CN114729949A - Thrombin solution, kit, method for stabilizing thrombin, detection reagent, method for measuring thrombin time, and use of antifoaming agent - Google Patents

Thrombin solution, kit, method for stabilizing thrombin, detection reagent, method for measuring thrombin time, and use of antifoaming agent Download PDF

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CN114729949A
CN114729949A CN202080068852.8A CN202080068852A CN114729949A CN 114729949 A CN114729949 A CN 114729949A CN 202080068852 A CN202080068852 A CN 202080068852A CN 114729949 A CN114729949 A CN 114729949A
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thrombin
antifoaming agent
buffer
solution
active ingredient
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王辰骥
徐文娟
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Shuishui Medical Technology Suzhou Co ltd
Sekisui Medical Co Ltd
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Shuishui Medical Technology Suzhou Co ltd
Sekisui Medical Co Ltd
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • 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

Abstract

A thrombin solution, a kit, a method for stabilizing thrombin, a detection reagent, and a method for measuring thrombin time. The thrombin solution contains thrombin and an antifoaming agent. The thrombin solution has improved stability, so that the thrombin can be kept stable for a long time under the condition of lower activity.

Description

Thrombin solution, kit, method for stabilizing thrombin, detection reagent, method for measuring thrombin time, and use of antifoaming agent Technical Field
The invention relates to the field of biotechnology, in particular to a thrombin solution, a kit, a thrombin stabilization method, a detection reagent, a thrombin time determination method and application of a silicone defoaming agent as a stabilizing agent in a thrombin solvent.
Background
Thrombin Time (TT) refers to the time at which blood coagulates after standardized thrombin has been added to plasma. Thrombin time measurement is a simple test to determine the function of the coagulation, anticoagulation and fibrinolysis systems, and in particular to know whether plasma fibrin contains sufficient fibrinogen and whether the results are normal.
The thrombin time is a common way for reflecting blood coagulation, fibrinogen is converted into insoluble fibrin under the action of thrombin, and the time required for coagulation is determined, namely the thrombin time of the plasma to be detected. The detection of thrombin time has certain clinical value.
The thrombin time is prolonged abnormally when plasma fibrinogen is reduced or structurally abnormal, heparin is increased or heparinoid is present, and in systemic lupus erythematosus, liver disease, kidney disease, fibrinogen degradation product increase, disseminated intravascular coagulation, (non) fibrinogen hematopathy, abnormal fibrinogen hematopathy (fibrinogen dysmechanistic hematopathy), abnormal globulin hematopathy or immunoglobulin increase and other diseases. In the case of abnormal fibrinogen disease, the presence of calcium ions in the blood or the acidity of the blood, the thrombin time is abnormally shortened. The detection of thrombin time can provide certain aids in the diagnosis of the above-mentioned diseases.
At present, most of the reagents sold in the domestic market are TT reagents sold by import manufacturers, most of the reagents are freeze-dried products, the price is high, and the reagents need to be re-dissolved before use. The liquid TT reagent can be directly used without re-dissolving. However, the TT reagent is easy to inactivate thrombin in liquid due to low enzyme activity of thrombin, and needs to consider stability of thrombin more. If the thrombin activity can be effectively and economically maintained, the liquid TT reagent meeting the current market requirements can be successfully obtained.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide a thrombin solution, a kit, a method for stabilizing thrombin, a detection reagent, and a method for measuring thrombin time, so as to improve the stability of thrombin solution and keep thrombin stable for a long time even when the activity of thrombin is low.
In addition, the invention provides a liquid thrombin reagent which does not need to be redissolved and can effectively reduce detection errors caused by redissolution.
In order to achieve the purpose, the invention adopts the following technical scheme.
1. A thrombin solution comprising thrombin and an antifoaming agent.
2. The thrombin solution according to 1, further comprising at least one selected from the group consisting of a buffer, a stabilizer and a preservative.
3. The thrombin solution according to 1 or 2, wherein the antifoaming agent is a silicone antifoaming agent.
4. The thrombin solution according to any one of claims 1 to 3, wherein the silicone antifoaming agent contains a polyalkylsiloxane as an active ingredient.
5. The thrombin solution according to any one of claims 1 to 4, wherein the silicone antifoaming agent comprises polydimethylsiloxane as an active ingredient.
