example of the one-reagent system, a reagent in which a PT measurement reagent and deficient plasma are mixed is formed, and a sample is reacted to perform measurement. A person skilled in the art can carry out by appropriately selecting which reagent configuration to take.

  The activity of each of the above factors can be examined by evaluating the time (coagulation time) until fibrin formation (coagulation) is observed or evaluating the time (%) relative to standard plasma. A person skilled in the art can select and implement an evaluation method as appropriate according to the purpose.

  EXAMPLES Hereinafter, the present invention will be specifically described by way of examples, but these do not limit the scope of the present invention.

Example 1: Preparation of PT measurement reagent
The PT measurement reagent was prepared by the following operation.
Tissue thromboplastin was prepared according to the following. First, a suspension of phospholipid mixture containing phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine (3.3 mg / mL), Triton X-100 (20%), and recombinant tissue factor (490 nmol / L) Was added and stirred. BioBeads SM-2 (BIO-RAD) was added thereto, and Triton X-100 was removed by adsorption. After stirring for 90 minutes after the addition of the biobeads, the biobeads were removed to obtain a tissue thromboplastin solution.
The prepared tissue thromboplastin solution was diluted with the following buffer solution so that the tissue factor concentration was 20 nmol / L to prepare a PT measurement reagent. Buffers were 100 mmol / L NaCl, 1 (W / V)% bovine serum albumin, 1 (W / V)% polyethylene glycol (molecular weight = 6000), 15 mmol / L CaCl ₂ , and 0.02 (W / V). The basic composition was 20 mmol / L HEPES buffer (pH 7.5) containing% sodium azide. Each PT measurement reagent was prepared by adding glycine, glycylglycine, glycylglycylglycine, alanylalanine, alanylglutamic acid, and glycylleucine to the basic composition to a final concentration of 10 mmol / L, respectively.

<< Example 2: Examination of storage stability of PT measurement reagent >>
The storage stability of the PT measurement reagent prepared according to Example 1 was examined. In each reagent immediately after preparation, PT of normal control plasma (manufactured by IL) was measured, and after storing each reagent at 37 ° C. for 4 weeks, PT of normal control plasma in the same lot was measured, and immediately after preparation of each reagent. The difference (seconds) between the PT and the PT after storage was determined.
PT measurement of the prepared reagent was performed using a blood coagulation analyzer, Corepresta (registered trademark) 2000 (Sekisui Medical). The difference between PT immediately after preparation and after storage is shown in FIG. As a result, in the case of no addition and the addition of glycine (Patent Document 7), which is an amino acid whose stabilizing effect is disclosed, an extension of PT exceeding 0.3 seconds as a reference was recognized. Thus, it was confirmed that all the PT measurement reagents to which oligopeptides were added had an extension of 0.3 seconds or less.
Further, glycylglycylglycine was the most effective, followed by alanylglutamic acid, then glycylglycine and glycylleucine, and then alanylalanine was effective.
Therefore, it was shown that the PT measurement reagent to which the oligopeptide was added was excellent in liquid stability and could be provided as a liquid reagent.

  In general, reagents for in-vitro diagnostics are required to have a stability of 12 months or longer at 4 ° C. In PT measurement reagents, fluctuations in coagulation time greatly affect the sensitivity and reliability of the reagents. Among these, fluctuations in PT (about 10 to 13 seconds) of normal plasma are extremely important, and therefore, fluctuations in coagulation time after 12 months at 4 ° C. are preferably within ± 0.3 seconds. In order to quickly obtain information on the stability after 12 months at 4 ° C., it is generally performed by applying the Arrhenius equation from the stability results at 37 ° C. In general, when the temperature rises by 10 ° C., the reaction rate constant doubles, so the stability of 4 ° C. stability is about 12 times that of 37 ° C. from the Arrhenius equation. That is, if liquid stability for one month at 37 ° C. can be confirmed, it can be estimated that liquid stability for one year at 4 ° C. can be secured. In other words, the acceptable condition for liquid stability is that the difference between the coagulation time on the first day of reagent preparation and the coagulation time after storage for 1 month at 37 ° C. is within ± 0.3 seconds. As a result of the above examination according to these conditions, by adding an oligopeptide to the PT measurement reagent, a liquid PT measurement reagent having a storage stability of at least one year can be prepared by storage at 4 ° C. It was found that a method and a reagent can be provided.

  The present invention can be used as a reagent for measuring blood coagulation ability. In particular, it can be used for the production of a liquid blood coagulation ability measuring reagent.

Claims (12)

A tissue thromboplastin-containing liquid reagent for measuring blood clotting ability , comprising an oligopeptide consisting of at least two amino acids (excluding a chromogenic substrate for thrombin) . The tissue thromboplastin-containing liquid reagent for measuring blood clotting ability according to claim 1, wherein the oligopeptide contains at least one amino acid selected from glycine, alanine, glutamic acid, and leucine. The tissue thromboplastin according to claim 1 or 2 , wherein the oligopeptide is at least one oligopeptide selected from glycylglycine, glycylglycylglycine, alanylalanine, alanylglutamic acid, and glycylleucine. Contains liquid reagent for measuring blood coagulation ability The tissue thromboplastin-containing liquid reagent for measuring blood clotting ability according to any one of claims 1 to 3 , wherein the concentration of the oligopeptide is 0.1 mmol / L to 100 mmol / L. The tissue thromboplastin-containing blood coagulability measuring liquid reagent according to any one of claims 1 to 4 , comprising serum albumin. The tissue thromboplastin-containing liquid coagulability measuring liquid reagent according to claim 5 , wherein the serum albumin concentration is 0.1 to 5 (W / V)%. The blood coagulation measurement reagent is proto b Nbin time measurement reagent, tissue thromboplastin-containing blood coagulation measuring liquid reagent according to any one of claims 1-6. The tissue thromboplastin-containing blood coagulation ability according to any one of claims 1 to 6 , wherein the blood coagulation ability measurement reagent is a complex factor measurement reagent for measuring coagulation factor II, coagulation factor VII, or coagulation factor X. Measuring liquid reagent. The tissue thromboplastin-containing liquid coagulation ability measurement liquid reagent according to any one of claims 1 to 6 , wherein the blood coagulation ability measurement reagent is a specific factor activity measurement reagent. A method for producing a tissue thromboplastin-containing liquid reagent for measuring blood clotting ability , comprising an oligopeptide comprising at least two amino acids (excluding a chromogenic substrate for thrombin) . A test sample, tissue thromboplastin, calcium ion, an oligopeptide consisting of at least two amino acids (excluding a chromogenic substrate for thrombin) in solution, and measuring the time from the start of contact until coagulation characterized in that it comprises a blood coagulation ability measuring method. Tissue thromboplastin-containing blood coagulation ability measurement liquid reagent characterized by coexisting an oligopeptide consisting of at least two amino acids (excluding a chromogenic substrate for thrombin) in a tissue thromboplastin-containing blood coagulation ability measurement liquid reagent Stabilization method.

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