A kit for antithrombin iii activity assay and its use

Abstract

The application relates to the technical field of biological medicine, and particularly discloses a kit for determining antithrombin III activity, which comprises a first reagent, a second reagent, a third reagent and a diluent; the first reagent at least comprises a first buffer, thrombin, a serine protease inhibitor and a first stabilizer; the second reagent at least comprises a second buffer, a chromogenic substrate and a second stabilizer; the third reagent at least comprises a third buffer, heparin, isopropyl alcohol and a third stabilizer; and the diluent at least comprises physiological saline and a fourth stabilizer. In the kit, the cost is low, the anti-interference capability is strong, the sensitivity is high, the stability is good, and the kit has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to a kit for determining the activity of antithrombin III and application thereof. BACKGROUND

[0002] Antithrombin III is a single-chain glycoprotein belonging to α2 globulin, and has the function of decomposing lipoprotein and inhibiting almost all active serine proteases in the coagulation system. As the most important inhibitor of active coagulation factors in blood, antithrombin III controls blood coagulation and fibrinolysis. The level of antithrombin III in blood changes depending on various diseases and symptoms. It decreases in disseminated intravascular coagulation (DIC), liver disease, nephrotic syndrome, etc. The decrease of antithrombin III in blood can cause the failure of heparin treatment. Therefore, it is important to grasp the activity of antithrombin III as an index for monitoring, pathological analysis, prognosis determination of such diseases, and heparin treatment or use of antithrombin III concentrate. SUMMARY

[0003] In view of the deficiencies of the prior art and actual needs, the present application provides a kit for determining the activity of antithrombin III and application thereof, which has low cost, strong anti-interference ability, high sensitivity, good stability, and wide application prospect.

[0004] To achieve the above purpose, the present application adopts the following technical solutions:

[0005] In a first aspect, the present application provides a kit for determining the activity of antithrombin III, comprising: a first reagent, a second reagent, a third reagent, and a diluent; the first reagent at least comprises: a first buffer, thrombin, a serine protease inhibitor, and a first stabilizer; the second reagent at least comprises: a second buffer, a chromogenic substrate, and a second stabilizer; the third reagent at least comprises: a third buffer, heparin, isopropyl alcohol, and a third stabilizer; and the diluent at least comprises: physiological saline and a fourth stabilizer.

[0006] The thrombin comprises bovine thrombin, and the concentration of the thrombin is 10-30 IU / mL.

[0007] The first reagent and the second reagent are in the form of lyophilized powder, and the third reagent and the diluent are in the form of liquid; the first reagent and the second reagent further comprise an excipient, wherein the excipient is selected from any one or several of mannitol, trehalose, polyethylene glycol, sucrose, cellulose, glycogen, sorbitol, polyvinylpyrrolidone, and dextran.

[0008] The heparin is heparin sodium, and the concentration of the heparin is 1-5 IU / mL.

[0009] The first buffer, the second buffer and / or the third buffer are selected from any one or several of Tris-HCl buffer, phosphate buffer, Tris-EDTA buffer, Mes buffer and HEPES buffer; and the concentration of the first buffer, the second buffer and / or the third buffer is 20-80 mmol / L.

[0010] The first stabilizer, the second stabilizer, the third stabilizer and / or the fourth stabilizer are selected from any one or several of bovine serum albumin and Tween-20.

[0011] The serine protease inhibitor is aprotinin, alpha 2-antiplasmin, alpha 2-macroglobulin, plasminogen activator inhibitor-1, plasminogen activator inhibitor-2, lysine analog or a combination thereof.

[0012] The first reagent, the second reagent, the third reagent and the diluent further comprise at least one of the following: a preservative.

[0013] The first reagent comprises the following components in the following final concentrations: 2-4% mannitol, 1-3% trehalose, 1-3% polyethylene glycol-6000, 0.5-1.5% bovine serum albumin, 10-30 IU / mL bovine thrombin, 1-5 IU / mL aprotinin, 20-80 mmol / L Tris-HCl buffer, and the balance being water, wherein the percentages are mass percentages of the total volume of the first reagent, and the pH value of the first reagent is 7.2-7.6; the second reagent comprises the following components in the following final concentrations: 3-5% mannitol, 0.5-1.5% trehalose, 0.5-1.5% polyethylene glycol-6000, 0.1-0.5% Tween-20, 0.03-0.05% preservative, 1-3 mmol / L chromogenic substrate, 20-80 mmol / L Tris-HCl buffer, and the balance being water, wherein the percentages are mass percentages of the total volume of the second reagent, and the pH value of the second reagent is 7.2-7.6; the third reagent comprises the following components in the following final concentrations: 1-3% isopropyl alcohol, 0.1-0.5% Tween-20, 0.03-0.05% preservative, 1-5 IU / mL heparin sodium, 20-80 mmol / L Tris-HCl buffer, and the balance being water, wherein the percentages are mass percentages of the total volume of the third reagent, and the pH value of the third reagent is 7.2-7.6; and the diluent comprises the following components in the following final concentrations: 0.9% sodium chloride, 0.5-1.5% bovine serum albumin, 0.03-0.05% preservative, and the balance being water, wherein the percentages are mass percentages of the total volume of the diluent, and the pH value of the diluent is 7.2-7.6.