6. The thrombin solution according to 4 or 5, wherein the concentration of the active ingredient of the antifoaming agent in the thrombin solution is 0.1 to 10 ppm.
7. The thrombin solution according to any one of claims 4 to 6, wherein the concentration of the active ingredient of the antifoaming agent in the thrombin solution is 1 to 10 ppm.
8. The thrombin solution according to any one of claims 4 to 7, wherein the concentration of the active ingredient of the antifoaming agent in the thrombin solution is 5 to 7 ppm.
9. The thrombin solution according to any one of claims 2 to 8, wherein the buffer is one selected from the group consisting of 5-100mM Bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane (Bis-Tris) buffer, Tris (hydroxymethyl) aminomethane (Tris) buffer, 3-morpholinopropanesulfonic acid (MOPS) buffer and 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) buffer.
10. The thrombin solution according to any one of claims 2-9, wherein the stabilizer is one or more selected from polyethylene glycol, Bovine Serum Albumin (BSA), calcium chloride (CaC12), Triton X-100, Tween, Brij-58, mannitol, fructose, glycerol, trehalose, maltose and gelatin.
11. A liquid thrombin time detection kit comprises thrombin and an antifoaming agent.
12. The liquid thrombin time detection kit according to claim 11, further comprising at least one selected from the group consisting of a buffer, a stabilizer and a preservative.
13. The liquid thrombin time detection kit according to 11 or 12, wherein the antifoaming agent is an organic silicon antifoaming agent.
14. The liquid thrombin time detection kit according to any one of claims 11 to 13, wherein the active ingredient of the silicone antifoaming agent is polyalkylsiloxane.
15. The liquid thrombin time detection kit according to any one of claims 11 to 14, wherein the active ingredient of the silicone antifoaming agent is polydimethylsiloxane.
16. The liquid thrombin time detection kit according to 14 or 15, wherein the concentration of the effective component of the antifoaming agent in the liquid thrombin time detection kit is 0.1-10 ppm.
17. The liquid thrombin time detection kit according to any one of claims 14 to 16, wherein the concentration of the active ingredient of the antifoaming agent in the liquid thrombin time detection kit is 1 to 10 ppm.
18. The liquid thrombin time detection kit according to any one of claims 14 to 17, wherein the concentration of the active ingredient of the antifoaming agent in the liquid thrombin time detection kit is 5 to 7 ppm.
19. The liquid thrombin time detection kit according to any one of claims 12 to 18, wherein the buffer is one selected from 5-100mM Bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane (Bis-Tris) buffer, Tris (hydroxymethyl) aminomethane (Tris) buffer, 3-morpholinopropanesulfonic acid (MOPS) buffer and 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) buffer.
20. The liquid thrombin time detection kit according to any one of claims 12-19, wherein the stabilizer is one or more selected from polyethylene glycol, Bovine Serum Albumin (BSA), calcium chloride (CaC12), Triton X-100, Tween, Brij-58, mannitol, fructose, glycerol, trehalose, maltose and gelatin.
21. A detection reagent comprising the thrombin solution according to any one of 1 to 10.
22. The detection reagent according to 21, which is a thrombin time detection reagent.
23. A method for determining thrombin time in plasma, the method comprising:
contacting plasma to be tested with the thrombin solution of any one of 1-10 or the detection reagent of 21 or 22, or,
contacting plasma to be detected with thrombin and an antifoaming agent contained in the liquid thrombin time detection kit according to any one of 11-20.
24. A method of stabilizing thrombin in a solution, the method comprising: an antifoaming agent is added to the thrombin-containing solution.
25. The stabilization method according to 24, wherein the solution further contains at least one selected from a buffer, a stabilizer, and a preservative.
26. The stabilization method according to 24 or 25, wherein the antifoaming agent is a silicone antifoaming agent.
27. The stabilization method according to any one of claims 24 to 26, wherein an active ingredient of the silicone antifoaming agent is a polyalkylsiloxane.
28. The stabilization method according to any one of claims 24 to 27, wherein an active ingredient of the silicone antifoaming agent is polydimethylsiloxane.
29. The stabilizing method according to 27 or 28, wherein the concentration of the active ingredient of the antifoaming agent in the thrombin-containing solution is 0.1 to 10 ppm.
