Thrombin solution, kit, method for stabilizing thrombin, detection reagent, method for determining thrombin time, and use of an antifoam

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 contains an antifoaming agent. The thrombin solution has improved stability, enabling the thrombin to remain stable for a long time even at a low activity.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a thrombin solution, a kit, a method for stabilizing thrombin, a detection reagent, a method for determining thrombin time, and the use of an organosilicon defoaming agent as a stabilizing agent in a thrombin solvent. BACKGROUND

[0002] Thrombin time (TT) refers to the time required for blood to coagulate after adding standardized thrombin to the plasma. Thrombin time determination is a simple test for detecting the function of the coagulation, anticoagulation and fibrinolysis system, and can particularly determine whether the fibrin in the plasma contains sufficient fibrinogen and whether the result is normal.

[0003] Thrombin time reflects the common pathway of coagulation, and fibrinogen is converted to insoluble fibrin under the action of thrombin. The time required for coagulation is the thrombin time of the plasma to be tested. The detection of thrombin time has certain clinical value.

[0004] When the plasma fibrinogen is reduced or structurally abnormal, heparin is increased or heparinoids exist, and in diseases such as systemic lupus erythematosus, liver disease, kidney disease, increased fibrinogen degradation products, disseminated intravascular coagulation, (no) fibrinogenemia, abnormal fibrinogenemia (fibrinogen mechanism poor anemia), abnormal globulinemia or increased immunoglobulin, the thrombin time will be abnormally prolonged. In abnormal fibrinogenemia, calcium ions exist in the blood or the blood is acidic, the thrombin time will be abnormally shortened. The detection of thrombin time can provide certain assistance for the diagnosis of the above diseases.

[0005] At present, the TT reagent sold on the domestic market is mainly sold by imported manufacturers. These reagents are mostly freeze-dried products, which are expensive and need to be reconstituted before use. Liquid TT reagent does not need to be reconstituted and can be used directly. However, due to the low enzyme activity of thrombin in the TT reagent, the thrombin in the liquid is easily inactivated, and the stability of thrombin needs to be considered more. If the activity of thrombin can be effectively and economically maintained, liquid TT reagent that meets the current market demand can be successfully obtained. SUMMARY

[0006] In view of this, the technical problem to be solved by the present application is to provide a thrombin solution, a kit, a method for stabilizing thrombin, a detection reagent, and a method for determining thrombin time, so as to improve the stability of the thrombin solution and enable thrombin to maintain stability for a long time even at low activity.

[0007] In addition, the technical problem to be solved by the present application is to provide a liquid thrombin reagent, which does not need to be reconstituted and can effectively reduce the detection error caused by reconstitution.

[0008] To achieve the above object, the present application adopts the following technical solution.

[0009] 1. A thrombin solution comprising thrombin and comprising an antifoaming agent.

[0010] 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.

[0011] 3. The thrombin solution according to claim 1 or 2, wherein the antifoaming agent is an organosilicon antifoaming agent.

[0012] 4. The thrombin solution according to any one of claims 1 to 3, wherein the effective component of the organosilicon antifoaming agent is a polyalkylsiloxane.

[0013] 5. The thrombin solution according to any one of claims 1 to 4, wherein the effective component of the organosilicon antifoaming agent is polydimethylsiloxane.

[0014] 6. The thrombin solution according to claim 4 or 5, wherein the concentration of the effective component of the antifoaming agent in the thrombin solution is 0.1 to 10 ppm.

[0015] 7. The thrombin solution according to any one of claims 4 to 6, wherein the concentration of the effective component of the antifoaming agent in the thrombin solution is 1 to 10 ppm.

[0016] 8. The thrombin solution according to any one of claims 4 to 7, wherein the concentration of the effective component of the antifoaming agent in the thrombin solution is 5 to 7 ppm.

