Disclosure of Invention
In order to overcome the defects in the prior art, the invention aims to provide a prothrombin time detection reagent which can effectively remove the prothrombin VII, so that the prepared thromboplastin does not contain the prothrombin VII, the prepared prothrombin time detection reagent has high sensitivity to the prothrombin VII, the detection accuracy is ensured, and the actual use effect of the prothrombin time detection reagent is improved.
In order to solve the problems, the technical scheme adopted by the invention is as follows:
A prothrombin time detection reagent is prepared from rabbit brain powder, a solvent, sodium chloride, a surfactant, metal salt, a reducing agent and calcium chloride.
Preferably, the solvent is deionized water.
Preferably, the amount of said rabbit brain powder is 45-65g/L compared to the volume of the solvent.
Preferably, the sodium chloride is used in an amount of 8-10g/L compared to the volume of the solvent.
Preferably, the surfactant is Tween 20, tween 80 or Triton X-100, and the amount of the surfactant is 0.1-1%V/V compared with the volume of the solvent.
Preferably, the metal salt is manganese chloride, copper chloride or magnesium sulfate, and the dosage of the metal salt is 0.01-8mmol/L compared with the volume of the solvent.
Preferably, the reducing agent is dithiothreitol, N-acetylcysteine or ascorbic acid, in an amount of 0.5-6g/L compared to the volume of the solvent.
Preferably, the calcium chloride is used in an amount of 4-6g/L compared to the volume of solvent.
The invention also includes a preparation method of the prothrombin time detection reagent according to any one of the above, comprising the steps of:
s101, preparing an extracting solution, namely adding rabbit brain powder, sodium chloride, a surfactant, metal salt and a reducing agent into a solvent, fully mixing, and extracting at constant temperature for 35-40min under the stirring condition to obtain the extracting solution;
S102, adding calcium chloride into the extracting solution, and fully mixing until the calcium chloride is completely dissolved, thus obtaining the calcium chloride.
Further, in the step S101, the constant temperature condition is specifically 40-45 ℃.
Compared with the prior art, the method has the beneficial effects that the method can effectively remove the blood coagulation factor VII, so that the prepared thromboplastin does not contain the blood coagulation factor VII, the prepared prothrombin time detection reagent has enough and determinable sensitivity to the blood coagulation factor VII, the stability and comparability of detection are ensured, and the actual use effect of the reagent is improved; in the invention, the metal ions and the reducing agent are effective factor VII inhibitors, and can deactivate factor VII in the extraction process, thereby achieving the purpose of removing the factor VII, and the surfactant can effectively protect the thromboplastin, so that the thromboplastin can not be lost in the process of removing the factor VII.
Detailed Description
In order to better understand the technical solution in the embodiments of the present invention and make the above objects, features and advantages of the embodiments of the present invention more comprehensible, the technical solution in the present invention is described in further detail below with reference to the embodiments.
The invention comprises a prothrombin time detection reagent which is prepared from rabbit brain powder, a solvent, sodium chloride, a surfactant, metal salt, a reducing agent and calcium chloride.
Specifically, the surfactant is Tween 20, tween 80 or Triton X-100, the metal salt is manganese chloride, copper chloride or magnesium sulfate, and 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 45-65g/L, the dosage of the sodium chloride is 8-10g/L, the dosage of the surfactant is 0.1-1%V/V, the dosage of the metal salt is 0.01-8mmol/L, the dosage of the reducing agent is 0.5-6g/L, and the dosage of the calcium chloride is 4-6g/L.
The invention also includes a preparation method of the prothrombin time detection reagent according to any one of the above, comprising the steps of:
s101, preparing an extracting solution, namely adding rabbit brain powder, sodium chloride, a surfactant, metal salt and a reducing agent into a solvent, fully mixing, and extracting at constant temperature for 35-40min under the stirring condition to obtain the extracting solution;
S102, adding calcium chloride into the extracting solution, and fully mixing until the calcium chloride is completely dissolved, thus obtaining the calcium chloride.
Further, in the step S101, the stirring speed is 500-800rpm/min, and the constant temperature condition is specifically 40-45 ℃.
Example 1
Preparation of prothrombin time detection reagent J-1
TABLE 1 Prothrombin time test reagent formulation
| Component (A) | Name of the name | Dosage of |
| Rabbit brain powder | Rabbit brain powder | 50g |
| Solvent(s) | Deionized water | 1L |
| Sodium chloride | Sodium chloride | 9g |
| Surface active agent | TritonX-100 | 5ml |
| Metal salts | Copper chloride | 2mmol |
| Reducing agent | Ascorbic acid | 3g |
| Calcium chloride | Calcium chloride | 5g |
According to the contents of the components in the table 1, adding the rabbit brain powder, sodium chloride, a surfactant, metal salt and a reducing agent into a solvent, fully mixing, extracting for 38min at a constant temperature of 42 ℃ under the condition of a stirring speed of 600rpm/min, adding calcium chloride into the extracting solution after obtaining the extracting solution, and fully mixing until the calcium chloride is fully dissolved, thus obtaining the prothrombin time detection reagent J-1.