30. A stabilizing method according to any one of claims 27 to 29, wherein the concentration of the active ingredient of the antifoaming agent in the thrombin-containing solution is 1 to 10 ppm.
31. The stabilizing method according to any one of claims 27 to 30, wherein the concentration of the active ingredient of the antifoaming agent in the thrombin-containing solution is 5 to 7 ppm.
32. The stabilization method according to any one of claims 25 to 31, wherein the buffer is one selected from the group consisting of 5 to 100mM Bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane (Bis-Tris) buffer, Tris (hydroxymethyl) aminomethane (Tris) buffer, 3-morpholinopropanesulfonic acid (MOPS) buffer, and 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) buffer.
33. The method according to any one of claims 25 to 32, wherein the stabilizer is one or more selected from the group consisting of polyethylene glycol, Bovine Serum Albumin (BSA), calcium chloride (CaC12), Triton X-100, Tween, Brij-58, mannitol, fructose, glycerol, trehalose, maltose, and gelatin.
34. Use of a silicone based antifoaming agent as a stabilizer in a thrombin solvent.
Technical effects of the invention
Through the technical scheme of the invention, the following technical effects can be realized.
By adding the antifoaming agent, the stability of the thrombin-containing reagent is improved on the original basis, so that the thrombin in the solution can be kept stable for a long time under the condition of low activity, and the method is very economical and effective.
In addition, the invention is a liquid reagent, does not need to be redissolved, and can effectively reduce the measurement error caused by redissolution.
Drawings
FIG. 1: normal plasma response curves in example 3 of the present application;
FIG. 2: reference reaction curves in example 3 of the present application.
Detailed Description
[ Thrombin solution ]
The invention provides a thrombin solution, which contains thrombin and an antifoaming agent. In the thrombin solution of the present invention, thrombin is a main functional component, and fibrinogen is converted into insoluble fibrin by the action of thrombin. In the present invention, by containing an antifoaming agent in a thrombin solvent, the stability of thrombin in the solvent can be improved, thereby allowing the thrombin to be stable in the solvent for a long period of time. In the present specification, the term "solution" does not mean a solid or semisolid state obtained by freezing a liquid (aqueous solution) at 0 ℃ or lower, but means a state of maintaining fluidity as a solution, for example, a state under refrigeration (for example, 2 to 8 ℃) when the solution is distributed as a reagent for clinical examination.
The thrombin solution of the present invention further contains at least one selected from the group consisting of a buffer, a stabilizer and a preservative, and preferably contains a buffer, a stabilizer and a preservative from the viewpoint of more remarkably obtaining the technical effects of the present invention. Since the thrombin solution of the present invention is a liquid type reagent containing a buffer or the like, it is not necessary to reconstitute the thrombin solution using a buffer or the like at the time of use, as in the case of conventional solid reagents or lyophilized reagents. The thrombin solution of the invention can be used directly without reconstitution, and is therefore very convenient to use.
In the thrombin solution of the present invention, the antifoaming agent is preferably an organosilicon antifoaming agent, and is preferably an aqueous emulsion type organosilicon antifoaming agent. The defoaming agent can be used for common food and medicine to prevent the generation of bubbles in the solution. The silicone defoamer is a defoamer having a silicone (polysiloxane) backbone. The active ingredient of the silicone antifoaming agent is preferably polyalkylsiloxane from the viewpoint of further improving the stability of thrombin. Specific examples of polyalkylsiloxanes include: polymethylsiloxane, polyethylsiloxane, polydimethylsiloxane, polydiethylsiloxane, polymethylethylsiloxane, and the like. The active ingredient of the silicone antifoaming agent is preferably polydimethylsiloxane from the viewpoint of further providing stability of thrombin.
Specific examples of the defoaming agent of the present invention include Antifoam B (manufactured by Sigma, a5757 or a6707) in which the active ingredient is polydimethylsiloxane, the content of the active ingredient in the defoaming agent is 10%, and the concentration range suggested in the product specification is 1 to 100 ppm.
In the present invention, the activity range of thrombin is not particularly limited, but is preferably 1 to 20NIH Units/mL, more preferably 1 to 10NIH Units/mL, and still more preferably 1 to 5NIH Units/mL. The technical effects of the present invention can be more exhibited when the activity range of thrombin is lower. That is, according to the present invention, even when thrombin activity is low, an excellent effect of maintaining stability for a long period of time can be achieved.