[0017] 9. The thrombin solution according to any one of claims 2 to 8, wherein the buffer is one selected from the group consisting of 5-100 mM Bis-Tris buffer, Tris buffer, MOPS buffer and HEPES buffer.

[0018] 10. The thrombin solution according to any one of claims 2 to 9, wherein the stabilizer is one or more selected from the group consisting of polyethylene glycol, bovine serum albumin (BSA), calcium chloride (CaCl2), Triton X-100, Tween, Brij-58, mannitol, fructose, glycerol, trehalose, maltose and gelatin.

[0019] 11. A liquid thrombin time test kit comprising thrombin and comprising an antifoaming agent.

[0020] 12. The liquid thromboplastin time test kit according to claim 11, further comprising at least one selected from the group consisting of a buffer, a stabilizer, and a preservative.

[0021] 13. The liquid thromboplastin time test kit according to claim 11 or 12, wherein the antifoaming agent is an organosilicon antifoaming agent.

[0022] 14. The liquid thromboplastin time test kit according to any one of claims 11 to 13, wherein the effective ingredient of the organosilicon antifoaming agent is a polyalkylsiloxane.

[0023] 15. The liquid thromboplastin time test kit according to any one of claims 11 to 14, wherein the effective ingredient of the organosilicon antifoaming agent is polydimethylsiloxane.

[0024] 16. The liquid thromboplastin time test kit according to claim 14 or 15, wherein the concentration of the effective ingredient of the antifoaming agent in the liquid thromboplastin time test kit is 0.1 to 10 ppm.

[0025] 17. The liquid thromboplastin time test kit according to any one of claims 14 to 16, wherein the concentration of the effective ingredient of the antifoaming agent in the liquid thromboplastin time test kit is 1 to 10 ppm.

[0026] 18. The liquid thromboplastin time test kit according to any one of claims 14 to 17, wherein the concentration of the effective ingredient of the antifoaming agent in the liquid thromboplastin time test kit is 5 to 7 ppm.

[0027] 19. The liquid thromboplastin time test kit according to any one of claims 12 to 18, wherein the buffer is one selected from the group consisting of 5-100 mM Bis-Tris buffer, Tris buffer, MOPS buffer, and HEPES buffer.

[0028] 20. The liquid thromboplastin time test kit according to any one of claims 12 to 19, wherein the stabilizer is one or more selected from the group consisting of polyethylene glycol, bovine serum albumin (BSA), calcium chloride (CaCl2), Triton X-100, Tween, Brij-58, mannitol, fructose, glycerol, trehalose, maltose, and gelatin.

[0029] 21. A test reagent comprising the thrombin solution according to any one of claims 1 to 10.

[0030] 22. The test reagent according to claim 21, which is a thromboplastin time test reagent.

[0031] 23. A method for measuring the thrombin time of plasma, the method comprising:

[0032] contacting the plasma to be measured with the thrombin solution according to any one of claims 1 to 10 or the detection reagent according to claim 21 or 22, or,

[0033] contacting the plasma to be measured with the thrombin and the defoaming agent contained in the liquid-type thrombin time detection kit according to any one of claims 11 to 20.

[0034] 24. A method for stabilizing thrombin in a solution, the method comprising: adding a defoaming agent to a solution containing thrombin.

[0035] 25. The method for stabilizing according to claim 24, wherein the solution further contains at least one selected from the group consisting of a buffer, a stabilizer, and a preservative.

[0036] 26. The method for stabilizing according to claim 24 or 25, wherein the defoaming agent is an organosilicon defoaming agent.

[0037] 27. The method for stabilizing according to any one of claims 24 to 26, wherein the effective ingredient of the organosilicon defoaming agent is a polyalkylsiloxane.

[0038] 28. The method for stabilizing according to any one of claims 24 to 27, wherein the effective ingredient of the organosilicon defoaming agent is polydimethylsiloxane.

[0039] 29. The method for stabilizing according to claim 27 or 28, wherein the concentration of the effective ingredient of the defoaming agent in the solution containing thrombin is 0.1 to 10 ppm.

