WO2021063392A1 - 凝血酶溶液、试剂盒、凝血酶的稳定化方法、检测试剂、凝血酶时间的测定方法以及消泡剂的用途 - Google Patents

凝血酶溶液、试剂盒、凝血酶的稳定化方法、检测试剂、凝血酶时间的测定方法以及消泡剂的用途 Download PDF

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WO2021063392A1
WO2021063392A1 PCT/CN2020/119362 CN2020119362W WO2021063392A1 WO 2021063392 A1 WO2021063392 A1 WO 2021063392A1 CN 2020119362 W CN2020119362 W CN 2020119362W WO 2021063392 A1 WO2021063392 A1 WO 2021063392A1
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thrombin
antifoaming agent
buffer
active ingredient
solution
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PCT/CN2020/119362
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English (en)
French (fr)
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王辰骥
徐文娟
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积水医疗科技(苏州)有限公司
积水医疗株式会社
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Priority to CN202080068852.8A priority Critical patent/CN114729949A/zh
Publication of WO2021063392A1 publication Critical patent/WO2021063392A1/zh

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/86—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood coagulating time or factors, or their receptors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes

Definitions

  • Thrombin time refers to the time for blood to clot after adding standardized thrombin to plasma. Thrombin time measurement is a simple test to detect the function of coagulation, anticoagulation and fibrinolysis system, especially to understand whether the fibrin in plasma contains sufficient amount of fibrinogen and whether the result is normal.
  • thrombin time is a common way to reflect blood coagulation. Under the action of thrombin, fibrinogen is converted to insoluble fibrin. The time required for coagulation is measured, which is the plasma thrombin time to be measured. The detection of thrombin time has certain clinical value.
  • heparin When plasma fibrinogen is reduced or structurally abnormal, heparin is increased or heparinoid substances are present, and in systemic lupus erythematosus, liver disease, nephropathy, fibrinogen degradation products increase and diffuse intravascular coagulation, (no) fibrinogenemia , Abnormal fibrinogenemia (hyperfibrinogen mechanism dysemia), abnormal globulinemia or increased immunoglobulin and other diseases, the thrombin time will be abnormally extended. In abnormal fibrinogenemia, when there are calcium ions in the blood or the blood is acidic, the thrombin time will be abnormally shortened. The detection of thrombin time can provide certain assistance in the diagnosis of the above-mentioned diseases.
  • TT reagents sold in the domestic market are TT reagents sold by imported manufacturers. These reagents are mostly freeze-dried products, which are expensive and need to be reconstituted before use.
  • the liquid TT reagent does not need to be reconstituted and can be used directly.
  • the thrombin in the liquid is easily inactivated, and more consideration is needed to consider the stability of thrombin. If the thrombin activity can be maintained effectively and economically, the liquid TT reagent that meets the current market demand can be successfully obtained.
  • the limited technical problem to be solved by the present invention is that the present invention provides a liquid thrombin reagent, which does not need to be reconstituted, and can effectively reduce the detection error caused by reconstituted.
  • the present invention adopts the following technical solutions.
  • a thrombin solution containing thrombin and an antifoaming agent 1.
  • thrombin solution according to any one of 1 to 3, wherein the active ingredient of the silicone antifoaming agent is polyalkylsiloxanes.
  • thrombin solution according to any one of 1 to 4, wherein the active ingredient of the silicone antifoaming agent is polydimethylsiloxane.
  • thrombin solution according to any one of 4 to 6, wherein the concentration of the active ingredient of the antifoaming agent in the thrombin solution is 1-10 ppm.
  • thrombin solution according to any one of 4 to 7, wherein the concentration of the active ingredient of the antifoaming agent in the thrombin solution is 5-7 ppm.
  • thrombin solution according to any one of 2 to 8, wherein the buffer is selected from 5-100mM bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane (Bis- One of Tris buffer, Tris buffer, 3-morpholinopropanesulfonic acid (MOPS) buffer, 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) buffer.
  • the buffer is selected from 5-100mM bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane (Bis- One of Tris buffer, Tris buffer, 3-morpholinopropanesulfonic acid (MOPS) buffer, 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) buffer.
  • thrombin solution according to any one of 2-9, wherein the stabilizer is selected polyethylene glycol, bovine serum albumin (BSA), calcium chloride (CaC12), Triton X-100, One or more of Tween, Brij-58, mannitol, fructose, glycerin, trehalose, maltose, and gelatin.
  • BSA bovine serum albumin
  • CaC12 calcium chloride
  • Triton X-100 Triton X-100
  • Tween mannitol
  • fructose fructose
  • glycerin glycerin
  • trehalose maltose
  • maltose and gelatin.
  • a liquid-type thrombin time detection kit which contains thrombin and an antifoaming agent.
  • liquid thrombin time detection kit which further contains at least one selected from a buffer, a stabilizer, and a preservative.
  • liquid thrombin time detection kit according to 11 or 12, wherein the antifoaming agent is a silicone antifoaming agent.
  • liquid thrombin time detection kit according to any one of 11 to 13, wherein the active ingredient of the silicone antifoaming agent is polyalkylsiloxanes.
  • liquid thrombin time detection kit according to any one of 11 to 14, wherein the active ingredient of the silicone antifoaming agent is polydimethylsiloxane.
  • liquid-type thrombin time detection kit according to 14 or 15, wherein the concentration of the active ingredient of the antifoaming agent in the liquid-type thrombin time detection kit is 0.1-10 ppm.
  • liquid-type thrombin time detection kit according to any one of 14 to 16, wherein the concentration of the active ingredient of the antifoaming agent in the liquid-type thrombin time detection kit is 1-10 ppm.
  • liquid thrombin time detection kit according to any one of 14 to 17, wherein the active ingredient of the antifoaming agent has a concentration of 5-7 ppm in the liquid thrombin time detection kit.
  • liquid thrombin time detection kit according to any one of 12 to 18, wherein the buffer is selected from the group consisting of 5-100 mM bis(2-hydroxyethyl)iminotris(hydroxymethyl) ) Methane (Bis-Tris) buffer, Tris buffer, 3-morpholinopropanesulfonic acid (MOPS) buffer, 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) buffer One of them.
  • the buffer is selected from the group consisting of 5-100 mM bis(2-hydroxyethyl)iminotris(hydroxymethyl) ) Methane (Bis-Tris) buffer, Tris buffer, 3-morpholinopropanesulfonic acid (MOPS) buffer, 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) buffer One of them.
  • liquid thrombin time detection kit according to any one of 12 to 19, wherein the stabilizer is selected from polyethylene glycol, bovine serum albumin (BSA), calcium chloride (CaC12), One or more of Triton X-100, Tween, Brij-58, mannitol, fructose, glycerin, trehalose, maltose, and gelatin.
  • BSA bovine serum albumin
  • CaC12 calcium chloride
  • Triton X-100 Triton X-100
  • Tween Brij-58
  • mannitol mannitol
  • fructose fructose
  • glycerin glycerin
  • trehalose maltose
  • maltose and gelatin.
  • a detection reagent comprising the thrombin solution described in any one of 1-10.
