A prothrombin time detection reagent and its preparation method

Abstract

The present invention discloses a prothrombin time detection reagent and a preparation method thereof. Among them, the prothrombin time detection reagent is prepared from rabbit brain powder, a solvent, sodium chloride, a surfactant, a metal salt, a reducing agent, and calcium chloride. It can effectively remove coagulation factor VII, so that the prepared thromboplastin does not contain coagulation factor VII, making the prepared prothrombin time detection reagent highly sensitive to coagulation factor VII, ensuring the detection accuracy and improving its actual use effect.

Description

Technical Field

[0001] The present invention belongs to the technical field of prothrombin time detection, and particularly relates to a prothrombin time detection reagent and a preparation method thereof. Background Art

[0002] Thromboplastin can be used to diagnose coagulation abnormalities, and the test used is called the prothrombin time test. In the presence of calcium, by adding the patient's plasma to tissue thromboplastin and measuring the time required for the formation of a clot, this time is the prothrombin time (PT). The main component in the prothrombin time detection reagent is thromboplastin. In the prior art, when using prothrombin time detection reagents from different manufacturers to test the same test material, there are significant differences in their sensitivity to individual coagulation factors, especially the sensitivity to factor VII, and the detection results are not comparable, which affects the practical application. Research has found that the sensitivity difference is mainly due to the fact that the preparation still contains trace amounts of coagulation factor VII. How to prepare a prothrombin time detection reagent that does not contain coagulation factor VII and improve its actual use effect is an urgent technical problem in this field. Summary of the Invention

[0003] In order to overcome the deficiencies of the prior art, the purpose of the present invention is to provide a prothrombin time detection reagent, which can effectively remove coagulation factor VII, so that the prepared thromboplastin does not contain coagulation factor VII, making the prepared prothrombin time detection reagent highly sensitive to coagulation factor VII, ensuring the detection accuracy and improving its actual use effect.

[0004] To solve the above problems, the technical solutions adopted by the present invention are as follows:

[0005] A prothrombin time detection reagent is prepared from rabbit brain powder, a solvent, sodium chloride, a surfactant, a metal salt, a reducing agent, and calcium chloride.

[0006] Preferably, the solvent is deionized water.

[0007] Preferably, compared with the volume of the solvent, the dosage of the rabbit brain powder is 45 - 65 g / L.

[0008] Preferably, compared with the volume of the solvent, the dosage of the sodium chloride is 8 - 10 g / L.

[0009] Preferably, the surfactant is Tween 20, Tween 80, or Triton X - 100, and compared with the volume of the solvent, its dosage is 0.1 - 1% V / V.

[0010] Preferably, the metal salt is manganese chloride, copper chloride, or magnesium sulfate, and compared with the volume of the solvent, its dosage is 0.01 - 8 mmol / L.

[0011] Preferably, the reducing agent is dithiothreitol, N-acetylcysteine or ascorbic acid, and its dosage is 0.5-6 g / L relative to the volume of the solvent.

[0012] Preferably, the dosage of calcium chloride is 4-6 g / L relative to the volume of the solvent.

[0013] The present invention also includes a preparation method of the prothrombin time detection reagent described in any one of the above, comprising the steps:

[0014] S101. Preparation of the extract: Add rabbit brain powder, sodium chloride, surfactant, metal salt and reducing agent into the solvent and mix well, and extract at a constant temperature for 35-40 min under stirring conditions to obtain the extract;

[0015] S102. Add calcium chloride to the extract and mix well until the calcium chloride is completely dissolved to obtain the product.

[0016] Further, in the step S101, the constant temperature condition is specifically 40-45 °C.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention can effectively remove coagulation factor VII, so that the prepared thromboplastin does not contain coagulation factor VII, and the prepared prothrombin time detection reagent has sufficient and determinable sensitivity to coagulation factor VII, ensuring the stability and comparability of the detection and improving its actual use effect; in the present invention, metal ions and reducing agents are effective inhibitors of coagulation factor VII, which can inactivate coagulation factor VII during the extraction process, so as to achieve the purpose of removing coagulation factor VII, while the surfactant can effectively protect thromboplastin, so that there is no loss of thromboplastin when removing coagulation factor VII; the preparation method in the present invention not only improves the sensitivity of thromboplastin to coagulation factor VII, but also does not affect the detection of PT within the normal range. Specific embodiments

[0018] In order to enable those skilled in the art to better understand the technical solutions in the embodiments of the present invention and make the above-mentioned objects, features and advantages of the embodiments of the present invention more obvious and understandable, the technical solutions in the present invention will be further described in detail below with reference to the embodiments.