[0040] 30. The method for stabilizing according to any one of claims 27 to 29, wherein the concentration of the effective ingredient of the defoaming agent in the solution containing thrombin is 1 to 10 ppm.

[0041] 31. The method for stabilizing according to any one of claims 27 to 30, wherein the concentration of the effective ingredient of the defoaming agent in the solution containing thrombin is 5 to 7 ppm.

[0042] 32. The method for stabilizing according to any one of claims 25 to 31, wherein the buffer is one selected from the group consisting of 5-100 mM Bis-Tris buffer, Tris buffer, MOPS buffer, and HEPES buffer.

[0043] 33. The stabilization method according to any one of claims 25 to 32, wherein the stabilizer is one or more of polyethylene glycol, bovine serum albumin (BSA), calcium chloride (CaCl2), Triton X-100, Tween, Brij-58, mannitol, fructose, glycerol, trehalose, maltose, and gelatin.

[0044] 34. Use of a silicone-based antifoaming agent as a stabilizer in a thrombin solvent.

[0045] Technical effects of the present application

[0046] The technical effects of the present application can be achieved by the technical solutions thereof.

[0047] By adding the antifoaming agent, the stability of the reagent containing thrombin is improved on the original basis, so that the thrombin in the solution can maintain stability for a long time even in a low activity condition, which is very economical and effective.

[0048] In addition, the present application is a liquid reagent, which does not need to be reconstituted, and can effectively reduce the measurement error caused by reconstitution. BRIEF DESCRIPTION OF DRAWINGS

[0049] Figure 1 : Normal plasma reaction curve in Example 3 of the present application;

[0050] Figure 2 : Reference product reaction curve in Example 3 of the present application. DETAILED DESCRIPTION

[0051] [Thrombin solution]

[0052] The present application provides a thrombin solution containing thrombin and an antifoaming agent. In the thrombin solution of the present application, thrombin is the main functional component, and fibrinogen is converted into insoluble fibrin under the action of thrombin. In the present application, by containing an antifoaming agent in the thrombin solvent, the stability of thrombin in the solvent can be improved, so that thrombin can be kept stable in the solvent for a long time. It should be noted that in the present specification, the term "solution" does not refer to the solid or semi-solid properties of a liquid (aqueous solution) frozen at 0°C or below, but refers to the properties of maintaining the flowability of the solution, for example, the properties under refrigeration (e.g. 2-8°C) when circulating as a reagent for clinical examination.

[0053] The thrombin solution of the present application 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 clearly obtaining the technical effects of the present application. Since the thrombin solution of the present application is a liquid-type reagent containing a buffer or the like, it does not need to be reconstituted with a buffer or the like at the time of use as in the case of the previous solid reagents or lyophilized reagents. The thrombin solution of the present application can be used directly without reconstitution, and thus is very convenient in use.

[0054] The thrombin solution of the present application contains the antifoaming agent, and the antifoaming agent is preferably an organosilicon antifoaming agent, and more preferably an organosilicon antifoaming agent of an emulsion type. The antifoaming agent can be used for general food and pharmaceutical products, and prevents the generation of bubbles in the solution. The organosilicon antifoaming agent is an antifoaming agent having an organosilicon (polysiloxane) skeleton. From the viewpoint of further improving the stability of thrombin, the effective component of the organosilicon antifoaming agent is preferably a polyalkylsiloxane. Specific examples of the polyalkylsiloxane can include polymethylsiloxane, polyethylsiloxane, polydimethylsiloxane, polydiethylsiloxane, polymethylethylsiloxane and the like. From the viewpoint of further providing the stability of thrombin, the effective component of the organosilicon antifoaming agent is preferably polydimethylsiloxane.