  • a method for measuring the thrombin time of plasma comprising:
  • the plasma to be tested is brought into contact with the thrombin solution described in any one of 1 to 10 or the detection reagent described in 21 or 22, or,
  • the plasma to be tested is brought into contact with the thrombin and antifoaming agent contained in the liquid thrombin time detection kit described in any one of 11-20.
  • a method for stabilizing thrombin in a solution comprising: adding an antifoaming agent to a solution containing thrombin.
  • the buffer is selected from 5-100mM bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane (Bis- One of Tris buffer, Tris buffer, 3-morpholinopropanesulfonic acid (MOPS) buffer, 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) buffer.
  • BSA bovine serum albumin
  • CaC12 calcium chloride
  • Triton X-100 Triton X-100
  • Tween mannitol
  • fructose fructose
  • glycerin glycerin
  • trehalose maltose
  • maltose and gelatin.
  • organosilicon defoamers as stabilizers in thrombin solvents.
  • the reagent containing thrombin is improved on the original basis, so that the thrombin in the solution can remain stable for a long time even when the activity is low, which is very economical and effective.
  • Figure 1 The normal plasma reaction curve in Example 3 of the present application.
  • Figure 2 The reaction curve of the reference product in Example 3 of the present application.
  • the present invention provides a thrombin solution containing thrombin and an antifoaming agent.
  • thrombin is the main functional component, and fibrinogen is converted into insoluble fibrin under the action of thrombin.
  • the antifoaming agent in the thrombin solvent by containing the antifoaming agent in the thrombin solvent, the stability of thrombin in the solvent can be improved, so that the thrombin can remain stable in the solvent for a long time.
  • solution does not refer to a solid or semi-solid state that is frozen when a liquid (aqueous solution) is placed below 0°C, but refers to maintaining fluidity as a solution
  • the sexual properties are, for example, properties under refrigeration (for example, 2 to 8°C) when distributed as a clinical test reagent.
  • the above-mentioned thrombin solution of the present invention further contains at least one selected from a buffer, a stabilizer, and a preservative. From the perspective of obtaining the technical effect of the present invention more clearly, it preferably contains a buffer, a stabilizer, and a preservative. Agent. Since the thrombin solution of the present invention is a liquid-type reagent containing a buffer, etc., it does not need to be reconstituted with a buffer or the like during use like the conventional solid reagent or lyophilized reagent. The thrombin solution of the present invention can be used directly without reconstitution, so it is very convenient in use.
  • the antifoaming agent is preferably a silicone antifoaming agent, preferably a water emulsion type silicone antifoaming agent.
  • the defoamer can be used for general food and medicine to prevent the generation of bubbles in the solution.
  • the silicone antifoaming agent is an antifoaming agent having a silicone (polysiloxane) skeleton.
  • the active ingredient of the silicone antifoaming agent is preferably polyalkylsiloxanes.
  • polyalkylsiloxanes include: polymethylsiloxane, polyethylsiloxane, polydimethylsiloxane, polydiethylsiloxane, polymethylethylsiloxane Oxyane and so on.
  • the active ingredient of the silicone antifoaming agent is preferably polydimethylsiloxane.
  • the defoaming agent of the present invention As a specific example of the defoaming agent of the present invention, one can cite, for example, Antifoam B (manufactured by Sigma, A5757 or A6707), the active ingredient of which is polydimethylsiloxane, and the content of the active ingredient in the defoaming agent is 10 %, the recommended concentration range in the product specification is 1-100ppm.
  • Antifoam B manufactured by Sigma, A5757 or A6707
  • the active ingredient of which is polydimethylsiloxane
  • the content of the active ingredient in the defoaming agent is 10 %
  • the recommended concentration range in the product specification is 1-100ppm.
  • the activity range of thrombin is not particularly limited, and is preferably 1 to 20 NIH Units/mL, particularly preferably 1 to 10 NIH Units/mL, and more preferably 1 to 5 NIH Units/mL.
  • the activity range of thrombin is low, the technical effect of the present invention can be better reflected. That is to say, through the technical solution of the present invention, even when the activity of thrombin is low, the excellent effect of maintaining stability for a long time can still be achieved.
  • the concentration of the active ingredient of the antifoaming agent in the thrombin solution may be 0.1-15 ppm, preferably 0.5-12 ppm, preferably 0.1-10 ppm, More preferably, it is 1-10 ppm, More preferably, it is 3-7 ppm, Especially preferably, it is 5-7 ppm.
  • the buffer is not particularly limited, but from the viewpoint of further improving the stability of thrombin, the buffer is preferably selected from 5-100 mM bis(2-hydroxyethyl)imino tris (Hydroxymethyl) methane (Bis-Tris) buffer, tris (Tris) buffer, 3-morpholinopropanesulfonic acid (MOPS) buffer, 4-hydroxyethylpiperazine ethanesulfonic acid ( HEPES) any of the buffers.
  • Bis-Tris bis(2-hydroxyethyl)imino tris
  • Tris tris
  • MOPS 3-morpholinopropanesulfonic acid
  • HEPES 4-hydroxyethylpiperazine ethanesulfonic acid
  • the buffer is HEPES with a concentration range of 10-100 mmol/L and pH 6.5-8.0; further preferably, the buffer is 25-75 mM HEPES, pH 7.1-7.5, even more preferably 50 mM HEPES, pH7.3.
  • the stabilizer is not particularly limited, but from the viewpoint of further improving the stability of thrombin, the stabilizer is selected from polyethylene glycol, bovine serum albumin (BSA), calcium chloride One or more of (CaC12), Triton X-100, Tween, Brij-58, mannitol, fructose, glycerin, trehalose, maltose, and gelatin.
  • BSA bovine serum albumin
  • CaC12 calcium chloride
  • Triton X-100 Triton X-100
  • Tween Brij-58
  • mannitol fructose
  • glycerin glycerin
  • trehalose maltose
  • maltose and gelatin.
  • the stabilizer is polyethylene glycol 4000 (PEG-4000), Tween-80, bovine serum albumin (BSA), and calcium chloride (CaC12).
  • the production method of the above-mentioned thrombin solution is not particularly limited, and the following production methods can be adopted.
  • the specific steps include: preparing 50 mM HEPES buffer, adjusting the pH to 7.3, adding preservatives, stabilizers, and antifoaming agents to the buffer, adding thrombin after mixing, and mixing after adjustment to obtain a thrombin solution.
  • the invention provides a liquid type thrombin time detection kit.
  • the kit includes the above-mentioned thrombin solution of the present invention and a package containing the above-mentioned thrombin solution.
  • the liquid-type thrombin time detection kit of the present invention contains thrombin and an antifoaming agent.
  • the above-mentioned kit of the present invention further contains at least one selected from a buffer, a stabilizer, and a preservative, and preferably contains a buffer, a stabilizer, and a preservative.
  • the thrombin time detection kit of the present invention is a liquid-type reagent containing a buffer and the like, it does not need to be reconstituted with a buffer or the like during use like the conventional solid reagent or lyophilized reagent.
  • the thrombin solution of the present invention can be used directly without reconstitution, so it is very convenient in use.