[0014] In a second aspect, the present application provides the use of the aforementioned kit in the preparation of a medical device for the determination of anti-thrombin III activity.

[0015] Compared with the prior art, the present application has the following beneficial effects:

[0016] (1) The present application can reduce the interference of heparin cofactor II and improve the linear range by adding isopropyl alcohol.

[0017] (2) The present application can not only reduce the interference of heparin cofactor II, but also has a certain stabilizing effect on thrombin and can improve the linear range by adding a stabilizer.

[0018] (3) The present application can reduce the interference of serine protease and improve the linear range by adding a serine protease inhibitor.

[0019] The kit of the present application has low cost, strong anti-interference ability, high sensitivity, good stability, improved detectable range, and wide application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed in the following embodiment description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without any creative effort based on these drawings. Among them:

[0021] Figure 1 is a linear correlation diagram between the measured value of the reagent kit for anti-thrombin III activity determination of the present application embodiment 1 and the measured value of the reference reagent;

[0022] Figure 2 is a linear correlation diagram between the measured value of the reagent kit for anti-thrombin III activity determination of the present application embodiment 2 and the measured value of the reference reagent;

[0023] Figure 3 is a linear correlation diagram between the measured value of the reagent kit for anti-thrombin III activity determination of the present application embodiment 3 and the measured value of the reference reagent. DETAILED DESCRIPTION

[0024] The technical solutions in the embodiments of the present application will be described clearly and completely in the following with the drawings in the embodiments of the present application. Obviously, the described embodiments are only some embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by those skilled in the art without any creative effort belong to the scope of protection of the present application.

[0025] The present inventors found in the long-term research that anti-thrombin III not only has anti-thrombin activity, but also has anti-serine protease (such as FXa, FIXa, FXIa, FXIIa, etc. coagulation factors and fibrinolysin, trypsin, kallikrein, etc.) activity. The arginine residue on anti-thrombin III can be combined with the serine residue of the serine protease, so that the serine protease in the sample will interfere with the activity determination result of anti-thrombin III. In addition, the present inventors also found that there is heparin cofactor II with anti-thrombin activity in the sample to be tested, which leads to that the residual thrombin activity determined by applying the chromogenic substrate method is the residual activity after the reaction of thrombin with anti-thrombin III and heparin cofactor II. In patients with normal heparin cofactor II level, the lack of anti-thrombin III will be likely to be covered due to the anti-thrombin activity of heparin cofactor II. Therefore, this method is easily interfered by heparin cofactor II in the sample, leading to inaccurate determination of anti-thrombin III, especially in the sample to be tested with lack of anti-thrombin III.

[0026] To this end, the application provides a kit for antithrombin III activity assay, which is based on a chromogenic substrate method, has the characteristics of high sensitivity, good accuracy, short detection time, and can be applied to various automatic analysis instruments.

[0027] Specifically, the kit of the application comprises a first reagent, a second reagent, a third reagent, and a diluent. The first reagent at least comprises a first buffer, thrombin, a serine protease inhibitor, and a first stabilizer. The second reagent at least comprises a second buffer, a chromogenic substrate, and a second stabilizer. The third reagent at least comprises a third buffer, heparin, isopropanol, and a third stabilizer. The diluent at least comprises physiological saline and a fourth stabilizer.

[0028] The physiological saline is a 0.8% to 1% sodium chloride solution, which can be used to adjust the osmotic pressure of the first reagent, thereby improving the efficiency of the first reagent in the determination of antithrombin III activity.

[0029] The detection principle of the kit of the application is that the thrombin in the first reagent is in excess relative to the antithrombin III in the plasma to be tested, and under the catalysis of heparin in the third reagent, the thrombin in the first reagent can react with the antithrombin III in the plasma to be tested to form an inactive complex, which partially inactivates the thrombin. The remaining thrombin will act on the chromogenic substrate in the second reagent to cleave a chromogenic group. The color depth is positively correlated with the amount of remaining thrombin, and negatively correlated with the activity of antithrombin III in the plasma.

[0030] The heparin can enhance the antithrombin activity of antithrombin III and reduce the influence of heparin cofactor II on the determination of antithrombin III activity.

[0031] The serine protease inhibitor in the first reagent has serine protease inhibition activity and good inhibition specificity, which can reduce the interference of serine protease on antithrombin III.

[0032] The isopropanol in the third reagent can effectively reduce the interference of heparin cofactor II on the determination of antithrombin III activity, thereby improving the accuracy of the determination of antithrombin III activity and reducing the false positive rate of the determination of antithrombin III activity.

[0033] The various stabilizers can reduce the interference of heparin cofactor II on the determination of antithrombin III activity while stabilizing thrombin, thereby improving the accuracy of the measurement.

[0034] The various buffers are used to adjust the pH value of the corresponding reagent.