[0055] As specific examples of the antifoaming agent of the present application, for example, Antifoam B (manufactured by Sigma, A5757 or A6707) can be given, which has polydimethylsiloxane as an effective component, and the content of the effective component in the antifoaming agent is 10%, and the concentration range recommended in the product specification is 1 to 100 ppm.

[0056] In the present application, 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 further preferably 1 to 5 NIH Units / mL. When the activity range of thrombin is lower, the technical effects of the present application can be more exhibited. That is, by the technical solution of the present application, even when the activity of thrombin is low, the excellent effect of maintaining stability for a long time can be achieved.

[0057] In the present application, from the viewpoint of further improving the stability of thrombin, the concentration of the effective component of the antifoaming agent in the thrombin solution can be 0.1 to 15 ppm, preferably 0.5 to 12 ppm, preferably 0.1 to 10 ppm, more preferably 1 to 10 ppm, further preferably 3 to 7 ppm, and particularly preferably 5 to 7 ppm.

[0058] In the present application, 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 5-100 mM Bis-Tris buffer, Tris buffer, MOPS buffer, and HEPES buffer.

[0059] Preferably, the buffer is HEPES at a concentration ranging from 10 to 100 mmol / L and at a pH of 6.5 to 8.0, further preferably, the buffer is 25 to 75 mM HEPES at a pH of 7.1 to 7.5, and more further preferably, the buffer is 50 mM HEPES at a pH of 7.3.

[0060] In the present application, the stabilizer is not particularly limited, but from the viewpoint of further improving the stability of thrombin, the stabilizer is selected from one or more of polyethylene glycol, bovine serum albumin (BSA), calcium chloride (CaCl2), Triton X-100, Tween, Brij-58, mannitol, fructose, glycerol, trehalose, maltose, and gelatin.

[0061] In a preferred embodiment of the present application, the stabilizer is polyethylene glycol 4000 (PEG-4000), Tween-80, bovine serum albumin (BSA), or calcium chloride (CaCl2).

[0062] The method for producing the above-described thrombin solution is not particularly limited, and the following production method can be used. The specific procedure includes: preparing 50 mM HEPES buffer, adjusting the pH to 7.3, adding a preservative, a stabilizer, and an antifoaming agent to the buffer, mixing them well, adding thrombin, and adjusting and mixing to obtain the thrombin solution.

[0063] [Liquid thrombin time test kit]

[0064] The present application provides a liquid thrombin time test kit. The kit includes the above-described thrombin solution of the present application and a package containing the above-described thrombin solution. Specifically, the liquid thrombin time test kit of the present application contains thrombin and an antifoaming agent.

[0065] The above-described kit of the present application further contains at least one selected from a buffer, a stabilizer, and a preservative, and preferably contains a buffer, a stabilizer, and a preservative. Since the thrombin time test kit of the present application is a liquid reagent containing a buffer and the like, it does not need to be reconstituted with a buffer and the like at the time of use as in the case of the previous solid reagent or lyophilized reagent. The thrombin solution of the present application can be used directly without reconstitution, and thus is very convenient to use.

[0066] In the above-mentioned kit of the present application, the antifoaming agent is preferably an organosilicon antifoaming agent, which can be the same antifoaming agent and the same effective component as the antifoaming agent in the above-mentioned thrombin solvent, and is preferably a polyalkylsiloxane, and more preferably a polydimethylsiloxane.

[0067] In the above-mentioned kit of the present application, the activity of thrombin is not particularly limited, and is preferably 1 to 20 NIH Units / mL, more preferably 1 to 10 NIH Units / mL, and further preferably 1 to 5 NIH Units / mL. When the activity of thrombin is low, the technical effects of the present application can be more effectively exhibited. That is, even when the activity of thrombin is low, the excellent effects of maintaining stability for a long time can be achieved by the technical solution of the present application.

[0068] In the present application, from the viewpoint of further improving the stability of thrombin, the concentration of the effective component of the antifoaming agent in the thrombin solution can be 0.1 to 15 ppm, preferably 0.5 to 12 ppm, more preferably 0.1 to 10 ppm, further preferably 1 to 10 ppm, more preferably 3 to 7 ppm, and particularly preferably 5 to 7 ppm.