  • the defoaming agent is preferably a silicone defoaming agent, and the silicone defoaming agent and the defoaming agent in the thrombin solvent described above may be the same defoaming agent and the same
  • the effective ingredients of, are preferably polyalkylsiloxanes, and more preferably polydimethylsiloxane.
  • the activity range of thrombin is not particularly limited, and is preferably 1-20 NIH Units/mL, particularly preferably 1-10 NIH Units/mL, and more preferably 1-5 NIH Units/mL.
  • the activity range of thrombin is low, the technical effect of the present invention can be better reflected. That is to say, through the technical solution of the present invention, even when the activity of thrombin is low, the excellent effect of maintaining stability for a long time can still be achieved.
  • the concentration of the active ingredient of the antifoaming agent in the thrombin solution may be 0.1-15 ppm, preferably 0.5-12 ppm, preferably 0.1-10 ppm, More preferably, it is 1-10 ppm, More preferably, it is 3-7 ppm, Especially preferably, it is 5-7 ppm.
  • the buffer and stabilizer are not particularly limited, and the same buffer and stabilizer as those in the thrombin solvent described above can be used under the same conditions.
  • An example of the detection reagent related to the present invention is a thrombin time detection reagent.
  • the thrombin time of the plasma is measured by contacting the plasma to be tested with the above-mentioned thrombin time detection reagent.
  • the above-mentioned thrombin detection reagent contains the above-mentioned thrombin solution of the present invention.
  • One type of the above-mentioned detection reagent of the present invention is the above-mentioned liquid-type thrombin time detection kit of the present invention, but it is not limited to this, and other types of detection reagents may also be used.
  • the detection reagent related to the present invention is a fibrinogen detection reagent, which converts the fibrinogen in the plasma into fibrin by contacting the plasma to be tested with the detection reagent containing thrombin and antifoaming agent.
  • the blood is coagulated and the amount of fibrinogen is measured.
  • the above-mentioned detection reagent containing thrombin and antifoaming agent contains the above-mentioned thrombin solution of the present invention.
  • One of the aforementioned detection reagents of the present invention is a fibrinogen detection kit containing the aforementioned detection reagent, but it is not limited to this, and other types of detection reagents may be used.
  • the present invention relates to a method for measuring the thrombin time of plasma.
  • the method includes:
  • the plasma to be tested is brought into contact with the thrombin and antifoaming agent contained in the liquid thrombin time detection kit of the present invention.
  • the invention relates to a method for stabilizing thrombin in a solution.
  • the method comprises: adding an antifoaming agent to a solution containing thrombin.
  • an antifoaming agent to the thrombin solvent, the stability of thrombin in the solvent can be improved for a long time, so that the thrombin can remain stable in the solvent for a long time.
  • the solution further contains at least one selected from a buffer, a stabilizer, and a preservative.
  • a buffer a stabilizer
  • a preservative a preservative
  • the defoaming agent is preferably a silicone defoaming agent.
  • the silicone defoaming agent and the defoaming agent in the thrombin solvent described above may be the same defoaming agent and the same effective ingredients, preferably Polyalkylsiloxanes are more preferably polydimethylsiloxane.
  • the activity range of thrombin is preferably 1-20 NIH Units/mL, particularly preferably 1-10 NIH Units/mL, and more preferably 1-5 NIH Units/mL.
  • the activity range of thrombin is low, the technical effect of the present invention can be better reflected.
  • the concentration of the active ingredient of the antifoaming agent in the solution containing thrombin may be 0.1-15 ppm, preferably 0.5-12 ppm, preferably 0.1-10 ppm, more preferably 1-10 ppm, further preferably 3-7 ppm, particularly preferably 5 ⁇ 7ppm.
  • the buffer and stabilizer are not particularly limited, and the same buffer and stabilizer as in the aforementioned thrombin solvent can be used under the same conditions.
  • the inventors of the present invention found that adding a silicone defoamer to a solution containing thrombin can improve the stability of thrombin in the solvent for a long time, especially when the activity range of thrombin is low. , It can improve the stability of thrombin in solvent. That is, the present inventors discovered a new use of silicone-based defoamers, that is, use as a stabilizer in a thrombin solvent.
  • the silicone-based defoamer of the present invention can stabilize thrombin in a solution. Therefore, the present invention is not limited to the use of thrombin time, and obviously can also be used in various blood clotting time methods, detection reagents, and kits containing thrombin as a component of the reagent (the detection method also includes fibrin Coagulation factors, anticoagulation factors, etc.), such as fibrinogen detection methods.
  • the active ingredient of the silicone antifoaming agent is preferably polyalkylsiloxanes, and more preferably polydimethylsiloxane.
  • the activity range of thrombin is preferably 1-20 NIH Units/mL, particularly preferably 1-10 NIH Units/mL, and more preferably 1-5 NIH Units/mL.
  • the concentration of the active ingredient of the defoamer in the solution containing thrombin may be 0.1-15 ppm, preferably 0.5-12 ppm, preferably 0.1-10 ppm, more preferably 1-10 ppm, further preferably 3-7 ppm, particularly preferably 5-7 ppm .
  • the raw material reagents or instruments used in the embodiments of the present invention are all common commercially available products, and all are available in the market.
  • the reagent of the present invention uses Sekisui Medical's CP series instrument for detection, and the operation steps are as follows:
  • Antifoam B (Sigma) was used as the antifoaming agent of the present invention to be added to the thrombin solution for research.
  • the specific active ingredients and related results are shown in Table 1.
  • thrombin time detection reagent To prepare the thrombin time detection reagent, first prepare 50mM HEPES buffer and adjust its pH to 7.3 with sodium hydroxide. Then add 30mM calcium chloride, 0.5% Tween-80, 0.5% PEG-4000, 1% BSA, 0.5% Proclin 300, 0ppm (Comparative Example 1) or 5ppm (Example 1) silicone defoamer (i.e. Antifoam B (Sigma Company)), after stirring well, dilute to the required volume. It should be noted that the content of the active ingredient in the Antifoam B is 10%, and the 5 ppm is the concentration of the active ingredient in the reagent. Subsequently, thrombin was added to make the actual thrombin concentration 2NIH Units/mL, and the thrombin time detection reagent was obtained after stirring evenly.
  • silicone defoamer i.e. Antifoam B (Sigma Company)
  • the prepared reagents were placed in an environment of 37°C.
  • the Sekisui Medical CP3000 automatic blood coagulometer was used to determine the value of the reference product, and the deviation of the seconds was compared.
  • Day 0 and 7 The comparison results of the measurement results of the day are shown in Table 1.
  • the initial detection value row shows the detection value on day 0, and the maximum detection value row shows the detection value on day 7.
  • the deviation of the detection value shows the difference between the detection value on the 0th day and the detection value on the 7th day, and the deviation ratio is shown as a percentage of the deviation of the detection value relative to the detection value on the 0th day.
  • the defoamer is added in the above (2) embodiment. Therefore, the second deviation of the detection result is significantly smaller than that of other reagents, indicating that the addition of defoamer can effectively improve the reagent’s performance. stability.
  • the above-mentioned technical effect does not exist in the comparative example (1) without adding a defoamer.
  • This example further explores the optimal concentration of the silicone defoamer whose active ingredient is polydimethylsiloxane.
