A method for preparing PT detection reagent

Abstract

The present invention discloses a method for preparing a PT detection reagent, belonging to the technical field of detection reagents. The method comprises the following steps: step 1, extracting rabbit brain powder; step 2, centrifuging to obtain a supernatant; step 3, taking the supernatant and adding immobilized trypsin, stirring; step 4, vacuum filtration, removing the immobilized trypsin, and obtaining a PT reagent. The method for preparing the PT detection reagent comprises adding immobilized trypsin to a rabbit brain powder extract and stirring, allowing it to fully act, and then using a Buchner funnel to separate and remove it by vacuum filtration. The trypsin fully acts on the active particles (containing tissue factor and phospholipids) wrapped in agglomerates in the extract, cutting them into uniform and fine active particles. The increase in the number of active particles increases the activity of the reagent and improves the sensitivity of the reagent. After the active particles are small, they are evenly dispersed and difficult to precipitate, thereby improving the stability and making the liquid clear.

Description

Technical Field

[0001] The present invention relates to the technical field of detection reagents, and in particular to a method for preparing a PT detection reagent. Background Art

[0002] Reagents, also known as biochemical reagents or test drugs, are mainly pure chemicals used to realize chemical reactions, analytical tests, research experiments, teaching experiments, and chemical formulas. They are generally divided into general reagents, high-purity reagents, analytical reagents, instrumental analysis reagents, clinical diagnostic reagents, biochemical reagents, inorganic ion colorimetric reagents, etc.

[0003] The prothrombin time (PT) test reagent is a classic technical method designed to measure the clot formation time in a sample by replacing the reactant with thromboplastin, mixing it with a plasma sample, initiating a coagulation reaction under calcium ion conditions to produce a clot, and then detecting it through a coagulometer. It is an important monitoring indicator for clinical anticoagulant therapy.

[0004] The main component of PT reagent is tissue thromboplastin (containing tissue factor and phospholipids). The existing simple preparation method usually extracts it from rabbit brain powder. Chinese Patent 201410093404.X discloses a preparation method for PT reagent. Fresh rabbit brain is processed to obtain rabbit brain powder, which is heat-activated at 37°C in physiological saline, then centrifuged to obtain a supernatant, and freeze-dried to obtain a freeze-dried preparation of rabbit brain tissue factor. However, the extract obtained by this method is turbid and contains unevenly dispersed large and small suspended particles, which are prone to aggregation and precipitation and have low activity.

[0005] Patent CN110887970A increases the repulsive force between the charges of phospholipids by adding external electrolytes, making the PT reagent less likely to precipitate. However, the invention does not address the problems of turbidity in the extract, uneven dispersion of suspended particles of varying sizes, and low activity. Therefore, a new process is needed to obtain a clear, stable, more active, and more sensitive PT reagent. Summary of the Invention

[0006] The present invention provides a method for preparing a PT detection reagent. The method comprises adding immobilized trypsin to a rabbit brain powder extract, stirring the mixture, allowing it to fully react, and then separating and removing the mixture using a Buchner funnel through vacuum filtration. The trypsin fully acts on the active particles (including tissue factor and phospholipids) encapsulated in the extract, cutting them into uniform, fine active particles. Increasing the number of active particles increases the activity of the reagent and improves the sensitivity of the reagent. Once the active particles are small, they are evenly dispersed, difficult to precipitate, and thus have improved stability and a clear liquid.

[0007] In order to achieve the above effects, the present invention provides the following technical solution: a method for preparing a PT detection reagent, comprising the following steps:

[0008] Step 1: Weigh the rabbit brain powder and set aside.

[0009] Step 2: Preheat the extraction buffer.

[0010] Step 3: Extract the rabbit brain powder in a water bath.

[0011] Step 4: Centrifuge the extract to obtain the supernatant.

[0012] Step 5: Take the supernatant and add immobilized trypsin, stirring.

[0013] Step 6: Vacuum filtration to remove the immobilized trypsin to obtain the PT reagent.

[0014] Step 7: Observe the clarity of the PT reagent liquid to verify its stability, and conduct comparative testing to verify its activity.

[0015] Furthermore, the method comprises the following steps: according to the operating steps in step 1, the extraction buffer comprises Hepes: 0.05 mol / L, NaCl: 4 / 10,000, NaN3: 0.5%, glycine: 5%, and trehalose 3%.

[0016] Further, the method comprises the following steps: according to the operation steps in step 1, the pH value of the extraction buffer is in the range of 7.3 to 7.5.

[0017] Further, the method includes the following steps: according to the operating steps in step 2, the extraction buffer is preheated to a temperature of 37 degrees Celsius.

[0018] Further, the method comprises the following steps: according to the operating steps in step 3, the rabbit brain powder is extracted in a water bath for 60 minutes.