[0069] In the above-mentioned kit of the present application, the buffer and the stabilizer are not particularly limited, and the same buffer and the same stabilizer as those in the above-mentioned thrombin solvent can be used.

[0070] [Detection reagent]

[0071] One example of the detection reagent according to the present application is a thrombin time detection reagent, which is used to measure the thrombin time of plasma by bringing the plasma to be measured into contact with the above-mentioned thrombin time detection reagent. The above-mentioned thrombin detection reagent contains the above-mentioned thrombin solution of the present application.

[0072] One type of the above-mentioned detection reagent of the present application is the above-mentioned liquid-type thrombin time detection kit of the present application, but is not limited thereto, and can be other forms of detection reagents.

[0073] In addition, another example of the detection reagent according to the present application is a fibrinogen detection reagent, which is used to measure the amount of fibrinogen by bringing the plasma to be measured into contact with the above-mentioned detection reagent containing thrombin and an antifoaming agent, and converting fibrinogen in the plasma into fibrin to coagulate blood. The above-mentioned detection reagent containing thrombin and an antifoaming agent contains the above-mentioned thrombin solution of the present application.

[0074] One type of the above-mentioned detection reagent of the present application is a fibrinogen detection reagent kit containing the above-mentioned detection reagent, but is not limited thereto, and can be other forms of detection reagents.

[0075] [Method for measuring thrombin time of plasma]

[0076] The present invention relates to a method for measuring thrombin time of plasma, the method comprising:

[0077] contacting the plasma to be measured with the above-mentioned thrombin solution or detection reagent of the present invention, or,

[0078] contacting the plasma to be measured with thrombin and an antifoaming agent contained in the liquid-type thrombin time detection reagent kit of the present invention.

[0079] By the above-mentioned detection method, the thrombin does not need to be reconstituted but can be used directly, thus greatly facilitating the detection.

[0080] [Method for stabilizing thrombin in solution]

[0081] The present invention relates to a method for stabilizing thrombin in solution, the method comprising: adding an antifoaming agent to a solution containing thrombin. By adding an antifoaming agent to the thrombin solvent, the stability of thrombin in the solvent can be improved for a long period of time, thus enabling thrombin to remain stable in the solvent for a long period of time.

[0082] 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 does not need to be reconstituted with a buffer or the like at the time of use as in the case of the previous solid reagent or lyophilized reagent. The thrombin solution of the present invention can be used directly without reconstitution, thus being very convenient in use.

[0083] Preferably, the antifoaming agent is preferably an organosilicon antifoaming agent, which can be the same antifoaming agent and the same effective component as the antifoaming agent in the above-mentioned thrombin solvent, preferably a polyalkylsiloxane, and more preferably a polydimethylsiloxane.

[0084] The activity range of thrombin is preferably 1 to 20 NIH Units / mL, particularly preferably 1 to 10 NIH Units / mL, and further preferably 1 to 5 NIH Units / mL. When the activity range of thrombin is low, the technical effects of the present invention are more exhibited.

[0085] Preferably, the concentration of the effective component of the antifoaming agent in the solution containing thrombin can be 0.1 to 15 ppm, preferably 0.5 to 12 ppm, preferably 0.1 to 10 ppm, more preferably 1 to 10 ppm, further preferably 3 to 7 ppm, and particularly preferably 5 to 7 ppm.

[0086] The buffer and stabilizer are not particularly limited and can be used in the same conditions as the same buffer and stabilizer as those described above in the thrombin solvent.