  • thrombin time detection reagent To prepare the thrombin time detection reagent, first prepare 50mM HEPES buffer and adjust its pH to 7.3 with sodium hydroxide. Then add 30mM calcium chloride, 0.5% Tween-80, 0.5% PEG-4000, 1% BSA, 0.5% Proclin 300, defoaming agent (Antifoam B) of each concentration (0ppm ⁇ 100ppm) shown in Table 2, effective The component content is 10%), after fully stirring, dilute to the required volume. Subsequently, thrombin was added to make the actual thrombin concentration 2NIH Units/mL, and the thrombin time detection reagent was obtained after stirring evenly.
  • defoaming agent Antifoam B
  • the prepared reagents were placed in an environment of 37°C.
  • the Sekisui Medical CP3000 automatic blood coagulometer was used to determine the value of the reference product, and the deviation of the seconds was compared.
  • Day 0 and 7 The comparison results of the measurement results of days are shown in Table 2.
  • Table 2 the meaning of one row of initial detection value, one row of maximum detection value, detection value deviation, and offset ratio are the same as those in Table 1.
  • the deviation of the seconds in the B to F reagents in the range of 1 ppm to 10 ppm of the active ingredient content is significantly smaller than that of the A reagent without the addition of a defoamer. It can be seen that when the effective component is in the range of 1 ppm to 10 ppm, a better stabilization effect can be obtained. Furthermore, the second deviation of the detection results of reagent D and reagent E is significantly smaller than that of other reagents. It can be seen that when the concentration of the active ingredient is 5-7 ppm, the stabilization effect can be further improved.
  • this embodiment further explores whether the addition of 50 ppm defoamer, that is, 5 ppm of active ingredient polydimethylsiloxane, will have a significant impact on the reactivity of the reagent.
  • thrombin time detection reagent To prepare the thrombin time detection reagent, first prepare 50mM HEPES buffer and adjust its pH to 7.3 with sodium hydroxide. Then add 30mM calcium chloride, 0.5% Tween-80, 0.5% PEG-4000, 1% BSA, 0.5% Proclin 300, antifoam (Antifoam B) (in terms of active ingredients, 5ppm in reagent B, or The content of reagent A is 0 ppm (no addition)). After stirring well, dilute the volume to the required volume. Subsequently, thrombin was added to make the actual thrombin concentration 2NIH Units/mL, and the thrombin time detection reagent was obtained after stirring evenly.
  • Antifoam B antifoam

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Abstract

一种凝血酶溶液、试剂盒、凝血酶的稳定化方法、检测试剂以及凝血酶时间的测定方法。凝血酶溶液含有凝血酶，并含有消泡剂。该凝血酶溶液具有提高的稳定性，使得凝血酶在活性较低的情况下也能够保持较长时间的稳定。

Description

凝血酶溶液、试剂盒、凝血酶的稳定化方法、检测试剂、凝血酶时间的测定方法以及消泡剂的用途 技术领域
本发明涉及生物技术领域，尤其涉及凝血酶溶液、试剂盒、凝血酶的稳定化方法、检测试剂、凝血酶时间的测定方法以及有机硅类消泡剂在凝血酶溶剂中作为稳定化剂的用途。
背景技术