[0035] In one embodiment, the thrombin comprises bovine thrombin, and the use of bovine thrombin instead of human thrombin can reduce the influence of heparin cofactor II on the anti-thrombin III detection.

[0036] Optionally, the concentration of thrombin is 10-30 IU / mL, for example, the concentration of thrombin is 10 IU / mL, 20 IU / mL or 30 IU / mL.

[0037] In one embodiment, the heparin can be provided in the form of a salt thereof, for example, it can be heparin sodium.

[0038] Optionally, the concentration of heparin is 1-5 IU / mL, at which concentration, the reaction between thrombin and anti-thrombin III can be effectively promoted, while the heparin cofactor II is not fully catalyzed, thereby reducing the interference of heparin cofactor II. For example, the concentration of heparin is 1 IU / mL, 3 IU / mL or 5 IU / mL. In this way, the reaction between thrombin and anti-thrombin III can be effectively promoted.

[0039] In one embodiment, the first buffer and / or the second buffer and / or the third buffer is selected from any one or several of Tris-HCl buffer, phosphate buffer, Tris-EDTA buffer, Mes buffer and HEPES buffer.

[0040] Optionally, the concentration of the first buffer and / or the second buffer and / or the third buffer is 20 mmol / L, the concentration of the first buffer and / or the second buffer and / or the third buffer is 50 mmol / L, and the concentration of the first buffer and / or the second buffer and / or the third buffer is 80 mmol / L.

[0041] In one embodiment, the first stabilizer and / or the second stabilizer and / or the third stabilizer and / or the fourth stabilizer is selected from any one or several of bovine serum albumin and Tween-20.

[0042] Since thrombin is unstable or has low activity at low concentrations, the addition of bovine serum albumin can play a protective or carrier role to greatly improve the activity of thrombin. Therefore, bovine serum albumin can be selected as a stabilizer in the first reagent and the diluent used to dilute the first reagent.

[0043] Tween 20 has the effect of stabilizing the chromogenic substrate and heparin, so Tween 20 can be selected as a stabilizer in the second reagent and the third reagent.

[0044] In the process of detecting the activity of antithrombin III by chromogenic substrate method, as the thrombin substrate, it is required to have high sensitivity and good water solubility, so as to effectively improve the sensitivity of detection, and has a wide linear range, so as to ensure the accuracy of the test results. If the solubility of the chromogenic substrate is too low, the reagent is close to the saturation state, and the precipitate is easy to produce in the detection process, which leads to the inaccuracy of the determination results. Therefore, the chromogenic substrate with good solubility: H-D-Phe-Pip-Arg-pNA·2HCl (S-2238) is selected in the application.

[0045] In an embodiment, the serine protease inhibitor can be a plasmin inhibitor, which has strong plasmin inhibitory activity and good inhibitory specificity, and can significantly inhibit the fibrinolytic effect of plasmin.

[0046] Optionally, the plasmin inhibitor is aprotinin, α2-antiplasmin, α2-macroglobulin, plasminogen activator inhibitor-1, plasminogen activator inhibitor-2, lysine analogues or combinations thereof.

[0047] In an embodiment, at least one of the first reagent, the second reagent, the third reagent and the diluent further comprises a preservative. Optionally, the preservative is any one or several of benzoic acid and its salts, sodium azide, sorbic acid and its salts, gentamicin, thiomersal, Proclin-300, etc. Thus, the storage time of the thrombin aqueous solution can be further improved.

[0048] In an embodiment, the first reagent comprises the following components with final concentrations: 0.5-1.5% bovine serum albumin, 10-30 IU / mL bovine thrombin, 1-5 IU / mL aprotinin, 20-80 mmol / L Tris-HCl buffer, and the balance is water, wherein the percentage is the mass percentage of the total volume of the first reagent, and the pH value of the first reagent is 7.2-7.6.

[0049] The second reagent comprises the following components with final concentrations: 0.1-0.5% Tween-20, 0.03-0.05% preservative, 1-3 mmol / L chromogenic substrate, 20-80 mmol / L Tris-HCl buffer, and the balance is water, wherein the percentage is the mass percentage of the total volume of the second reagent, and the pH value of the second reagent is 7.2-7.6.

[0050] The third reagent includes the following components in the following final concentrations: 1-3% isopropyl alcohol, 0.1-0.5% Tween-20, 0.03-0.05% preservative, 1-5 IU / mL sodium heparin, 20-80 mmol / L Tris-HCl buffer, and the balance being water, the percentages being mass / volume percentages of the total volume of the third reagent, the pH of the third reagent being 7.2-7.6.

[0051] The diluent includes the following components in the following final concentrations: 0.9% sodium chloride, 0.5-1.5% bovine serum albumin, 0.03-0.05% preservative, and the balance being water, the percentages being mass / volume percentages of the total volume of the diluent, the pH of the diluent being 7.2-7.6.

[0052] As a further improvement on the above scheme, the first reagent and the second reagent are packaged in the reagent kit in the form of lyophilized powder, and the third reagent and the diluent are packaged in the reagent kit in the form of liquid. Thus, the first reagent and the second reagent further include an excipient, wherein the excipient is selected from any one or several of mannitol, trehalose, polyethylene glycol, sucrose, cellulose, glycogen, sorbitol, polyvinylpyrrolidone, and dextran.