[0087] [Use of silicone-based defoaming agent as stabilizer in thrombin solvent]

[0088] The inventors of the present application have found that the addition of a silicone-based defoaming agent to a solution containing thrombin can improve the stability of thrombin in the solvent for a long period of time, particularly in cases where the activity range of thrombin is low, and can further improve the stability of thrombin in the solvent. That is, the inventors of the present application have found a new use of a silicone-based defoaming agent, i.e., as a stabilizer in a thrombin solvent.

[0089] As described above, the silicone-based defoaming agent of the present application can stabilize thrombin in a solution. Thus, the present application is not limited to the use of thrombin time, and is obviously also useful in various blood coagulation time methods, detection reagents, kits (including detection of coagulation factors such as fibrinogen and anticoagulation factors in the detection methods and the like), detection methods of, for example, fibrinogen, and the like, which contain thrombin as a reagent constituent.

[0090] The effective component of the silicone defoaming agent is preferably a polyalkylsiloxane, and more preferably a polydimethylsiloxane.

[0091] The activity range of thrombin is preferably 1 to 20 NIH Units / mL, particularly preferably 1 to 10 NIH Units / mL, and further preferably 1 to 5 NIH Units / mL.

[0092] The concentration of the effective component of the defoaming agent in the solution containing thrombin can be 0.1 to 15 ppm, preferably 0.5 to 12 ppm, preferably 0.1 to 10 ppm, more preferably 1 to 10 ppm, further preferably 3 to 7 ppm, and particularly preferably 5 to 7 ppm.

[0093] Examples

[0094] The raw reagents or instruments used in the examples of the present application are all ordinary commercially available products and can be purchased on the market.

[0095] The reagents of the present application were detected using the CP series instruments of Shimadzu Medical Systems, and the operation steps were as follows:

[0096]

[0097] The present application will be further described below with reference to examples:

[0098] Example 1

[0099] In this example, Antifoam B (Sigma) was used as the defoaming agent of the present application and was added to the thrombin solution. The specific effective component and related results are shown in Table 1.

[0100] The thrombin time detection reagent was prepared by first preparing a 50 mM HEPES buffer, and adjusting the pH to 7.3 using sodium hydroxide. Then 30 mM calcium chloride, 0.5% Tween-80, 0.5% PEG-4000, 1% BSA, 0.5% Proclin 300, 0 ppm (Comparative Example 1) or 5 ppm (Example 1) silicone defoaming agent (i.e. Antifoam B (Sigma)) were added, and the mixture was stirred until uniform, and then diluted to the required volume. It should be noted that the effective component content in the Antifoam B is 10%, and the 5 ppm is the concentration of the effective component in the reagent. Then thrombin was added to give an actual thrombin concentration of 2 NIH Units / mL, and the mixture was stirred until uniform to obtain the thrombin time detection reagent.

[0101] The prepared reagents were placed in a 37°C environment, and the reference values were measured on day 0 and day 7 using the CP3000 automatic blood coagulation instrument from Kyowa Medical Devices, and the second offset was compared. The comparison results of the measurement results on day 0 and day 7 are shown in Table 1. 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. 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 offset ratio is shown in the form of the percentage of the detection value deviation relative to the detection value on day 0.

[0102] Table 1 Effect of using a defoaming agent or not on stability

[0103]

[0104]

[0105] According to the offset ratio of the number of seconds, in the above (2) example, the defoaming agent was added, and therefore the number of seconds of the detection result was significantly smaller than that of the other reagents, indicating that the addition of the defoaming agent can effectively improve the stability of the reagent. However, in the (1) comparative example without adding the defoaming agent, the above technical effect does not exist.

[0106] Example 2

[0107] This example further explores the most suitable concentration of the silicone defoaming agent with the effective component of polydimethylsiloxane.

[0108] The thrombin time test reagent was prepared by first preparing 50 mM HEPES buffer, adjusting the pH to 7.3 using sodium hydroxide, then adding 30 mM calcium chloride, 0.5% Tween-80, 0.5% PEG-4000, 1% BSA, 0.5% Proclin 300, each concentration (0 ppm to 100 ppm) of antifoam agent (Antifoam B, active ingredient content 10%) shown in Table 2, and stirring until uniform, then adding thrombin to a final thrombin concentration of 2 NIH Units / mL, and stirring until uniform to obtain the thrombin time test reagent.