In the present invention, the concentration of the active ingredient of the antifoaming agent in the thrombin solution may be 0.1 to 15ppm, preferably 0.5 to 12ppm, preferably 0.1 to 10ppm, more preferably 1 to 10ppm, further preferably 3 to 7ppm, and particularly preferably 5 to 7ppm, from the viewpoint of further improving the stability of thrombin.
In the present invention, the buffer is not particularly limited, but from the viewpoint of further improving the stability of thrombin, the buffer is preferably selected from any one of 5 to 100mM Bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane (Bis-Tris) buffer, Tris buffer, 3-morpholinopropanesulfonic acid (MOPS) buffer, and 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) buffer.
Preferably, the buffer solution is HEPES, the concentration range is 10-100 mmol/L, and the pH value is 6.5-8.0; more preferably, the buffer is 25-75 mM HEPES, pH 7.1-7.5, and even more preferably 50mM HEPES, pH 7.3.
In the present invention, the stabilizer is not particularly limited, but is selected from one or more of polyethylene glycol, Bovine Serum Albumin (BSA), calcium chloride (CaC12), Triton X-100, Tween, Brij-58, mannitol, fructose, glycerol, trehalose, maltose, and gelatin, from the viewpoint of further improving the stability of thrombin.
In a preferred embodiment of the invention, the stabilizer is polyethylene glycol 4000(PEG-4000), Tween-80, Bovine Serum Albumin (BSA), calcium chloride (CaC 12).
The method for producing the thrombin solution is not particularly limited, and the following production method can be employed. The specific procedures comprise: preparing 50mM HEPES buffer solution, adjusting the pH value to 7.3, adding a preservative, a stabilizer and an antifoaming agent into the buffer solution, uniformly mixing, adding thrombin, and uniformly mixing after adjustment to obtain the thrombin solution.
[ liquid type thrombin time detection kit ]
The invention provides a liquid thrombin time detection kit. The kit comprises the thrombin solution of the present invention and a package for containing the thrombin solution. Specifically, the liquid thrombin time detection kit of the present invention contains thrombin and an antifoaming agent.
The kit of the present invention further comprises at least one selected from the group consisting of a buffer, a stabilizer and a preservative, and preferably comprises a buffer, a stabilizer and a preservative. Since the thrombin time measuring kit of the present invention is a liquid type reagent containing a buffer or the like, it is not necessary to reconstitute the reagent using a buffer or the like at the time of use, as in the case of conventional solid reagents or lyophilized reagents. The thrombin solution of the invention can be used directly without reconstitution, and is therefore very convenient to use.
In the kit of the present invention, the antifoaming agent is preferably a silicone antifoaming agent, and the silicone antifoaming agent may be the same antifoaming agent and the same active ingredient as the antifoaming agent in the thrombin solvent, and is preferably a polyalkylsiloxane, and more preferably a polydimethylsiloxane.
In the kit of the present invention, the activity range of thrombin is not particularly limited, but is preferably 1 to 20NIH Units/mL, more preferably 1 to 10NIH Units/mL, and still more preferably 1 to 5NIH Units/mL. The technical effect of the invention can be better embodied when the activity range of the thrombin is lower. That is, according to the present invention, even when thrombin activity is low, an excellent effect of maintaining stability for a long period of time can be achieved.
In the present invention, the concentration of the active ingredient of the antifoaming agent in the thrombin solution may be 0.1 to 15ppm, preferably 0.5 to 12ppm, preferably 0.1 to 10ppm, more preferably 1 to 10ppm, further preferably 3 to 7ppm, and particularly preferably 5 to 7ppm, from the viewpoint of further improving the stability of thrombin.
In the kit of the present invention, the buffer and the stabilizer are not particularly limited, and the same buffer and stabilizer as those in the thrombin solvent described above may be used under the same conditions.
[ detection reagent ]
An example of the detection reagent according to the present invention is a thrombin time detection reagent, and the thrombin time in plasma is measured by bringing plasma to be measured into contact with the thrombin time detection reagent. The thrombin assay reagent contains the thrombin solution of the present invention.