[0053] In one embodiment, the polyethylene glycol used in the above first reagent and second reagent has a molecular weight of 2000-20000 (e.g., 2000, 6000, 10000, 20000), whereby the stability of the first reagent and the second reagent in the form of lyophilized powder can be further improved.

[0054] Specifically, the method for preparing the lyophilized powder of the first reagent is as follows: 1) preparing the first reagent, the first reagent including the following components in the following final concentrations: 2%-4% mannitol, 1%-3% trehalose, 1%-3% polyethylene glycol-6000, 0.5-1.5% bovine serum albumin, 10-30 IU / mL bovine thrombin, 1-5 IU / mL aprotinin, 20-80 mmol / L Tris-HCl buffer, and the balance being water, the percentages being mass / volume percentages of the total volume of the first reagent, the pH of the first reagent being 7.2-7.6; 2) subjecting the first reagent in step 1) to low-temperature pre-freezing and freeze-drying, thereby obtaining the first reagent lyophilized powder.

[0055] The preparation method of the freeze-dried powder of the second reagent is as follows: 1) preparing the second reagent, the second reagent comprising the following components at the final concentrations: 3%-5% mannitol, 0.5-1.5% trehalose, 0.5-1.5% polyethylene glycol-6000, 0.1-0.5% Tween-20, 0.03-0.05% preservative, 1-3 mmol / L chromogenic substrate, 20-80 mmol / L Tris-HCl buffer, and the balance being water, wherein the percentages are mass percentages of the total volume of the second reagent, and the pH value of the second reagent is 7.2-7.6; 2) pre-freezing and freeze-drying the second reagent in step 1) to obtain the freeze-dried powder of the second reagent.

[0056] The third reagent comprises the following components at the final concentrations: 1-3% isopropyl alcohol, 0.1-0.5% Tween-20, 0.03-0.05% preservative, 1-5 IU / mL sodium heparin, 20-80 mmol / L Tris-HCl buffer, and the balance being water, wherein the percentages are mass percentages of the total volume of the third reagent, and the pH value of the third reagent is 7.2-7.6.

[0057] The diluent comprises the following components at the final concentrations: 0.9% sodium chloride, 0.5-1.5% bovine serum albumin, 0.03-0.05% preservative, and the balance being water, wherein the percentages are mass percentages of the total volume of the diluent, and the pH value of the diluent is 7.2-7.6.

[0058] The first reagent and the second reagent in the form of freeze-dried powder are packaged in the kit, which can effectively protect the activities of thrombin, aprotinin, bovine serum albumin and chromogenic substrate, has good stability, and can effectively monitor the performance of the reagents in clinical applications. The method has the advantages of low cost, simple operation, large batch production and long-term preservation.

[0059] The application also provides an anti-thrombin III activity determination method based on the kit for anti-thrombin III activity determination, which comprises the following steps: taking a sample to be tested, diluting the sample with the diluent, and incubating at 37°C; taking the diluted sample, adding the first reagent reconstituted with the diluent, and incubating at 37°C; adding the second reagent and the third reagent reconstituted with the diluent, and incubating at 37°C; and determining the absorbance difference (△OD) at the first time point and the second time point at 405 nm by using a coagulation analyzer. The determined absorbance difference (△OD) is substituted into a standard curve (five-point calibration) to calculate the activity (%) of anti-thrombin III in the sample to be tested.

[0060] The application also provides the use of the kit in the preparation of medical devices for anti-thrombin III activity determination.

[0061] The technical solutions of the present application are described and explained further below through specific examples. If not otherwise specified, the raw materials used in the examples of the present application are all the commonly used raw materials in the field, and the methods used in the examples are all the conventional methods in the field.

[0062] Example 1

[0063] The kit for anti-thrombin III activity determination of Example 1 of the present application comprises the following components:

[0064] The first reagent, after pre-freezing and freeze-drying at low temperature, is in the form of a freeze-dried powder and is contained in the reagent kit, and the first reagent comprises the following components at the following final concentrations: 3% mannitol, 2% trehalose, 2% polyethylene glycol-6000, 1% bovine serum albumin, 20 IU / mL bovine thrombin, 3 IU / mL aprotinin, 50 mmol / L Tris-HCl buffer, and the balance being water, wherein the percentages are mass percentages of the total volume of the first reagent, and the pH value of the first reagent is 7.4.

[0065] The second reagent, after pre-freezing and freeze-drying at low temperature, is in the form of a freeze-dried powder and is contained in the reagent kit, and the second reagent comprises the following components at the following final concentrations: 4% mannitol, 1% trehalose, 1% polyethylene glycol-6000, 0.3% Tween-20, 0.04% Proclin-300, 2 mmol / L chromogenic substrate S-2238, 50 mmol / L Tris-HCl buffer, and the balance being water, wherein the percentages are mass percentages of the total volume of the second reagent, and the pH value of the second reagent is 7.4.