[0109] The prepared reagents were placed in a 37°C environment, and the reference values were measured on day 0 and day 7 using the CP3000 automatic blood coagulation instrument from Hydris, and the second offset was compared. The comparison results of the measurement results on day 0 and day 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 in Table 1.

[0110] Table 2 Effect of adding different concentrations of polydimethylsiloxane antifoam on thrombin stability

[0111]

[0112] According to the second offset, the second offset of reagents B to F with an active ingredient content of 1 ppm to 10 ppm was significantly smaller than that of reagent A without the addition of antifoam agent. It can be seen that when the active ingredient is in the range of 1 ppm to 10 ppm, a better stabilization effect can be obtained. Further, the second offset of the detection results of reagents D and E was significantly smaller than that of other reagents, indicating that when the concentration of the active ingredient is 5-7 ppm, the stabilization effect can be further improved.

[0113] Example 3

[0114] Based on the above experiment, this example further investigates whether the addition of 50 ppm antifoam agent, i.e. 5 ppm active ingredient polydimethylsiloxane, will have a significant effect on the reactivity of the reagent.

[0115] The thrombin time test reagent was prepared by first preparing 50 mM HEPES buffer, adjusting the pH to 7.3 using sodium hydroxide, and then adding 30 mM calcium chloride, 0.5% Tween-80, 0.5% PEG-4000, 1% BSA, 0.5% Proclin 300, and antifoam agent (Antifoam B, 5 ppm in Reagent B or 0 ppm (not added) in Reagent A (effective ingredient)) to the required volume after thorough mixing. Then, thrombin was added to give an actual thrombin concentration of 2 NIH Units / mL, and the thrombin time test reagent was obtained after thorough mixing.

[0116] After preparation of the thrombin time reagent A or B, the reagents were used to detect the reference and normal plasma samples on the CP3000 automatic coagulation instrument of Hydris Medical, and the reaction curves were obtained. The results of the measured thrombin time are shown in Table 3, the reference detection results row, and the normal plasma detection results row. In addition, the takeoff point of the reaction curve indicating the coagulation point of the normal plasma can be seen from Figure 1 , and the takeoff point of the reaction curve indicating the coagulation point of the reference can be seen from Figure 2 .

[0117] Table 3 Effect of the addition of polydimethylsiloxane antifoam agent on reactivity

[0118] Reagent A B Antifoam No addition Addition Reference sample detection result (sec) 19.9 19.7 Normal plasma detection result (sec) 16.8 17.1

[0119] From the comparison results of Table 3 above and Figure 1 and Figure 2 , it can be seen that the addition of the antifoam agent has no significant effect on the detection results of the samples. First, from Table 3, it can be seen that the results of Reagent A and Reagent B in the reference detection results row and the results of Reagent A and Reagent B in the normal plasma detection results row are similar, and whether the antifoam agent is added or not, it has no substantial effect on the detection results in seconds. Second, in Figure 1 and Figure 2 , in terms of being able to confirm the time of the coagulation reaction (takeoff point) and the scattered light intensity at the saturation of the coagulation reaction from the change in scattered light intensity, Reagent A and Reagent B are similar, which is also the same in the normal plasma ( Figure 1 ) and the reference ( Figure 2 ).

[0120] From the results shown in Table 3 above, Figure 1 , Figure 2 , it can be considered that the antifoam agent of the present application has no effect on the detection results of the thrombin time test reagent.