凝血酶时间(TT)是指血浆中加入标准化的凝血酶后血液凝固的时间。凝血酶时间测定是检测凝血、抗凝及纤维蛋白溶解系统功能的一个简便试验，尤其可了解血浆中的纤维蛋白是否含有足够量的纤维蛋白原及其结果是否正常。
凝血酶时间是反映凝血的共同途径，在凝血酶作用下纤维蛋白原转变为不溶性纤维蛋白，测定凝固所需的时间，即为待测血浆凝血酶时间。凝血酶时间的检测具有一定的临床价值。
当血浆纤维蛋白原减少或结构异常、肝素增多或类肝素物质存在，以及在系统性红斑狼疮，肝病，肾病，纤维蛋白原降解产物增多及弥漫性血管内凝血、(无)纤维蛋白原血症、异常纤维蛋白原血症(纤维蛋白原机制不良血症)，异常球蛋白血症或免疫球蛋白增多等疾病中，凝血酶时间会异常延长。而在异常纤维蛋白原血症，血液中有钙离子存在或血液呈酸性时，凝血酶时间会异常缩短。凝血酶时间的检测能够对上述疾病的诊断提供一定的辅助。
目前，国内市场上销售的多为进口厂家销售的TT试剂，这些试剂多为冻干品，价格昂贵，且在使用前，需要对试剂进行复溶。而液体型TT试剂则不需复溶，直接使用即可。但是TT试剂由于其中凝血酶的酶活偏低，液体中凝血酶容易失活，需要更多考虑凝血酶的稳定性。若能有效并经济的实现凝血酶活性的保持，便可成功获取满足当前市场需求的液体TT试剂。
发明内容
鉴于此，本发明要解决的技术问题在于提供一种凝血酶溶液、试剂盒、凝血酶的稳定化方法、检测试剂、凝血酶时间的测定方法，从而能够提高凝血酶溶液的稳定性，使得凝血酶在活性较低的情况下也能够保持较长时间的稳定。
另外，本发明要解决的限定性技术问题是，本发明提供一种液体型凝血酶试剂，该试剂无需复溶，可以有效减少由于复溶带来的检测误差。
为实现上述目的，本发明采用如下技术方案。
1、一种凝血酶溶液，其含有凝血酶，并含有消泡剂。
2、根据1所述的凝血酶溶液，其进一步含有选自缓冲液、稳定剂和防腐剂中的至少一种。
3、根据1或2所述的凝血酶溶液，其中，所述消泡剂为有机硅消泡剂。
4、根据1～3中任一项所述的凝血酶溶液，其中，所述有机硅消泡剂的有效成分为聚烷基硅氧烷类。
5、根据1～4中任一项所述的凝血酶溶液，其中，所述有机硅消泡剂的有效成分为聚二甲基硅氧烷。
6、根据4或5所述的凝血酶溶液，其中，所述消泡剂的有效成分在凝血酶溶液中的浓度为0.1～10ppm。
7、根据4～6中任一项所述的凝血酶溶液，其中，所述消泡剂的有效成分在凝血酶溶液中的浓度为1～10ppm。
8、根据4～7中任一项所述的凝血酶溶液，其中，所述消泡剂的有效成分在凝血酶溶液中的浓度为5～7ppm。
9、根据2～8中任一项所述的凝血酶溶液，其中，所述缓冲液为选自5-100mM二(2-羟乙基)亚胺基三(羟甲基)甲烷(Bis-Tris)缓冲液、三羟甲基氨基甲烷(Tris)缓冲液、3-吗啉丙磺酸(MOPS)缓冲液、4-羟乙基哌嗪乙磺酸(HEPES)缓冲液中的一种。
10、根据2～9中任一项所述的凝血酶溶液，其中，所述稳定剂为选择聚乙二醇、牛血清白蛋白(BSA)、氯化钙(CaC12)、Triton X-100、吐温、Brij-58、甘露醇、果糖、甘油、海藻糖、麦芽糖、明胶中的一种或几种。
11、一种液体型凝血酶时间检测试剂盒，其含有凝血酶，并含有消泡剂。
12、根据11所述的液体型凝血酶时间检测试剂盒，其进一步含有选自缓冲液、稳定剂和防腐剂中的至少一种。
13、根据11或12所述的液体型凝血酶时间检测试剂盒，其中，所述消泡剂为有机硅消泡剂。
14、根据11～13中任一项所述的液体型凝血酶时间检测试剂盒，其中，所述有机硅消泡剂的有效成分为聚烷基硅氧烷类。
15、根据11～14中任一项所述的液体型凝血酶时间检测试剂盒，其中，所述有机硅消泡剂的有效成分为聚二甲基硅氧烷。
16、根据14或15所述的液体型凝血酶时间检测试剂盒，其中，所述消泡剂的有效成分在液体型凝血酶时间检测试剂盒中的浓度为0.1～10ppm。
17、根据14～16中任一项所述的液体型凝血酶时间检测试剂盒，其中，所述消泡剂的有效成分在液体型凝血酶时间检测试剂盒中的浓度为1～10ppm。
18、根据14～17中任一项所述的液体型凝血酶时间检测试剂盒，其中，所述消泡剂的有效成分在液体型凝血酶时间检测试剂盒中的浓度为5～7ppm。
19、根据12～18中任一项所述的液体型凝血酶时间检测试剂盒，其中，所述缓冲液为选自5-100mM二(2-羟乙基)亚胺基三(羟甲基)甲烷(Bis-Tris)缓冲液、三羟甲基氨基甲烷(Tris)缓冲液、3-吗啉丙磺酸(MOPS)缓冲液、4-羟乙基哌嗪乙磺酸(HEPES)缓冲液中的一种。
20、根据12～19中任一项所述的液体型凝血酶时间检测试剂盒，其中，所述稳定剂为选择聚乙二醇、牛血清白蛋白(BSA)、氯化钙(CaC12)、Triton X-100、吐温、Brij-58、甘露醇、果糖、甘油、海藻糖、麦芽糖、明胶中的一种或几种。
21、一种检测试剂，其含有1～10中任一项所述的凝血酶溶液。
22、根据21所述的检测试剂，其是凝血酶时间检测试剂。
23、一种血浆的凝血酶时间的测定方法，该方法包括：
将待测血浆与1～10中任一项所述的凝血酶溶液或21或22所述的检测试剂接触，或者，
将待测血浆与11～20中任一项所述的液体型凝血酶时间检测试剂盒中所含的凝血酶和消泡剂接触。
24、一种溶液中凝血酶的稳定化方法，该方法包括：在含有凝血酶的溶液中添加消泡剂。
25、根据24所述的稳定化方法，其中，所述溶液进一步含有选自缓冲液、稳定剂和防腐剂中的至少一种。
26、根据24或25所述的稳定化方法，其中，所述消泡剂为有机硅消泡剂。
27、根据24～26中任一项所述的稳定化方法，其中，所述有机硅消泡剂的有效成分为聚烷基硅氧烷类。
28、根据24～27中任一项所述的稳定化方法，其中，所述有机硅消泡剂的有效成分为聚二甲基硅氧烷。
29、根据27或28所述的稳定化方法，其中，所述消泡剂的有效成分在含有凝血酶的溶液中的浓度为0.1～10ppm。
30、根据27～29中任一项所述的稳定化方法，其中，所述消泡剂的有效成分在含有凝血酶的溶液中的浓度为1～10ppm。
31、根据27～30中任一项所述的稳定化方法，其中，所述消泡剂的有效成分在含有凝血酶的溶液中的浓度为5～7ppm。
32、根据25～31中任一项所述的稳定化方法，其中，所述缓冲液为选自5-100mM二(2-羟乙基)亚胺基三(羟甲基)甲烷(Bis-Tris)缓冲液、三羟甲基氨基甲烷(Tris)缓冲液、3-吗啉丙磺酸(MOPS)缓冲液、4-羟乙基哌嗪乙磺酸(HEPES)缓冲液中的一种。
33、根据25～32中任一项所述的稳定化方法，其中，所述稳定剂为选择聚乙二醇、牛血清白蛋白(BSA)、氯化钙(CaC12)、Triton X-100、吐温、Brij-58、甘露醇、果糖、甘油、海藻糖、麦芽糖、明胶中的一种或几种。
34、有机硅类消泡剂在凝血酶溶剂中作为稳定化剂的用途。
本发明的技术效果
通过本发明的技术方案，可以实现以下技术效果。
通过加入消泡剂，使含有凝血酶的试剂在原有基础上提高了稳定性，使得溶液中凝血酶在活性较低的情况下也能保持较长时间的稳定，十分经济有效。