[0066] The third reagent comprises the following components at the following final concentrations: 2% isopropyl alcohol, 0.3% Tween-20, 0.04% Proclin-300, 3 IU / mL heparin sodium, 50 mmol / L Tris-HCl buffer, and the balance being water, wherein the percentages are mass percentages of the total volume of the third reagent, and the pH value of the third reagent is 7.4.

[0067] The diluent comprises the following components at the following final concentrations: 0.9% sodium chloride, 1% bovine serum albumin, 0.04% Proclin-300, and the balance being water, wherein the percentages are mass percentages of the total volume of the diluent, and the pH value of the diluent is 7.4.

[0068] Example 2

[0069] The kit for anti-thrombin III activity determination of Example 2 of the present application comprises the following components:

[0070] The first reagent, after low-temperature pre-freezing and freeze-drying, is in the form of a freeze-dried powder in the reagent box, and the first reagent comprises the following components at the following final concentrations: 2% mannitol, 1% trehalose, 1% polyethylene glycol-6000, 0.5% bovine serum albumin, 10 IU / mL bovine thrombin, 1 IU / mL aprotinin, 20 mmol / L Tris-HCl buffer, and the balance being water, wherein the percentages are mass percentages of the total volume of the first reagent, and the pH value of the first reagent is 7.2.

[0071] The second reagent, after low-temperature pre-freezing and freeze-drying, is in the form of a freeze-dried powder in the reagent box, and the second reagent comprises the following components at the following final concentrations: 3% mannitol, 0.5% trehalose, 0.5% polyethylene glycol-6000, 0.1% Tween-20, 0.03% Proclin-300, 1 mmol / L chromogenic substrate S-2238, 20 mmol / L Tris-HCl buffer, and the balance being water, wherein the percentages are mass percentages of the total volume of the second reagent, and the pH value of the second reagent is 7.2.

[0072] The third reagent comprises the following components at the following final concentrations: 1% isopropyl alcohol, 0.1% Tween-20, 0.03% Proclin-300, 1 IU / mL sodium heparin, 20 mmol / L Tris-HCl buffer, and the balance being water, wherein the percentages are mass percentages of the total volume of the third reagent, and the pH value of the third reagent is 7.4.

[0073] The diluent comprises the following components at the following final concentrations: 0.9% sodium chloride, 0.5% bovine serum albumin, 0.03% Proclin-300, and the balance being water, wherein the percentages are mass percentages of the total volume of the diluent, and the pH value of the diluent is 7.4.

[0074] Example 3

[0075] The kit for anti-thrombin III activity determination of Example 3 of the present application comprises the following components:

[0076] The first reagent, after low-temperature pre-freezing and freeze-drying, is in the form of a freeze-dried powder in the reagent box, and the first reagent comprises the following components at the following final concentrations: 4% mannitol, 3% trehalose, 3% polyethylene glycol-6000, 1.5% bovine serum albumin, 30 IU / mL bovine thrombin, 5 IU / mL aprotinin, 80 mmol / L Tris-HCl buffer, and the balance being water, wherein the percentages are mass percentages of the total volume of the first reagent, and the pH value of the first reagent is 7.6.

[0077] The second reagent, which is pre-frozen at low temperature and freeze-dried, is contained in the reagent box in the form of a freeze-dried powder, and the second reagent comprises the following components at the following final concentrations: 5% mannitol, 1.5% trehalose, 1.5% polyethylene glycol-6000, 0.5% Tween-20, 0.05% Proclin-300, 3 mmol / L chromogenic substrate S-2238, 80 mmol / L Tris-HCl buffer, and the balance being water, wherein the percentages are mass percentages of the total volume of the second reagent, and the pH value of the second reagent is 7.4.

[0078] The third reagent comprises the following components at the following final concentrations: 3% isopropanol, 0.5% Tween-20, 0.05% Proclin-300, 5 IU / mL sodium heparin, 80 mmol / L Tris-HCl buffer, and the balance being water, wherein the percentages are mass percentages of the total volume of the third reagent, and the pH value of the third reagent is 7.6.

[0079] The diluent comprises the following components at the following final concentrations: 0.9% sodium chloride, 1.5% bovine serum albumin, 0.05% Proclin-300, and the balance being water, wherein the percentages are mass percentages of the total volume of the diluent, and the pH value of the diluent is 7.6.

[0080] Comparative Example 1

[0081] Comparative Example 1 The first reagent of Comparative Example 1 differs from Example 1 in that the first reagent of Comparative Example 1 does not contain aprotinin, and the second reagent, the third reagent, and the diluent are all the same as in Example 1.

[0082] Comparative Example 2

[0083] Comparative Example 2 The first reagent and the diluent of Comparative Example 2 differ from Example 1 in that the first reagent and the diluent of Comparative Example 2 do not contain bovine serum albumin, and the second reagent and the third reagent are all the same as in Example 1.

[0084] Comparative Example 3

[0085] Comparative Example 3 The second reagent and the third reagent of Comparative Example 3 differ from Example 1 in that the second reagent and the third reagent of Comparative Example 3 do not contain Tween-20, and the first reagent and the diluent are all the same as in Example 1.