[0121] The above merely is the preferred embodiment of the present application, it should be pointed out that, for ordinary skilled in the art, without departing from the principles of the present application, can also make a number of improvements and refinements, these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A thrombin solution comprising thrombin and a defoaming agent, the defoaming agent is a silicone defoaming agent, and an effective component of the silicone defoaming agent is polydimethylsiloxane, and the concentration of the polydimethylsiloxane in the thrombin solution is 1 to 10 ppm. 2.The thrombin solution according to claim 1, further comprising at least one selected from a buffer, a stabilizer, and a preservative.

3. The thrombin solution according to claim 1 or 2, wherein, the concentration of the effective component of the defoaming agent in the thrombin solution is 5 to 7 ppm.

4. The thrombin solution of claim 2, wherein, the buffer is one selected from 5-100 mM Bis-Tris buffer, Tris buffer, MOPS buffer, and HEPES buffer.

5. The thrombin solution of claim 2, wherein, the stabilizer is one or more selected from polyethylene glycol, bovine serum albumin (BSA), calcium chloride (CaCl 2), Triton X-100, Tween, Brij-58, mannitol, fructose, glycerol, trehalose, maltose, and gelatin. 6.A liquid thrombin time test kit comprising thrombin and a defoaming agent, the defoaming agent is a silicone defoaming agent, and an effective component of the silicone defoaming agent is polydimethylsiloxane, and the concentration of the polydimethylsiloxane in the liquid thrombin time test kit is 1 to 10 ppm.

8. The liquid thromboplastin time test kit according to claim 6 or 7, wherein, 7.The liquid thrombin time test kit according to claim 6, further comprising at least one selected from a buffer, a stabilizer, and a preservative.

9. The liquid thromboplastin time test kit according to claim 7, wherein, the concentration of the effective component of the defoaming agent in the liquid thrombin time test kit is 5 to 7 ppm.

10. The liquid thromboplastin time test kit according to claim 7, wherein, the buffer is one selected from 5-100 mM Bis-Tris buffer, Tris buffer, MOPS buffer, and HEPES buffer. the stabilizer is one or more selected from polyethylene glycol, bovine serum albumin (BSA), calcium chloride (CaCl 2), Triton X-100, Tween, Brij-58, mannitol, fructose, glycerol, trehalose, maltose, and gelatin. 11.A test reagent comprising the thrombin solution according to any one of claims 1 to 5. 12.The test reagent according to claim 11, which is a thrombin time test reagent. 13.A method for measuring the thrombin time of plasma, the method comprising: contacting the plasma to be measured with the thrombin solution according to any one of claims 1 to 5 or the test reagent according to claim 11 or 12, or contacting the plasma to be measured with the thrombin and the defoaming agent contained in the liquid thrombin time test kit according to any one of claims 6 to 10.

14. A method of stabilizing thrombin in solution, the method comprising: A defoaming agent is added to a solution containing thrombin, the defoaming agent being a silicone defoaming agent, an effective component of the silicone defoaming agent being polydimethylsiloxane, the concentration of the polydimethylsiloxane in the solution containing thrombin being 1 to 10 ppm.

15. The method of stabilizing according to claim 14, wherein, The solution further contains at least one selected from a buffer, a stabilizer, and a preservative.

16. The method of stabilizing according to claim 14 or 15, wherein, The concentration of the effective component of the defoaming agent in the solution containing thrombin is 5 to 7 ppm.

17. The method of stabilizing according to claim 15, wherein, The buffer is one selected from 5-100 mM Bis-Tris buffer, Tris buffer, MOPS buffer, and HEPES buffer.

18. The method of stabilizing according to claim 15, wherein, The stabilizer is one or more selected from polyethylene glycol, bovine serum albumin (BSA), calcium chloride (CaCl2), Triton X-100, Tween, Brij-58, mannitol, fructose, glycerol, trehalose, maltose, and gelatin.

19. Use of a silicone defoaming agent as a stabilizing agent in a thrombin solvent, wherein, an effective component of the silicone defoaming agent is polydimethylsiloxane, the concentration of the polydimethylsiloxane in the thrombin solvent being 1 to 10 ppm.

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