另外，本发明为液体型试剂，无需复溶，可有效减少由复溶带来的测量误差。
附图说明
图1：本申请实施例3中的正常血浆反应曲线；
图2：本申请实施例3中的参考品反应曲线。
具体实施方式
[凝血酶溶液]
本发明提供一种凝血酶溶液，其含有凝血酶，并含有消泡剂。在本发明的凝血酶溶液中，凝血酶是主要功能性成分，在凝血酶的作用下纤维蛋白原被转变为不溶性纤维蛋白。在本发明中，通过在凝血酶溶剂中含有消泡剂，可以提高凝血酶在溶剂中的稳定性，从而，使得凝血酶能够长时间地在溶剂中保持稳定。需要说明的是，在本说明书中，“溶液”这一用语并不指将液体(水溶液)放置在0℃以下时被冻结而成的固体或半固体的性状，而是指保持作为溶液的流动性的性状，例如，作为临床检查用试剂而流通时的冷藏(例如2～8℃)下的性状。
本发明的上述凝血酶溶液中进一步含有选自缓冲液、稳定剂和防腐剂中的至少一种，从更加明显地获得本发明的技术效果的角度上来看，优选包含缓冲液、稳定剂和防腐剂。由于本发明的凝血酶溶液是含有缓冲剂等的液体型试剂，因此，不需要像以前的固体试剂或冻干试剂那样需要在使用时采用缓冲剂等进行复溶。本发明的凝血酶溶液可以无需复溶而直接使用，因此在使用上非常方便。
本发明的上述凝血酶溶液中，所述消泡剂优选为有机硅消泡剂，优选水乳剂型有机硅消泡剂。该消泡剂可用于一般的食品及药品，防止溶液中气泡的产生。所述有机硅消泡剂是具有有机硅(聚硅氧烷)骨架的消泡剂。从能够 进一步提高凝血酶的稳定性的观点来看，所述有机硅消泡剂的有效成分优选聚烷基硅氧烷类。作为聚烷基硅氧烷类的具体例子可以列举：聚甲基硅氧烷、聚乙基硅氧烷、聚二甲基硅氧烷、聚二乙基硅氧烷、聚甲基乙基硅氧烷等。从能够更加提供凝血酶的稳定性的观点来看，所述有机硅消泡剂的有效成分优选聚二甲基硅氧烷。
作为本发明的消泡剂的具体例子，可以列举，例如Antifoam B(Sigma公司制造，A5757或A6707)，其有效成分为聚二甲基硅氧烷，该消泡剂中有效成分的含量为10％，其产品说明书中建议的浓度范围为1～100ppm。
在本发明中，凝血酶的活性范围没有特别的限定，优选1～20NIH Units/mL，特别优选1～10NIH Units/mL，进一步优选1～5NIH Units/mL。当凝血酶的活性范围较低时，更能体现本发明的技术效果。也就是说，通过本发明的技术方案，即使在凝血酶的活性较低时，仍然能够实现长时间保持稳定性的优异效果。
在本发明中，从能够进一步提高凝血酶的稳定性的观点来看，所述消泡剂的有效成分在凝血酶溶液中的浓度可以为0.1～15ppm，优选0.5～12ppm，优选0.1～10ppm，更加优选1～10ppm，进一步优选3～7ppm，特别优选5～7ppm。
在本发明中，所述缓冲液没有特别的限定，但从进一步提高凝血酶的稳定性的观点来看，所述缓冲液优选选自5-100mM二(2-羟乙基)亚胺基三(羟甲基)甲烷(Bis-Tris)缓冲液、三羟甲基氨基甲烷(Tris)缓冲液、3-吗啉丙磺酸(MOPS)缓冲液、4-羟乙基哌嗪乙磺酸(HEPES)缓冲液中的任一种。
优选地，所述缓冲液为HEPES，浓度范围为10～100mmol/L，pH 6.5-8.0；进一步优选地，所述缓冲液为25～75mM HEPES，pH 7.1-7.5，更进一步优选为50mM HEPES，pH7.3。
在本发明中，所述稳定剂没有特别的限定，但从进一步提高凝血酶的稳定性的观点来看，所述稳定剂选自聚乙二醇、牛血清白蛋白(BSA)、氯化钙(CaC12)、Triton X-100、吐温、Brij-58、甘露醇、果糖、甘油、海藻糖、麦芽糖、明胶中的一种或几种。
在本发明的优选实施例中，稳定剂为聚乙二醇4000(PEG-4000)、吐温-80、牛血清白蛋白(BSA)、氯化钙(CaC12)。
上述凝血酶溶液的制造方法没有特别的限定，可以采用以下的制造方法。具体工序包括：配制50mM的HEPES缓冲液，调节pH至7.3，向该缓冲液中加入防腐剂、稳定剂、消泡剂，混合均匀后添加凝血酶，调节后混匀即得凝血酶溶液。
[液体型凝血酶时间检测试剂盒]
本发明提供一种液体型凝血酶时间检测试剂盒。该试剂盒包括本发明的上述凝血酶溶液、以及收纳上述凝血酶溶液的包装。具体而言，本发明的液体型凝血酶时间检测试剂盒含有凝血酶，并含有消泡剂。
本发明的上述试剂盒中进一步含有选自缓冲液、稳定剂和防腐剂中的至少一种，优选包含缓冲液、稳定剂和防腐剂。由于本发明的凝血酶时间检测试剂盒是含有缓冲剂等的液体型试剂，因此，不需要像以前的固体试剂或冻干试剂那样需要在使用时采用缓冲剂等进行复溶。本发明的凝血酶溶液可以无需复溶而直接使用，因此在使用上非常方便。
本发明的上述试剂盒中，所述消泡剂优选为有机硅消泡剂，所述有机硅消泡剂与前面所述的凝血酶溶剂中的消泡剂可以是相同的消泡剂以及相同的有效成分，优选聚烷基硅氧烷类，更加优选聚二甲基硅氧烷。
在本发明的上述试剂盒中，凝血酶的活性范围没有特别的限定，优选1～20NIH Units/mL，特别优选1～10NIH Units/mL，进一步优选1～5NIH Units/mL。当凝血酶的活性范围较低时，更能体现本发明的技术效果。也就是说，通过本发明的技术方案，即使在凝血酶的活性较低时，仍然能够实现长时间保持稳定性的优异效果。
在本发明中，从能够进一步提高凝血酶的稳定性的观点来看，所述消泡剂的有效成分在凝血酶溶液中的浓度可以为0.1～15ppm，优选0.5～12ppm，优选0.1～10ppm，更加优选1～10ppm，进一步优选3～7ppm，特别优选5～7ppm。
在本发明的上述试剂盒中，所述缓冲液和稳定剂没有特别的限定，可以以相同的条件使用与前面所述的凝血酶溶剂中相同缓冲液和稳定剂。
[检测试剂]
本发明涉及的检测试剂的一个例子是凝血酶时间检测试剂，通过使待测血浆与上述凝血酶时间检测试剂相接触，来测定血浆的凝血酶时间。上述凝血酶检测试剂中含有本发明的上述凝血酶溶液。
本发明的上述检测试剂的一种类型是本发明的上述液体型凝血酶时间检测试剂盒，但并不限定于此，也可以是其他形式的检测试剂。
另外，本发明涉及的检测试剂的另一个例子是纤维蛋白原检测试剂，其通过将待测血浆与上述含有凝血酶和消泡剂的检测试剂接触，使血浆中的纤维蛋白原转变为纤维蛋白而使血液凝固，并测定纤维蛋白原的量。上述含有凝血酶和消泡剂的检测试剂中含有本发明的上述凝血酶溶液。
本发明的上述检测试剂的一种是含有上述检测试剂的纤维蛋白原检测试剂盒，但并不限定于此，还可以是其他样式的检测试剂。
[血浆的凝血酶时间的测定方法]
本发明涉及一种血浆的凝血酶时间的测定方法，该方法包括：
将待测血浆与本发明的上述凝血酶溶液或检测试剂接触，或者，
将待测血浆与本发明的液体型凝血酶时间检测试剂盒中所含的凝血酶和消泡剂接触。
通过以上的检测方法，不需要对凝血酶进行复溶，而是直接使用即可，因此，极大地方便了检测。
[溶液中凝血酶的稳定化方法]