[0086] Comparative Example 4

[0087] Comparative Example 4 The third reagent of Comparative Example 4 differs from Example 1 in that the third reagent of Comparative Example 4 does not contain isopropanol, and the first reagent, the second reagent, and the diluent are all the same as in Example 1.

[0088] Comparative Example 5

[0089] Comparative Example 5 The third reagent of Comparative Example 5 differs from that of Example 1 in that the concentration of heparin sodium in the third reagent of Comparative Example 4 is 10 IU / mL, and the first reagent, the second reagent and the diluent are the same as in Example 1.

[0090] Comparative Example 6

[0091] Comparative Example 6 The first reagent of Comparative Example 6 differs from that of Example 1 in that the first reagent of Comparative Example 4 contains 20 IU / mL of human thrombin and does not contain bovine thrombin, and the second reagent, the third reagent and the diluent are the same as in Example 1.

[0092] Example 4

[0093] The anti-thrombin III assay kits of Examples 1 to 3 and Comparative Examples 1 to 4 were subjected to accuracy, blank limit, minimum detection limit, linear range, repeatability, stability, anti-heparin cofactor II interference ability and correlation tests.

[0094] (1) Accuracy test and its results

[0095] According to the aforementioned anti-thrombin III activity assay method, the anti-thrombin III activity (%) of WHO International Standard (NIBSC code: 08 / 258d) was measured using the anti-thrombin III assay kits of Examples 1 to 3 and Comparative Examples 1 to 6, the test was repeated 3 times, the relative deviation was calculated according to the following Formulas (1) and (2), and the test results are shown in Table 1. The test results should meet the requirements: the relative deviation should be within ±8%.

[0096] Formula (1): Formula (2):

[0097] In the formula:

[0098] x i is the measured value (%) of anti-thrombin III activity for each test;

[0099] is the average value of the measured values;

[0100] n is the number of tests;

[0101] i is the serial number of the test;

[0102] B is the relative deviation;

[0103] T is the labeled value of the WHO International Standard.

[0104] Table 1 Accuracy test results of the anti-thrombin III assay kits of Examples 1 to 3 and Comparative Examples 1 to 6

[0105]

[0106] From the test results of Table 1, it is found that the relative deviations of Examples 1-3 are low, proving that Examples 1-3 all have good accuracy. The relative deviations of Comparative Examples 1-6 are not within the range of ±8%, Comparative Example 1 is interfered by the serine protease in the sample to be measured, Comparative Examples 2-3 do not contain stabilizers, resulting in the decrease of the activities of thrombin, chromogenic substrate and heparin, and Comparative Examples 4-6 are interfered by the heparin cofactor II in the sample to be measured, thus the accuracy of Comparative Examples 1-6 is not as good as that of Examples 1-3.

[0107] (2) Test of blank limit and its results

[0108] According to the aforementioned method for determining the activity of antithrombin III, the antithrombin III determination kit of Examples 1-3 was used to determine the blank sample for 20 times, and the average value was calculated according to the aforementioned formula (1) and formula (3) standard deviation (SD) and blank limit The test results are shown in Table 2. The test results should meet the requirements: the blank limit should be ≤5%.

[0109] Formula (3): In the formula, SD is the standard deviation.

[0110] Table 2 Test results of blank limit of antithrombin III determination kit of Examples 1-3

[0111]

[0112] From the test results of Table 2, it is found that Examples 1-3 all meet the requirement of blank limit ≤5%.

[0113] (3) Test of minimum detection limit and its results

[0114] According to the aforementioned method for determining the activity of antithrombin III, the antithrombin III determination kit of Examples 1-3 was used to detect 5 low-value samples with a concentration of approximately 8%, each sample was detected for 5 times, and the measured value x i was sorted in size, and the test results are shown in Table 3. The test results should meet: ① the measured value x i should be ≤5%; ② the measured value x i should all be ≤80%.

[0115] Table 3 Test results of minimum detection limit of antithrombin III determination kit of Examples 1-3

[0116]

[0117] The test results in Table 3 show that Examples 1-3 all meet the minimum detection limit requirements mentioned above.

[0118] (4) Linear range test and its results

[0119] The high-concentration sample, close to the upper limit of the linear range, was diluted into six samples of different concentrations. Following the aforementioned method for determining antithrombin III activity, each concentration was measured three times using the antithrombin III assay kits described in Examples 1-3. The average of the three test results was calculated. A linear regression was performed with the theoretical value (xi) of the sample as the independent variable and the average value (yi) of the actual measured results of the sample as the dependent variable. The linear regression correlation coefficient r was calculated according to formula (4). The test results are shown in Table 4. The test results should meet the following requirements: the linear range should be within 10% to 170%, and the linear correlation coefficient r should be ≥ 0.98.

[0120] Formula (4)

[0121] Table 4. Linear range test results of the antithrombin III assay kits in Examples 1-3.

[0122]

[0123] The test results in Table 4 show that Examples 1-3 all meet the above-mentioned linear range requirements.