[0019] The present invention includes a prothrombin time detection reagent, which is prepared from rabbit brain powder, solvent, sodium chloride, surfactant, metal salt, reducing agent and calcium chloride.

[0020] Specifically, the surfactant is Tween 20, Tween 80 or Triton X-100; the metal salt is manganese chloride, copper chloride or magnesium sulfate; the reducing agent is dithiothreitol, N-acetylcysteine or ascorbic acid. Compared with the volume of the solvent, the dosage of the rabbit brain powder is 45 - 65 g / L, the dosage of sodium chloride is 8 - 10 g / L, the dosage of the surfactant is 0.1 - 1% V / V, the dosage of the metal salt is 0.01 - 8 mmol / L, the dosage of the reducing agent is 0.5 - 6 g / L, and the dosage of calcium chloride is 4 - 6 g / L.

[0021] The present invention also includes a preparation method of the prothrombin time detection reagent according to any one of the above, comprising the steps:

[0022] S101. Preparation of the extract: Add rabbit brain powder, sodium chloride, surfactant, metal salt and reducing agent into the solvent and mix well, and perform constant-temperature extraction for 35 - 40 min under stirring conditions to obtain the extract;

[0023] S102. Add calcium chloride to the extract and mix well until the calcium chloride is completely dissolved to obtain the product.

[0024] Further, in the step S101, the stirring rate is 500 - 800 rpm / min, and the constant-temperature condition is specifically 40 - 45 °C.

[0025] Example 1

[0026] Prepare the prothrombin time detection reagent J-1

[0027] Table 1 Prothrombin time detection reagent formulation table

[0028] Component Name Dosage Rabbit brain powder Rabbit brain powder 50g Solvent Deionized water 1L Sodium chloride Sodium chloride 9g Surfactant Triton X-100 5ml Metal salt Copper chloride 2 mmol Reducing agent Ascorbic acid 3g Calcium chloride Calcium chloride 5g

[0029] According to the content of each component in Table 1 above, add rabbit brain powder, sodium chloride, surfactant, metal salt and reducing agent into the solvent and mix well. Under the condition of a stirring rate of 600 rpm / min, perform constant-temperature extraction at 42 °C for 38 min. After obtaining the extract, add calcium chloride to the extract and mix well until the calcium chloride is completely dissolved to obtain the prothrombin time detection reagent J-1.

[0030] Example 2

[0031] Prepare the prothrombin time detection reagent J-2

[0032] Table 2 Prothrombin time detection reagent formulation table

[0033] Component Name Dosage Rabbit brain powder Rabbit brain powder 65g Solvent Deionized water 1L Sodium chloride Sodium chloride 9g Surfactant Tween 80 8ml Metal salt Magnesium sulfate 3 mmol Reducing agent Ascorbic acid 3g Calcium chloride Calcium chloride 5g

[0034] According to the content of each component in Table 2 above, add rabbit brain powder, sodium chloride, surfactant, metal salt and reducing agent into the solvent and mix well. Under the condition of a stirring rate of 600 rpm / min, extract at a constant temperature of 40 °C for 40 min. After obtaining the extract, add calcium chloride to the extract and mix well until the calcium chloride is completely dissolved, then the prothrombin time detection reagent J-2 is obtained.

[0035] Example 3

[0036] Prepare prothrombin time detection reagent J-3

[0037] Table 3 Prothrombin Time Detection Reagent Formulation Table

[0038] Component Name Dosage Rabbit brain powder Rabbit brain powder 45g Solvent Deionized water 1L Sodium chloride Sodium chloride 9g Surfactant Tween 20 8ml Metal salt Manganese chloride 2.5 mmol Reducing agent Dithiothreitol 2g Calcium chloride Calcium chloride 5g

[0039] According to the content of each component in Table 3 above, add rabbit brain powder, sodium chloride, surfactant, metal salt and reducing agent into the solvent and mix well. Under the condition of a stirring rate of 700 rpm / min, extract at a constant temperature of 44 °C for 37 min. After obtaining the extract, add calcium chloride to the extract and mix well until the calcium chloride is completely dissolved, then the prothrombin time detection reagent J-3 is obtained.