本发明涉及一种溶液中凝血酶的稳定化方法，该方法包括：在含有凝血酶的溶液中添加消泡剂。通过在凝血酶溶剂中添加消泡剂，可以长时间地提高凝血酶在溶剂中的稳定性，从而，使得凝血酶能够长时间地在溶剂中保持稳定。
优选地，所述溶液进一步含有选自缓冲液、稳定剂和防腐剂中的至少一种。由于凝血酶溶液是含有缓冲剂等的液体型试剂，因此，不需要像以前的固体试剂或冻干试剂那样需要在使用时采用缓冲剂等进行复溶。本发明的凝血酶溶液可以无需复溶而直接使用，因此在使用上非常方便。
优选地，所述消泡剂优选为有机硅消泡剂，所述有机硅消泡剂与前面所述的凝血酶溶剂中的消泡剂可以是相同的消泡剂以及相同的有效成分，优选聚烷基硅氧烷类，更加优选聚二甲基硅氧烷。
凝血酶的活性范围优选1～20NIH Units/mL，特别优选1～10NIH Units/mL，进一步优选1～5NIH Units/mL。当凝血酶的活性范围较低时，更能体现本发明的技术效果。
优选地，所述消泡剂的有效成分在含有凝血酶的溶液中的浓度可以为0.1～15ppm，优选0.5～12ppm，优选0.1～10ppm，更加优选1～10ppm，进一步优选3～7ppm，特别优选5～7ppm。
所述缓冲液和稳定剂没有特别的限定，可以以相同的条件使用与前面所述的凝血酶溶剂中相同缓冲液和稳定剂。
[有机硅类消泡剂在凝血酶溶剂中作为稳定化剂的用途]
本发明的发明人发现，将有机硅类消泡剂添加于含有凝血酶的溶液中，可以长时间地提高凝血酶在溶剂中的稳定性，特别是在凝血酶的活性范围较低的情况下，更能够提高凝血酶在溶剂中的稳定性。即，本发明人发现了有机硅类消泡剂的一个新型用途，即在凝血酶溶剂中作为稳定化剂的用途。
如上所述，本发明的有机硅类消泡剂可以在溶液中对凝血酶进行稳定化。由此，本发明并不限于凝血酶时间的用途，显然也可用于含有凝血酶作为试剂构成成分的各种血液凝固时间方法、检测用试剂、试剂盒(所述检测方法等中也包括纤维蛋白原等的凝血因子、抗凝因子的检测等)、例如纤维蛋白原的检测方法等。
所述有机硅消泡剂的有效成分优选聚烷基硅氧烷类，更加优选聚二甲基硅氧烷。
凝血酶的活性范围优选1～20NIH Units/mL，特别优选1～10NIH Units/mL，进一步优选1～5NIH Units/mL。
所述消泡剂的有效成分在含有凝血酶的溶液中的浓度可以为0.1～15ppm，优选0.5～12ppm，优选0.1～10ppm，更加优选1～10ppm，进一步优选3～7ppm，特别优选5～7ppm。
实施例
本发明的实施例中所采用的原料试剂或仪器皆为普通市售品，皆可于市场购得。
本发明的试剂使用积水医疗的CP系列仪器进行检测，操作步骤如下：
Figure PCTCN2020119362-appb-000001
下面结合实施例，进一步阐述本发明：
实施例1
本实施例中，采用Antifoam B(Sigma公司)作为本发明的消泡剂添加于凝血酶溶液中，进行了研究。具体的有效成分和相关结果如表1所示。
配制凝血酶时间检测试剂，首先配制50mM的HEPES缓冲液，使用氢氧化钠调节其pH至7.3。随后添加30mM氯化钙、0.5％吐温-80、0.5％PEG-4000、1％BSA、0.5％Proclin 300、0ppm(比较例1)或5ppm(实施例1)有机硅消泡剂(即Antifoam B(Sigma公司))，经充分搅拌均匀后定容至所需要的体积。需要说明的是，所述Antifoam B中的有效成分含量为10％，所述5ppm为该有效成分在试剂中的浓度。随后加入凝血酶使得实际凝血酶浓度为2NIH Units/mL，经搅拌均匀后即获取凝血酶时间检测试剂。
配制完成的各试剂放置于37℃环境下，于第0天、第7天使用积水医疗CP3000全自动血凝仪测定参考品的数值，比较其秒数偏移情况，第0天和第7天的测定结果的对比结果如表1所示。表1中，初始检测值一行显示了第0天的检测值，最大检测值一行显示了第7天的检测值。另外，检测值偏差显示了第0天的检测值与第7天的检测值之差，偏移比例以检测值的偏差相对于第0天的检测值的百分比的形式示出。
表1使用消泡剂与否对稳定性的影响
Figure PCTCN2020119362-appb-000002
Figure PCTCN2020119362-appb-000003
根据秒数偏移比例的情况来看，在上述(2)实施例中加入了消泡剂，因而，其检测结果的秒数偏移明显小于其他试剂，说明添加消泡剂可有效提升试剂的稳定性。但是没有添加消泡剂的(1)比较例中不存在上述技术效果。
实施例2
本实施例进一步探究有效成分为聚二甲基硅氧烷的有机硅消泡剂最适宜的浓度。
配制凝血酶时间检测试剂，首先配制50mM的HEPES缓冲液，使用氢氧化钠调节其pH至7.3。随后添加30mM氯化钙、0.5％吐温-80、0.5％PEG-4000、1％BSA、0.5％Proclin 300、表2所示的各浓度(0ppm～100ppm)的消泡剂(Antifoam B，有效成分含量为10％)，经充分搅拌均匀后，定容至所需要的体积。随后加入凝血酶使得实际凝血酶浓度为2NIH Units/mL，经搅拌均匀后获取凝血酶时间检测试剂。
配制完成的各试剂放置于37℃环境下，于第0天、第7天使用积水医疗CP3000全自动血凝仪测定参考品的数值，比较其秒数偏移情况，第0天和第7天的测定结果的对比结果如表2所示。表2中，初始检测值一行、最大检测值一行、检测值偏差、偏移比例的意义与表1相同。
表2不同浓度聚二甲基硅氧烷消泡剂的加入给凝血酶稳定性带来的影响
Figure PCTCN2020119362-appb-000004
根据秒数偏移的情况来看，在有效成分含量为1ppm～10ppm的范围内的B～F试剂中，秒数的偏移明显小于未添加消泡剂的A试剂。可以得知，当有 效成分在1ppm～10ppm的范围内时，能够得到更佳的稳定化效果。进一步地，D试剂和E试剂的检测结果的秒数偏移明显较其他试剂更小，由此可知，其有效成分的浓度为5-7ppm时，能够进一步提高稳定化效果。
实施例3
在上述实验基础上，本实施例进一步探究50ppm消泡剂，即5ppm有效成分聚二甲基硅氧烷的加入对于试剂的反应性是否会有显著的影响。
配制凝血酶时间检测试剂，首先配制50mM的HEPES缓冲液，使用氢氧化钠调节其pH至7.3。随后添加30mM氯化钙、0.5％吐温-80、0.5％PEG-4000、1％BSA、0.5％Proclin 300、消泡剂(Antifoam B)(就有效成分而言，试剂B中为5ppm，或者试剂A中为0ppm(不添加))，经充分搅拌均匀后，定容至所需要的体积。随后加入凝血酶使得实际凝血酶浓度为2NIH Units/mL，经搅拌均匀后获取凝血酶时间检测试剂。
凝血酶时间试剂A或B配制完成后使用这些试剂于积水医疗CP3000全自动血凝仪上检测参考品与正常血浆样本，获取反应曲线。测得的凝血酶时间的结果示于表3的参考品的检测结果一行、正常血浆的检测结果一行。另外，正常血浆的表示凝固点的反应曲线的起跳点可以从图1中看出，而参考品的表示凝固点的反应曲线的起跳点可以从图2中看出。