[0124] (5) Repeatability tests and their results

[0125] Following the aforementioned method for determining antithrombin III activity, the antithrombin III assay kits described in Examples 1-3 were used to measure samples with high concentrations (80-120%), medium concentrations (30-50%), and low concentrations (<30%). Each test was performed 10 times, and the average value of the test results was calculated according to formulas (1) and (3). The coefficient of variation (CV) was calculated using formula (5) and standard deviation (SD). The test results are shown in Table 5. The test results should meet the following requirements: the CV for high-concentration samples should be ≤6%, the CV for medium-concentration samples should be ≤6%, and the CV for low-concentration samples should be ≤8%.

[0126] Formula (5): In the formula: CV is the coefficient of variation.

[0127] Table 5. Repeatability test results of the antithrombin III assay kits in Examples 1-3

[0128]

[0129] The test results in Table 4 show that Examples 1-3 all meet the above repeatability requirements.

[0130] (6) Stability test and its results

[0131] ①Reconstitution stability: The first reagent and the second reagent in the antithrombin III assay kit of Example 1-3 and Comparative Example 2-3 were reconstituted with diluent and stored at 2-8°C. On day 0, day 10, day 20, day 25, day 30, day 33, day 35, the reconstituted antithrombin III assay kit was used to perform accuracy test, and the test results are shown in Table 6. The test results should meet the requirements: the first reagent and the second reagent can be stable for 30 days after reconstitution and storage at 2-8°C, i.e. the relative deviation should be within ±8%.

[0132] Table 6 Reconstitution stability test results of the antithrombin III assay kit of Example 1-3 and Comparative Example 2-3

[0133]

[0134]

[0135] From the test results in Table 6, it is found that Example 1-3 meets the above reconstitution stability requirements, and Comparative Example 2-3 without adding a stabilizer does not meet the above reconstitution stability requirements. Compared with Comparative Example 2-3, the kit of Example 1-3 has higher reconstitution stability.

[0136] ②Accelerated stability: The antithrombin III assay kit of Example 1-3 and Comparative Example 2-3 was stored at 37°C in an unopened state. On day 0, day 8, day 12, day 16, day 20, day 22, day 24, the reconstituted antithrombin III assay kit was used to perform accuracy test, and the test results are shown in Table 7. The test results should meet the requirements: the unopened state can be stable for 20 days at 37°C, i.e. the relative deviation should be within ±8%.

[0137] Table 7 Accelerated stability test results of the antithrombin III assay kit of Example 1-3 and Comparative Example 2-3

[0138]

[0139] From the test results in Table 7, it is found that Example 1-3 meets the above accelerated stability requirements, and Comparative Example 2-3 without adding a stabilizer does not meet the above accelerated stability requirements. Compared with Comparative Example 2-3, the kit of Example 1-3 has higher accelerated stability.

[0140] (7) Anti-heparin cofactor II interference ability test and its results

[0141] The anti-thrombin III assay reagent kits of Examples 1-3 and Comparative Examples 4-6 were used to determine the plasma samples to be tested with heparin cofactor II concentration of 0 U / mL, 1 U / mL, 2 U / mL, 4 U / mL, 6 U / mL according to the aforementioned anti-thrombin III activity determination method, and the test results are shown in Table 8. The test results should meet the requirements: the relative deviation of the test results of the plasma samples to be tested with heparin cofactor II concentration less than or equal to 6 U / ml should be within ±8%.

[0142] Table 7 Test results of anti-heparin cofactor II interference ability of anti-thrombin III assay reagent kits of Examples 1-3 and Comparative Examples 4-6

[0143]

[0144] From the test results in Table 7, it is found that Examples 1-3 have anti-heparin cofactor II interference ability, as the relative deviation of the test results of the plasma samples to be tested with heparin cofactor II concentration less than or equal to 6 U / ml is within ±8%; while Comparative Examples 4-6 are interfered by heparin cofactor II in the samples to be tested, as the relative deviation of the test results of the plasma samples to be tested with heparin cofactor II concentration greater than 1 U / ml and less than or equal to 6 U / ml is not within ±8%.

[0145] (8) Correlation test and its results

[0146] A group of clinical samples (40 cases) covering the linear range were simultaneously tested by using the reference kit (anti-thrombin III (AT-III) assay reagent kit (chromogenic substrate method), DIAGNOSTICA STAGO, national medical instrument injection 20162405280) and the anti-thrombin III assay reagent kits of Examples 1-3, with the measured value of the reference kit as the x-axis and the measured value of the reagent kits of Examples 1-3 as the y-axis, linear regression analysis was performed, and the test results are shown in Table 8 and Figures 1-3. The test results should meet the requirements: the linear regression analysis should meet the slope k of the linear regression equation between 0.9 and 1.1 and the correlation coefficient r≥0.975.

[0147] Table 8 Correlation test results of Examples 1-3

[0148]

[0149]

[0150] From the test results of Table 8, the results of testing clinical samples by the kits of Examples 1-3 and the reference kit are subjected to linear regression analysis, the slope k and correlation r of the linear regression equation both meet the requirements, proving that the kits of Examples 1-3 and the reference kit have good correlation.