[0019] Further, the method includes the following steps: according to the operating steps in step 4, the centrifugal parameters are a rotation speed of 4000 revolutions per minute and a centrifugal time of 10 minutes.

[0020] Further, the method comprises the following steps: according to the operation steps in step 5, the solid phase carrier is 4B.

[0021] Furthermore, the method comprises the following steps: according to the operation steps in step five, the concentration range of the immobilized trypsin is 0.05 to 0.20 mg / dL.

[0022] Further, the method includes the following steps: according to the operation steps in step six, vacuum filtration is performed using a Buchner funnel.

[0023] The present invention provides a method for preparing a PT detection reagent, which has the following beneficial effects: the PT detection reagent preparation method comprises the following steps: adding immobilized trypsin to a rabbit brain powder extract, stirring the mixture, allowing it to fully act, and then separating and removing the mixture using a Buchner funnel through vacuum filtration; the trypsin fully acts on active particles (including tissue factor and phospholipids) encapsulated in the extract and cuts them into uniform and fine active particles; the increase in the number of active particles increases the activity of the reagent, and the sensitivity of the reagent is also improved; after the active particles are small, they are evenly dispersed and difficult to precipitate, thereby improving stability and making the liquid clear. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is a schematic diagram of the preparation process of the present invention, DETAILED DESCRIPTION

[0025] The technical solutions in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative work shall fall within the scope of protection of the present invention.

[0026] Example 1: A method for preparing a PT detection reagent, comprising the following steps:

[0027] Step 1: Weigh rabbit brain powder for use. The extraction buffer consists of Hepes: 0.05 mol / L, NaCl: 4 / 10,000, NaN3: 0.5%, glycine: 5% and trehalose 3%. The pH of the extraction buffer is adjusted to 7.3-7.5 using sodium hydroxide. 100 ml of the extraction buffer is preheated to 37 degrees Celsius. Step 2: Preheat the extraction buffer. Step 3: Extract the rabbit brain powder under water bath conditions. The rabbit brain powder is extracted in a water bath at 37 degrees Celsius for 60 minutes. Step 4: Centrifuge the extract to separate and obtain the supernatant. The centrifugation parameters are a speed of 4000 rpm and a centrifugation time of 10 minutes. Step 5: Take the supernatant and add immobilized trypsin and stir. Step 6: Vacuum filtration to remove the immobilized trypsin. , obtain PT reagent, step seven, observe the clarity of PT reagent liquid, verify its stability, comparative test, verify its activity, comparative test required test plasma: take fresh venous blood to be tested, the instrument used in the blood collection process is a disposable plastic instrument, the test plasma and 0.109 mol per liter of sodium citrate are quickly mixed at a volume ratio of 9:1 to obtain a mixed solution, the mixed solution is added with buffer solution and mixed, and then placed in a low-temperature centrifuge, centrifuged at 3000 rpm for 15 minutes, the plasma is allowed to stand, and the plasma is allowed to stand for stratification. The upper layer of plasma is taken as the test plasma with a plastic pipette, the pia mater and vascular network of the fresh rabbit brain are completely removed, washed with physiological saline, and ground in a mortar to remove impurities that cannot be ground, and 3 times the amount of acetone is added and ground for 0.5 min ( Be careful not to grind for too long, which will cause it to become jelly-like and acetone to be difficult to separate. If it has become jelly-like, add a small amount of acetone and mix gently to separate it). After standing for a few minutes, pour off the supernatant and add an appropriate amount of acetone. Repeat this 5 to 6 times to completely dehydrate the brain tissue into a grayish-white fine powder. Filter it with filter paper. If possible, squeeze out the acetone, spread the brain powder, and dry it in the air to form a non-sticky granular powder (you can also use a vacuum pump or place it in a 37°C incubator for 1 hour to dry it). After the dry brain powder is made, it should be packaged and sealed and stored in an ordinary refrigerator at 4°C. Glycine (Glycine, abbreviated as Gly), also known as aminocaproic acid, has the chemical formula C2H5NO2. It is a white solid at room temperature and pressure. It is the simplest structure in the amino acid series and is not a human molecule. An essential amino acid, it possesses both acidic and basic functional groups within its molecule. It ionizes in water and is highly hydrophilic, yet it is non-polar. It is soluble in polar solvents but poorly soluble in non-polar solvents. It also has high boiling and melting points. Adjusting the pH of its aqueous solution can produce different molecular forms of glycine. Trehalose has been successfully used in medicine as a stabilizer for bioactive substances such as blood products, vaccines, lymphocytes, and cell tissues. Trehalose can be stored dry at room temperature and can also be used to preserve research biological reagents, such as various enzyme tools, cell membranes, organelles, antibodies, antigens, and viruses, making life science research more convenient, efficient, and effective. (Camilo et al., University of Camilo, UK)C et al. conducted a detailed study on the protective effects of trehalose on DNA restriction endonucleases, DNA ligase, and DNA polymerase. The results showed that all enzyme samples dried with trehalose maintained their activity after storage at 70°C for 35 days or at 37°C for 9 months, and were still able to accurately truncate DNA. Three diagnostic enzymes used for human serum cholesterol determination, prepared by the Institute of Microbiology, Chinese Academy of Sciences, showed activity retention rates exceeding 90% after long-term storage at room temperature. These enzymes have now been successfully used in clinical practice, an effect currently unattainable by other types of protective agents. Using trehalose as a stabilizer and protective agent for diagnostic enzymes and other biological reagents, they can be dried and stored at room temperature.