Example 2
Preparation of prothrombin time detection reagent J-2
TABLE 2 Prothrombin time test reagent formulation
| Component (A) | Name of the name | Dosage of |
| Rabbit brain powder | Rabbit brain powder | 65g |
| Solvent(s) | Deionized water | 1L |
| Sodium chloride | Sodium chloride | 9g |
| Surface active agent | Tween 80 | 8ml |
| Metal salts | Magnesium sulfate | 3mmol |
| Reducing agent | Ascorbic acid | 3g |
| Calcium chloride | Calcium chloride | 5g |
According to the contents of the components in the table 2, adding the rabbit brain powder, sodium chloride, a surfactant, metal salt and a reducing agent into a solvent, fully mixing, extracting for 40min at a constant temperature of 40 ℃ under the condition of a stirring speed of 600rpm/min, adding calcium chloride into the extracting solution after obtaining the extracting solution, and fully mixing until the calcium chloride is fully dissolved, thus obtaining the prothrombin time detection reagent J-2.
Example 3
Preparation of prothrombin time detection reagent J-3
TABLE 3 Prothrombin time test reagent formulation
| Component (A) | Name of the name | Dosage of |
| Rabbit brain powder | Rabbit brain powder | 45g |
| Solvent(s) | Deionized water | 1L |
| Sodium chloride | Sodium chloride | 9g |
| Surface active agent | Tween 20 | 8ml |
| Metal salts | Manganese chloride | 2.5mmol |
| Reducing agent | Dithiothreitol | 2g |
| Calcium chloride | Calcium chloride | 5g |
According to the contents of the components in the table 3, adding the rabbit brain powder, sodium chloride, a surfactant, metal salt and a reducing agent into a solvent, fully mixing, extracting for 37 minutes at the constant temperature of 44 ℃ under the condition of the stirring speed of 700rpm/min, adding calcium chloride into the extracting solution after obtaining the extracting solution, and fully mixing until the calcium chloride is fully dissolved, thus obtaining the prothrombin time detection reagent J-3.
Example 4
Preparation of prothrombin time detection reagent J-4
TABLE 4 Prothrombin time test reagent formulation
| Component (A) | Name of the name | Dosage of |
| Rabbit brain powder | Rabbit brain powder | 45g |
| Solvent(s) | Deionized water | 1L |
| Sodium chloride | Sodium chloride | 8g |
| Surface active agent | Tween 20 | 6ml |
| Metal salts | Magnesium sulfate | 0.05mmol |
| Reducing agent | N-acetylcysteine | 0.5g |
| Calcium chloride | Calcium chloride | 4g |
According to the contents of the components in the table 4, adding the rabbit brain powder, sodium chloride, the surfactant, the metal salt and the reducing agent into a solvent, fully mixing, extracting for 38min at the constant temperature of 43 ℃ under the condition of the stirring speed of 700rpm/min, adding calcium chloride into the extracting solution after obtaining the extracting solution, and fully mixing until the calcium chloride is fully dissolved, thus obtaining the prothrombin time detection reagent J-4.
Example 5
Preparation of prothrombin time detection reagent J-5
TABLE 5 Prothrombin time test reagent formulation
According to the contents of the components in the table 5, adding the rabbit brain powder, sodium chloride, a surfactant, metal salt and a reducing agent into a solvent, fully mixing, extracting for 35min at a constant temperature of 45 ℃ under the condition of a stirring speed of 800rpm/min, adding calcium chloride into the extracting solution after obtaining the extracting solution, and fully mixing until the calcium chloride is fully dissolved, thus obtaining the prothrombin time detection reagent J-5.
Experiment 1
Detection of the reagents prepared in examples 1 to 5
1. Collecting 8 normal samples and 4 samples with prolonged PT time, and numbering, wherein the numbers 1-8 are normal samples, and the numbers 9-12 are samples with prolonged PT time;
2. Samples were tested on a Sysmex CS-1600 fully automatic hemagglutination analyzer using the reagents prepared in the examples above (reagents prepared in examples 1-5), control A (well known brand 1) and control B (well known brand 2) reagents, and the test results are shown in Table 8 below.
Table 8 test data table
By the test results in Table 8, the reagent of the present invention is consistent with the test results of the control prothrombin reagent, and samples with abnormal PT time can be normally distinguished.
Experiment 2
Comparison of the inventive reagent with PT values from other manufacturers
PT values of samples lacking factor VII were measured with the test group (reagents prepared in examples 1-5), control group a (well-known brand 1) and control group B (well-known brand 2) reagents, respectively, and the measurement results are shown in table 9.
Table 9 comparative reagent data sheet
The factor VII deletion samples were purchased commercially, and it can be seen from table 9 that the reagent prepared according to the present invention showed longer clotting time than the PT detection results of the other two products, i.e., the reagent prepared according to the present invention had higher sensitivity to factor VII deletion samples.
It will be apparent to those skilled in the art from this disclosure that various other changes and modifications can be made which are within the scope of the invention as defined in the appended claims.