表3聚二甲基硅氧烷消泡剂的加入给反应性带来的影响
试剂 A B
消泡剂 未添加 添加
参考品检测结果(秒) 19.9 19.7
正常血浆检测结果(秒) 16.8 17.1
由上述表3以及图1和图2的对比结果可以看出，消泡剂的添加对于样本的检测结果未产生明显的影响。首先，从表3可以看出，就参考品检测结果一行中试剂A、B的结果、正常血浆检测结果一行中试剂A、B的结果而言，试剂A与试剂B之间都是相近的，无论是否添加消泡剂，其对于检测结果的秒数并没有实质性的影响。其次，在图1和图2中，就能够以散射光强度的变 化来确认凝固反应的时间(起跳点)与凝固反应饱和时的散射光强度而言，试剂A与试剂B之间都是相近的，这一点在正常血浆(图1)和参考品(图2)中也是同样的。
根据上述表3、图1、图2所示的结果可以认为，本发明的消泡剂对于凝血酶时间检测试剂的检测结果没有影响。
以上仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (34)

  1. 一种凝血酶溶液，其含有凝血酶，并含有消泡剂。
  2. 根据权利要求1所述的凝血酶溶液，其进一步含有选自缓冲液、稳定剂和防腐剂中的至少一种。
  3. 根据权利要求1或2所述的凝血酶溶液，其中，所述消泡剂为有机硅消泡剂。
  4. 根据权利要求1～3中任一项所述的凝血酶溶液，其中，所述有机硅消泡剂的有效成分为聚烷基硅氧烷类。
  5. 根据权利要求1～4中任一项所述的凝血酶溶液，其中，所述有机硅消泡剂的有效成分为聚二甲基硅氧烷。
  6. 根据权利要求4或5所述的凝血酶溶液，其中，所述消泡剂的有效成分在凝血酶溶液中的浓度为0.1～10ppm。
  7. 根据权利要求4～6中任一项所述的凝血酶溶液，其中，所述消泡剂的有效成分在凝血酶溶液中的浓度为1～10ppm。
  8. 根据权利要求4～7中任一项所述的凝血酶溶液，其中，所述消泡剂的有效成分在凝血酶溶液中的浓度为5～7ppm。
  9. 根据权利要求2～8中任一项所述的凝血酶溶液，其中，所述缓冲液为选自5-100mM二(2-羟乙基)亚胺基三(羟甲基)甲烷(Bis-Tris)缓冲液、三羟甲基氨基甲烷(Tris)缓冲液、3-吗啉丙磺酸(MOPS)缓冲液、4-羟乙基哌嗪乙磺酸(HEPES)缓冲液中的一种。
  10. 根据权利要求2～9中任一项所述的凝血酶溶液，其中，所述稳定剂为选择聚乙二醇、牛血清白蛋白(BSA)、氯化钙(CaC1 2)、Triton X-100、吐温、Brij-58、甘露醇、果糖、甘油、海藻糖、麦芽糖、明胶中的一种或几种。
  11. 一种液体型凝血酶时间检测试剂盒，其含有凝血酶，并含有消泡剂。
  12. 根据权利要求11所述的液体型凝血酶时间检测试剂盒，其进一步含有选自缓冲液、稳定剂和防腐剂中的至少一种。
  13. 根据权利要求11或12所述的液体型凝血酶时间检测试剂盒，其中，所述消泡剂为有机硅消泡剂。
  14. 根据权利要求11～13中任一项所述的液体型凝血酶时间检测试剂盒，其中，所述有机硅消泡剂的有效成分为聚烷基硅氧烷类。
  15. 根据权利要求11～14中任一项所述的液体型凝血酶时间检测试剂盒，其中，所述有机硅消泡剂的有效成分为聚二甲基硅氧烷。
  16. 根据权利要求14或15所述的液体型凝血酶时间检测试剂盒，其中，所述消泡剂的有效成分在液体型凝血酶时间检测试剂盒中的浓度为0.1～10ppm。
  17. 根据权利要求14～16中任一项所述的液体型凝血酶时间检测试剂盒，其中，所述消泡剂的有效成分在液体型凝血酶时间检测试剂盒中的浓度为1～10ppm。
  18. 根据权利要求14～17中任一项所述的液体型凝血酶时间检测试剂盒，其中，所述消泡剂的有效成分在液体型凝血酶时间检测试剂盒中的浓度为5～7ppm。
  19. 根据权利要求12～18中任一项所述的液体型凝血酶时间检测试剂盒，其中，所述缓冲液为选自5-100mM二(2-羟乙基)亚胺基三(羟甲基)甲烷(Bis-Tris)缓冲液、三羟甲基氨基甲烷(Tris)缓冲液、3-吗啉丙磺酸(MOPS)缓冲液、4-羟乙基哌嗪乙磺酸(HEPES)缓冲液中的一种。
  20. 根据权利要求12～19中任一项所述的液体型凝血酶时间检测试剂盒，其中，所述稳定剂为选择聚乙二醇、牛血清白蛋白(BSA)、氯化钙(CaC1 2)、Triton X-100、吐温、Brij-58、甘露醇、果糖、甘油、海藻糖、麦芽糖、明胶中的一种或几种。
  21. 一种检测试剂，其含有权利要求1～10中任一项所述的凝血酶溶液。
  22. 权利要求21所述的检测试剂，其是凝血酶时间检测试剂。
  23. 一种血浆的凝血酶时间的测定方法，该方法包括：
    将待测血浆与权利要求1～10中任一项所述的凝血酶溶液或权利要求21或22所述的检测试剂接触，或者，
    将待测血浆与权利要求11～20中任一项所述的液体型凝血酶时间检测试剂盒所含的凝血酶和消泡剂接触。
  24. 一种溶液中凝血酶的稳定化方法，该方法包括：在含有凝血酶的溶 液中添加消泡剂。
  25. 根据权利要求24所述的稳定化方法，其中，所述溶液进一步含有选自缓冲液、稳定剂和防腐剂中的至少一种。
  26. 根据权利要求24或25所述的稳定化方法，其中，所述消泡剂为有机硅消泡剂。
  27. 根据权利要求24～26中任一项所述的稳定化方法，其中，所述有机硅消泡剂的有效成分为聚烷基硅氧烷类。
  28. 根据权利要求24～27中任一项所述的稳定化方法，其中，所述有机硅消泡剂的有效成分为聚二甲基硅氧烷。
  29. 根据权利要求27或28所述的稳定化方法，其中，所述消泡剂的有效成分在含有凝血酶的溶液中的浓度为0.1～10ppm。
  30. 根据权利要求27～29中任一项所述的稳定化方法，其中，所述消泡剂的有效成分在含有凝血酶的溶液中的浓度为1～10ppm。
  31. 根据权利要求27～30中任一项所述的稳定化方法，其中，所述消泡剂的有效成分在含有凝血酶的溶液中的浓度为5～7ppm。
  32. 根据权利要求25～31中任一项所述的稳定化方法，其中，所述缓冲液为选自5-100mM二(2-羟乙基)亚胺基三(羟甲基)甲烷(Bis-Tris)缓冲液、三羟甲基氨基甲烷(Tris)缓冲液、3-吗啉丙磺酸(MOPS)缓冲液、4-羟乙基哌嗪乙磺酸(HEPES)缓冲液中的一种。
  33. 根据权利要求25～32中任一项所述的稳定化方法，其中，所述稳定剂为选择聚乙二醇、牛血清白蛋白(BSA)、氯化钙(CaC1 2)、Triton X-100、吐温、Brij-58、甘露醇、果糖、甘油、海藻糖、麦芽糖、明胶中的一种或几种。
  34. 有机硅类消泡剂在凝血酶溶剂中作为稳定化剂的用途。
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