One type of the detection reagent of the present invention is the liquid thrombin time detection kit of the present invention, but the detection reagent is not limited thereto, and may be other types of detection reagents.
In addition, another example of the detection reagent according to the present invention is a fibrinogen detection reagent in which fibrinogen in plasma to be tested is brought into contact with the above-mentioned detection reagent containing thrombin and an antifoaming agent to convert fibrinogen in the plasma into fibrin, thereby coagulating the blood and measuring the amount of fibrinogen. The thrombin solution of the present invention is contained in the detection reagent containing thrombin and an antifoaming agent.
One of the detection reagents of the present invention is a fibrinogen detection kit containing the detection reagent, but the present invention is not limited thereto, and other types of detection reagents may be used.
[ method for measuring thrombin time in plasma ]
The invention relates to a method for measuring thrombin time of plasma, which comprises the following steps:
the plasma to be tested is contacted with the thrombin solution or the detection reagent of the present invention as described above, or,
the plasma to be measured is contacted with thrombin and an antifoaming agent contained in the liquid thrombin time detection kit of the present invention.
By the detection method, thrombin does not need to be redissolved, and the thrombin can be directly used, so that the detection is greatly facilitated.
[ method for stabilizing thrombin in solution ]
The invention relates to a method for stabilizing thrombin in a solution, which comprises the following steps: an antifoaming agent is added to the thrombin-containing solution. By adding an antifoaming agent to a thrombin solvent, the stability of thrombin in the solvent can be improved for a long period of time, thereby enabling the thrombin to be stable in the solvent for a long period of time.
Preferably, the solution further contains at least one selected from the group consisting of a buffer, a stabilizer and a preservative. Since the thrombin solution is a liquid type reagent containing a buffer or the like, it is not necessary to reconstitute the thrombin solution with a buffer or the like at the time of use, as in the case of conventional solid reagents or lyophilized reagents. The thrombin solution of the invention can be used directly without reconstitution, and is therefore very convenient to use.
Preferably, the anti-foaming agent is a silicone anti-foaming agent, which may be the same anti-foaming agent and the same active ingredient as the anti-foaming agent in the thrombin solvent described above, preferably a polyalkylsiloxane, more preferably a polydimethylsiloxane.
The thrombin activity is preferably 1 to 20NIH Units/mL, more preferably 1 to 10NIH Units/mL, and still more preferably 1 to 5NIH Units/mL. The technical effects of the present invention can be more exhibited when the activity range of thrombin is lower.
Preferably, the concentration of the active ingredient of the antifoaming agent in the thrombin-containing solution may be 0.1 to 15ppm, preferably 0.5 to 12ppm, preferably 0.1 to 10ppm, more preferably 1 to 10ppm, further preferably 3 to 7ppm, and particularly preferably 5 to 7 ppm.
The buffer and the 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.
[ use of Silicone-based antifoaming agent as stabilizer in Thrombin solvent ]
The inventors of the present invention have found that the stability of thrombin in a solvent can be improved for a long period of time by adding a silicone-based antifoaming agent to a solution containing thrombin, and particularly, the stability of thrombin in a solvent can be improved even when the activity range of thrombin is low. That is, the present inventors have found a novel use of a silicone-based antifoaming agent, i.e., a use as a stabilizer in a thrombin solvent.
As described above, the silicone-based antifoaming agent of the present invention can stabilize thrombin in a solution. Thus, the present invention is not limited to the use of thrombin time, and can be obviously applied to various blood coagulation time methods, detection reagents, kits (the detection methods and the like also include detection of a coagulation factor such as fibrinogen and an anticoagulation factor), for example, a method for detecting fibrinogen, and the like, which contain thrombin as a reagent component.
The active ingredient of the silicone antifoaming agent is preferably polyalkylsiloxane, and polydimethylsiloxane is more preferably.
The activity range of thrombin is preferably 1 to 20NIH Units/mL, particularly preferably 1 to 10NIH Units/mL, and further preferably 1 to 5NIH Units/mL.
The concentration of the active ingredient of the antifoaming agent in the thrombin-containing solution may be 0.1 to 15ppm, preferably 0.5 to 12ppm, more preferably 0.1 to 10ppm, still more preferably 1 to 10ppm, further preferably 3 to 7ppm, and particularly preferably 5 to 7 ppm.