[0151] In summary, the present application has the following advantages:

[0152] (1) The present application can reduce the interference of heparin cofactor II and improve the linear range by adding isopropyl alcohol.

[0153] (2) The present application can not only reduce the interference of heparin cofactor II, but also has a certain stabilizing effect on thrombin and can improve the linear range by adding a stabilizer.

[0154] (3) The present application can reduce the interference of serine protease and improve the linear range by adding a serine protease inhibitor.

[0155] The kit of the present application has low cost, strong anti-interference ability, high sensitivity, good stability, improved detectable range, and wide application prospect.

[0156] In addition, the technical scope of the present application is not exhausted at the point value, and the new technical solutions formed by the same or multiple technical features in the embodiment technical solutions are also within the scope of the present application; at the same time, all the enumerated or unenumerated embodiments of the present application scheme, the parameters in the same embodiment only represent one example (i.e. a feasible scheme) of the technical solution.

[0157] The specific embodiments described herein are merely illustrative of the present application. Those skilled in the art can make various modifications or supplements to the described specific embodiments or replace them with similar ways, without departing from the spirit of the present application or exceeding the scope defined by the appended claims.

Claims

1. A kit for the determination of antithrombin III activity, characterized in that, The kit comprises: a first reagent, a second reagent, a third reagent, and a diluent; the first reagent at least comprises: a first buffer, thrombin, a serine protease inhibitor, and a first stabilizer; the second reagent at least comprises: a second buffer, a chromogenic substrate, and a second stabilizer; the third reagent at least comprises: a third buffer, heparin, isopropanol, and a third stabilizer; the diluent at least comprises: physiological saline and a fourth stabilizer; the thrombin comprises bovine thrombin; the first stabilizer comprises bovine serum albumin, the second stabilizer comprises Tween-20, the third stabilizer comprises Tween-20, and the fourth stabilizer comprises bovine serum albumin; the concentration of the thrombin is 10-30 IU / mL, the concentration of the serine protease inhibitor is 1-5 IU / mL, the concentration of the heparin is 1-5 IU / mL, and the concentration of the isopropanol is 1-3%.

2. The kit of claim 1, wherein The first reagent and the second reagent are in the form of lyophilized powder, and the third reagent and the diluent are in the form of liquid; the first reagent and the second reagent further comprise an excipient, wherein the excipient is selected from any one or several of mannitol, trehalose, polyethylene glycol, sucrose, cellulose, glycogen, sorbitol, polyvinylpyrrolidone, and dextran.

3. The kit of claim 1, wherein The heparin is sodium heparin.

4. The kit of claim 1, wherein the first buffer and / or the second buffer and / or the third buffer is selected from any one or several of Tris-HCl buffer, phosphate buffer, Tris-EDTA buffer, Mes buffer, and HEPES buffer; the concentration of the first buffer and / or the second buffer and / or the third buffer is 20-80 mmol / L.

5. The kit of claim 1, wherein The serine protease inhibitor is aprotinin, α2-antiplasmin, α2-macroglobulin, plasminogen activator inhibitor-1, plasminogen activator inhibitor-2, a lysine analogue, or a combination thereof.

6. The kit of claim 1, wherein At least one of the first reagent, the second reagent, the third reagent, and the diluent further comprises a preservative.

7. The kit of claim 1, wherein the first reagent comprises the following components at the final concentrations: 2%-4% mannitol, 1%-3% trehalose, 1%-3% polyethylene glycol-6000, 0.5-1.5% bovine serum albumin, 10-30 IU / mL bovine thrombin, 1-5 IU / mL aprotinin, 20-80 mmol / L Tris-HCl buffer, and the balance is water, wherein the percentages are mass percentages of the total volume of the first reagent, and the pH value of the first reagent is 7.2-7.

6. The second reagent comprises the following components at the following final concentrations: 3-5% mannitol, 0.5-1.5% trehalose, 0.5-1.5% polyethylene glycol-6000, 0.1-0.5% Tween-20, 0.03-0.05% preservative, 1-3 mmol / L chromogenic substrate, 20-80 mmol / L Tris-HCl buffer, and the balance being water, wherein the percentages are mass / volume percentages of the total volume of the second reagent, and the pH of the second reagent is 7.2-7.6; The third reagent comprises the following components at the following final concentrations: 1-3% isopropyl alcohol, 0.1-0.5% Tween-20, 0.03-0.05% preservative, 1-5 IU / mL heparin sodium, 20-80 mmol / L Tris-HCl buffer, and the balance being water, wherein the percentages are mass / volume percentages of the total volume of the third reagent, and the pH of the third reagent is 7.2-7.6; The diluent comprises the following components at the following final concentrations: 0.9% sodium chloride, 0.5-1.5% bovine serum albumin, 0.03-0.05% preservative, and the balance being water, wherein the percentages are mass / volume percentages of the total volume of the diluent, and the pH of the diluent is 7.2-7.

6.

8. Use of the kit according to any one of claims 1-7 in the preparation of a medical device for the determination of antithrombin III activity.

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