[0028] Example 2:

[0029] The extraction method of the present invention is used to extract thromboplastin;

[0030] Weigh two portions of rabbit brain powder and add 100 ml of preheated extraction buffer to each portion. After mixing, heat-activate at 37°C and centrifuge to obtain the supernatant: ① lyophilize to obtain lyophilized rabbit brain tissue factor preparation PT1. ② Add immobilized trypsin to the supernatant and remove the immobilized trypsin by vacuum filtration to obtain a clear liquid. Dilute the supernatant 3.2-fold with buffer and lyophilize to obtain lyophilized rabbit brain tissue factor preparation PT2.

[0031] 1. Activity:

[0032] Normal human plasma and abnormal human plasma were tested using freeze-dried reagents PT1 and PT2, respectively. Test plasma was prepared by obtaining fresh venous blood for testing using a disposable plastic device. The venous blood was rapidly mixed with 0.109 mol / L sodium citrate at a volume ratio of 9:1. Buffer B was added and mixed thoroughly, followed by centrifugation at 3000 rpm for 15 minutes in a refrigerated centrifuge. The upper layer of plasma was then collected using a plastic pipette as the test plasma. The results are as follows:

[0033] Table 1:

[0034]

[0035] For raw materials of the same mass, the amount of PT reagent obtained by the present invention is three times that of the original method.

[0036] 2. Repeatability:

[0037] The freeze-dried reagent obtained by the method of the present invention and the Siemens freeze-dried PT reagent were used to detect Siemens normal (abnormal) value quality control plasma. The test was repeated 10 times, and the results are as follows:

[0038] Table 2: Normal value quality control plasma test results:

[0039]

[0040] Table 3: Abnormal value quality control plasma test results:

[0041]

[0042] 3. Sensitivity

[0043] The freeze-dried reagent obtained by the method of the present invention and the Siemens freeze-dried PT reagent were used to detect Siemens quality control plasma L1, L2 and L3, and the detection was repeated 10 times. The results are as follows:

[0044] <![CDATA[L1]]> <![CDATA[L2]]> <![CDATA[L3]]> Siemens PT 11.5 39.8 65.4 PT of the present invention 11.2 38.9 64.8

[0045] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a PT detection reagent, characterized in that: The following steps are involved: S1. Weigh rabbit brain powder and set aside; S2, preheating the extraction buffer; S3. Extract the rabbit brain powder under water bath conditions; S4, centrifuging the extract to obtain a supernatant; S5. Take the supernatant and add immobilized trypsin, stirring; S6. Vacuum filtration to remove the immobilized trypsin to obtain the PT reagent; S7. Observe the clarity of the PT reagent liquid to verify its stability, and conduct comparative testing to verify its activity; The extraction buffer comprises Hepes: 0.05 mol / L, NaCl: 0.4 g / L, NaN3: 0.5%, glycine: 5%, and trehalose 3%.

2. The method for preparing a PT detection reagent according to claim 1, characterized in that: The following steps are involved: According to the operating steps in S1, the pH value of the extraction buffer ranges from 7.3 to 7.

5.

3. The method for preparing a PT detection reagent according to claim 1, characterized in that: The method comprises the following steps: according to the operation steps in S2, the extraction buffer is preheated to a temperature of 37 degrees Celsius.

4. The method for preparing a PT detection reagent according to claim 1, wherein: The following steps are involved: According to the operating steps in S3, the rabbit brain powder was extracted in a water bath for 60 minutes.

5. The method for preparing a PT detection reagent according to claim 1, characterized in that: The following steps are involved: According to the operation steps in S4, the centrifugation parameters are a rotation speed of 4000 rpm and a centrifugation time of 10 minutes.

6. The method for preparing a PT detection reagent according to claim 1, characterized in that: The method comprises the following steps: according to the operation steps in S5, the solid phase carrier is Sepharose® 4B.

7. The method for preparing a PT detection reagent according to claim 1, characterized in that: The method comprises the following steps: according to the operation steps in S5, the concentration range of the immobilized trypsin is 0.05-0.20 mg / dL.

8. The method for preparing a PT detection reagent according to claim 1, characterized in that: The following steps are involved: Vacuum filter using a Buchner funnel according to the procedure in S6.

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