Examples
The raw material reagents or instruments used in the examples of the present invention are all commercially available products, and are all commercially available.
The reagent of the invention uses a CP series instrument for hydrops treatment to carry out detection, and the operation steps are as follows:
Figure PCTCN2020119362-APPB-000001
the invention is further illustrated by the following examples:
example 1
In this example, an examination was conducted by adding Antifoam B (Sigma) as an antifoaming agent of the present invention to a thrombin solution. Specific active ingredients and related results are shown in table 1.
The thrombin time detection reagent was prepared by first preparing 50mM HEPES buffer and adjusting the pH to 7.3 with sodium hydroxide. Then 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 Antifoam (i.e., Antifoam B (Sigma Co.)), stirred well and then brought to the desired volume. The content of the active ingredient in the aforementioned Antifoam B was 10%, and the aforementioned 5ppm was the concentration of the active ingredient in the reagent. Then adding thrombin to make the actual thrombin concentration be 2NIH Units/mL, and uniformly stirring to obtain the thrombin time detection reagent.
The prepared reagents were placed in an environment of 37 ℃ and measured for reference values on days 0 and 7 using a hydro-therapeutic CP3000 fully automatic coagulometer, and the second deviation was compared, and the comparison results between the measurement results on days 0 and 7 are shown in table 1. In table 1, the row of the initial measurement values shows the measurement values of day 0, and the row of the maximum measurement values shows the measurement values of day 7. In addition, the detection value deviation shows the difference between the detection value on day 0 and the detection value on day 7, and the deviation ratio is shown as the percentage of the deviation of the detection value with respect to the detection value on day 0.
TABLE 1 Effect of Using antifoam on stability
Figure PCTCN2020119362-APPB-000002
Figure PCTCN2020119362-APPB-000003
According to the second deviation proportion, the defoaming agent is added in the embodiment (2), so that the second deviation of the detection result is obviously smaller than that of other reagents, and the addition of the defoaming agent can effectively improve the stability of the reagents. However, the above-mentioned technical effects do not exist in the comparative example (1) in which no defoaming agent is added.
Example 2
This example further explores the optimum concentration of silicone defoamer with an active ingredient being polydimethylsiloxane.
The thrombin time detection reagent was prepared by first preparing 50mM HEPES buffer and adjusting its pH to 7.3 with sodium hydroxide. Then, 30mM calcium chloride, 0.5% Tween-80, 0.5% PEG-4000, 1% BSA, 0.5% Proclin 300, and an antifoaming agent (Antifoam B, active ingredient content: 10%) at each concentration (0ppm to 100ppm) shown in Table 2 were added thereto, and after sufficiently and uniformly stirred, the volume was adjusted to the desired volume. Then adding thrombin to make the actual thrombin concentration be 2NIH Units/mL, and obtaining the thrombin time detection reagent after stirring uniformly.
The prepared reagents were placed in an environment of 37 ℃ and measured for reference values on days 0 and 7 using a hydro-therapeutic CP3000 fully automatic coagulometer, and the second deviation was compared, and the comparison results between the measurement results on days 0 and 7 are shown in table 2. In table 2, the meanings of the initial detection value row, the maximum detection value row, the detection value deviation, and the offset ratio are the same as those in table 1.
TABLE 2 Effect of the addition of different concentrations of polydimethylsiloxane antifoam on thrombin stability
Figure PCTCN2020119362-APPB-000004
In the case of the shift in seconds, the shift in seconds is significantly smaller in the reagents B to F having an active ingredient content in the range of 1ppm to 10ppm than in the reagent A to which no defoaming agent is added. It was found that a more excellent stabilizing effect was obtained when the effective component was in the range of 1ppm to 10 ppm. Further, the second number of shifts in the detection results of the D reagent and the E reagent were significantly smaller than those of the other reagents, and it was found that the stabilization effect was further improved when the concentration of the active ingredient was 5 to 7 ppm.
Example 3
Based on the above experiments, this example further investigated whether the addition of 50ppm defoamer, i.e. 5ppm active ingredient polydimethylsiloxane, would have a significant effect on the reactivity of the reagent.