[0040] Example 4

[0041] Prepare prothrombin time detection reagent J-4

[0042] Table 4 Prothrombin Time Detection Reagent Formulation Table

[0043] Component Name Dosage Rabbit brain powder Rabbit brain powder 45g Solvent Deionized water 1L Sodium chloride Sodium chloride 8g Surfactant Tween 20 6ml Metal salt Magnesium sulfate 0.05 mmol Reducing agent N-acetylcysteine 0.5g Calcium chloride Calcium chloride 4g

[0044] According to the content of each component in Table 4 above, add rabbit brain powder, sodium chloride, surfactant, metal salt and reducing agent into the solvent and mix well. Under the condition of a stirring rate of 700 rpm / min, extract at a constant temperature of 43 °C for 38 min. After obtaining the extract, add calcium chloride to the extract and mix well until the calcium chloride is completely dissolved, then the prothrombin time detection reagent J-4 is obtained.

[0045] Example 5

[0046] Prepare prothrombin time detection reagent J-5

[0047] Table 5 Prothrombin Time Detection Reagent Formulation Table

[0048]

[0049] According to the content of each component in Table 5 above, add rabbit brain powder, sodium chloride, surfactant, metal salt and reducing agent into the solvent and mix well. Under the condition of a stirring rate of 800 rpm / min, extract at a constant temperature of 45 °C for 35 min. After obtaining the extract, add calcium chloride to the extract and mix well until the calcium chloride is completely dissolved, then the prothrombin time detection reagent J-5 is obtained.

[0050] Experiment 1

[0051] Detection of the reagents prepared in Examples 1-5

[0052] 1. Collect 8 normal samples and 4 samples with prolonged PT time, and number them. Among them, samples No. 1-8 are normal samples, and samples No. 9-12 are samples with prolonged PT time;

[0053] 2. Use the reagents prepared in the above examples (the reagents prepared in Examples 1-5), control group A (well-known brand 1) and control group B (well-known brand 2) reagents to detect the samples on a Sysmex CS-1600 type automatic blood coagulation analyzer. The test results are shown in Table 8 below.

[0054] Table 8 Detection data table

[0055]

[0056] Through the test results in Table 8, the detection results of the reagent of the present invention are consistent with those of the control prothrombin reagent, and samples with abnormal PT time can be normally distinguished.

[0057] Experiment 2

[0058] Comparison of the PT values of the reagent of the present invention with those of other manufacturers

[0059] Use the reagents of the experimental group (the reagents prepared in Examples 1-5), control group A (well-known brand 1) and control group B (well-known brand 2) to detect the PT values of samples lacking factor VII respectively. The detection results are shown in Table 9.

[0060] Table 9 Reagent comparison data table

[0061]

[0062] The samples lacking factor VII are purchased on the market. According to Table 9 above, it can be seen that the reagent prepared by the present invention shows a longer sample coagulation time in the PT detection results compared with the products of the other two companies, that is, the reagent prepared by the present invention has higher sensitivity to samples lacking factor VII.

[0063] For those skilled in the art, various corresponding changes and deformations can be made according to the technical solutions and concepts described above, and all these changes and deformations should fall within the protection scope of the claims of the present invention.

Claims

1. A prothrombin time detection reagent, characterized in that, It is prepared from rabbit brain powder, solvent, sodium chloride, surfactant, metal salt, reducing agent and calcium chloride; The reducing agent is dithiothreitol, N-acetylcysteine or ascorbic acid, and its dosage is 0.5-6 g / L compared with the volume of the solvent; The metal salt is manganese chloride, copper chloride or magnesium sulfate, and its dosage is 0.01-8 mmol / L compared with the volume of the solvent; The surfactant is Tween 20, Tween 80 or Triton X-100, and its dosage is 0.1-1% V / V compared with the volume of the solvent.

2. The prothrombin time detection reagent according to claim 1, wherein The solvent is deionized water.

3. The prothrombin time detection reagent according to claim 1, characterized in that, The dosage of the rabbit brain powder is 45-65 g / L compared with the volume of the solvent.

4. The prothrombin time detection reagent according to claim 1, characterized in that, The dosage of the sodium chloride is 8-10 g / L compared with the volume of the solvent.

5. The prothrombin time detection reagent according to claim 1, characterized in that, The dosage of the calcium chloride is 4-6 g / L compared with the volume of the solvent.

6. The preparation method of the prothrombin time detection reagent according to any one of claims 1-5, comprising the steps: S101. Preparation of the extract: Add rabbit brain powder, sodium chloride, surfactant, metal salt and reducing agent into the solvent and mix well, and perform constant-temperature extraction for 35-40 min under stirring conditions to obtain the extract; S102. Add calcium chloride to the extract and mix well until the calcium chloride is completely dissolved to obtain the product.

7. The preparation method according to claim 6, characterized in that, In the step S101, the constant-temperature condition is specifically 40-45°C.

Images