The thrombin time detection reagent was prepared by first preparing 50mM HEPES buffer and adjusting its pH to 7.3 with sodium hydroxide. Then, 30mM calcium chloride, 0.5% Tween-80, 0.5% PEG-4000, 1% BSA, 0.5% Proclin 300, and an antifoaming agent (antifoaming agent B) (5 ppm in reagent B or 0ppm in reagent A (not added) as an effective component) were added thereto, and the mixture was sufficiently stirred to be uniform and then the volume was adjusted to the desired volume. Then adding thrombin to make the actual thrombin concentration be 2NIH Units/mL, and obtaining the thrombin time detection reagent after stirring uniformly.
After the thrombin time reagent A or B is prepared, the reagents are used for detecting a reference substance and a normal plasma sample on a hydrops medical CP3000 full-automatic hemagglutination instrument, and a reaction curve is obtained. The results of measuring the thrombin time are shown in Table 3 in the test results of the reference sample and in the test results of the normal plasma. In addition, the jump point of the response curve indicating the freezing point of normal plasma can be seen from fig. 1, and the jump point of the response curve indicating the freezing point of the reference substance can be seen from fig. 2.
TABLE 3 Effect of the addition of Dimethicone antifoam on reactivity
Reagent A B
Defoaming agent Without adding Adding
Reference test result (seconds) 19.9 19.7
Normal plasma test results (seconds) 16.8 17.1
As can be seen from the above table 3 and the comparison results of fig. 1 and fig. 2, the addition of the antifoaming agent did not have a significant effect on the detection results of the samples. First, as can be seen from table 3, the results of the reagent A, B in the row of the reference product test results and the results of the reagent A, B in the row of the normal plasma test results are similar to each other between the reagent a and the reagent B, and the addition of the antifoaming agent does not substantially affect the number of seconds of the test results. Next, in fig. 1 and 2, the time (trip point) for the coagulation reaction and the scattered light intensity at the time of saturation of the coagulation reaction can be confirmed as changes in the scattered light intensity, and this is similar between the reagent a and the reagent B, and is also similar in the normal plasma (fig. 1) and the reference product (fig. 2).
From the results shown in Table 3, FIG. 1 and FIG. 2, it is considered that the antifoaming agent of the present invention has no influence on the detection result of the thrombin time detecting reagent.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.

Claims (34)

  1. A thrombin solution comprising thrombin and an antifoaming agent.
  2. The thrombin solution according to claim 1, further comprising at least one selected from the group consisting of a buffer, a stabilizer and a preservative.
  3. The thrombin solution according to claim 1 or 2, wherein the antifoaming agent is a silicone antifoaming agent.
  4. The thrombin solution according to any one of claims 1 to 3, wherein the silicone antifoaming agent comprises a polyalkylsiloxane as an active ingredient.
  5. The thrombin solution according to any one of claims 1 to 4, wherein the silicone antifoaming agent comprises polydimethylsiloxane as an active ingredient.
  6. The thrombin solution according to claim 4 or 5, wherein the concentration of the active ingredient of the antifoaming agent in the thrombin solution is 0.1-10 ppm.
  7. The thrombin solution according to any one of claims 4 to 6, wherein the concentration of the active ingredient of the antifoaming agent in the thrombin solution is 1 to 10 ppm.
  8. The thrombin solution according to any one of claims 4 to 7, wherein the concentration of the active ingredient of the antifoaming agent in the thrombin solution is 5 to 7 ppm.
  9. The thrombin solution according to any one of claims 2 to 8, wherein the buffer is one selected from the group consisting of 5-100mM Bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane (Bis-Tris) buffer, Tris buffer, 3-morpholinopropanesulfonic acid (MOPS) buffer, 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) buffer.
  10. The thrombin solution according to any one of claims 2 to 9, wherein the stabilizer is selected from the group consisting of polyethylene glycol, Bovine Serum Albumin (BSA), calcium chloride (CaC 1)2) One or more of Triton X-100, Tween, Brij-58, mannitol, fructose, glycerol, trehalose, maltose and gelatin.
  11. A liquid thrombin time detection kit comprises thrombin and an antifoaming agent.
  12. The liquid thrombin time detection kit according to claim 11, further comprising at least one selected from the group consisting of a buffer, a stabilizer and a preservative.
  13. The liquid thrombin time detection kit according to claim 11 or 12, wherein the antifoaming agent is a silicone antifoaming agent.
  14. The liquid thrombin time detection kit according to any one of claims 11 to 13, wherein the silicone antifoaming agent comprises polyalkylsiloxanes as an active ingredient.
  15. The liquid thrombin time detection kit according to any one of claims 11 to 14, wherein the silicone antifoaming agent comprises polydimethylsiloxane as an active ingredient.
  16. The liquid thrombin time detection kit according to claim 14 or 15, wherein the concentration of the active ingredient of said antifoaming agent in said liquid thrombin time detection kit is 0.1-10 ppm.
  17. The liquid thrombin time detection kit according to any one of claims 14 to 16, wherein the concentration of the active ingredient of the antifoaming agent in the liquid thrombin time detection kit is 1 to 10 ppm.
  18. The liquid thrombin time detection kit according to any one of claims 14 to 17, wherein the concentration of the active ingredient of the antifoaming agent in the liquid thrombin time detection kit is 5 to 7 ppm.
  19. The liquid thrombin time detection kit according to any one of claims 12 to 18, wherein the buffer is one selected from the group consisting of 5-100mM Bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane (Bis-Tris) buffer, Tris (hydroxymethyl) aminomethane (Tris) buffer, 3-morpholinopropanesulfonic acid (MOPS) buffer and 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) buffer.
  20. The liquid thrombin time detection kit according to any one of claims 12 to 19, wherein the stabilizer is selected from the group consisting of polyethylene glycol, Bovine Serum Albumin (BSA), calcium chloride (CaC 1)2) One or more of Triton X-100, Tween, Brij-58, mannitol, fructose, glycerol, trehalose, maltose and gelatin.
  21. A detection reagent comprising the thrombin solution according to any one of claims 1 to 10.
  22. The detection reagent according to claim 21, which is a thrombin time detection reagent.
  23. A method for determining thrombin time in plasma, the method comprising:
    contacting the plasma to be tested with the thrombin solution according to any one of claims 1 to 10 or the detection reagent according to claim 21 or 22, or,
    contacting plasma to be tested with thrombin and an antifoaming agent contained in the liquid thrombin time detection kit according to any one of claims 11 to 20.
  24. A method of stabilizing thrombin in a solution, the method comprising: an antifoaming agent is added to the solution containing thrombin.
  25. The stabilization method according to claim 24, wherein the solution further contains at least one selected from a buffer, a stabilizer, and a preservative.
  26. A stabilization method according to claim 24 or 25, wherein the defoamer is a silicone defoamer.
  27. A stabilization method according to any one of claims 24 to 26, wherein the active ingredient of the silicone antifoam agent is a polyalkylsiloxane.
  28. The stabilization method according to any one of claims 24 to 27, wherein an active ingredient of the silicone antifoaming agent is polydimethylsiloxane.
  29. The stabilizing method according to claim 27 or 28, wherein the concentration of the active ingredient of the antifoaming agent in the thrombin-containing solution is 0.1 to 10 ppm.
  30. The stabilizing method according to any one of claims 27 to 29, wherein the concentration of the active ingredient of the antifoaming agent in the thrombin-containing solution is 1 to 10 ppm.
  31. The stabilizing method according to any one of claims 27 to 30, wherein the concentration of the active ingredient of the antifoaming agent in the thrombin-containing solution is 5 to 7 ppm.
  32. The stabilization method according to any one of claims 25 to 31, wherein the buffer is one selected from 5 to 100mM Bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane (Bis-Tris) buffer, Tris buffer, 3-morpholinopropanesulfonic acid (MOPS) buffer, 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) buffer.
  33. A method according to any one of claims 25 to 32, wherein the stabilising agent is selected from the group consisting of polyethylene glycol, Bovine Serum Albumin (BSA), calcium chloride (CaC 1)2) One or more of Triton X-100, Tween, Brij-58, mannitol, fructose, glycerol, trehalose, maltose and gelatin.
  34. Use of a silicone based antifoaming agent as a stabilizer in a thrombin solvent.
CN202080068852.8A 2019-09-30 2020-09-30 Thrombin solution, kit, method for stabilizing thrombin, detection reagent, method for measuring thrombin time, and use of antifoaming agent Pending CN114729949A (en)

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