CN106591267B - Thromboplastin, extraction method thereof and PT reagent - Google Patents
Thromboplastin, extraction method thereof and PT reagent Download PDFInfo
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Abstract
The invention relates to the technical field of biochemical detection, and discloses thromboplastin, an extraction method thereof and a PT reagent. The extraction method of the invention adds buffer solution with low metal chelating ability and non-ionic surfactant into rabbit brain powder, and then the rabbit brain powder is oscillated, extracted and centrifuged, and the obtained supernatant is thromboplastin. In the process of extracting the thromboplastin by taking the rabbit brain powder as the raw material, the specific buffer solution and the nonionic surfactant are selected for extraction, the extracted thromboplastin is short in time and large in extraction amount, and only blood plasma to be detected needs to be incubated for 1min when the PT is detected.
Description
Technical Field
The invention relates to the technical field of biochemical detection, in particular to thromboplastin, an extraction method thereof and a PT reagent.
Background
Prothrombin Time (PT) is a screening test for examining extrinsic coagulation factors, and is widely used for examining factors before surgery and in various extrinsic coagulation pathways, quantitatively detecting coagulation factors, and detecting oral anticoagulant therapy. Such as reduced levels of fibrinogen (factor I), prothrombin (factor II), V, VII, and X factors, there is a sensitive change in PT time.
The Prothrombin Time (PT) reagent mainly comprises three parts of thromboplastin, phospholipid mixture and calcium ions. The most important component of the reagent is thromboplastin, factor iii, commonly referred to as Tissue Factor (TF). TF is prepared by a genetic engineering method, the process is complex, the process is difficult to control, and phosphatidylethanolamine, phosphatidylcholine and phosphatidylserine are added in the later period for esterification.
The existing simple preparation method is usually obtained by extracting rabbit brain powder, such as chinese patent 201410093404.X, which discloses a technical scheme for extracting rabbit brain tissue factor (thromboplastin) from rabbit brain, but in the process from rabbit brain powder to rabbit brain tissue factor, the patent technology needs to incubate rabbit brain powder in physiological saline at 37 ℃ for 60min, the extraction time is long, and the extraction efficiency is low; chinese patent 200510030621.5 discloses an in vitro diagnostic kit for clinical examination of prothrombin time, which relates to the technical scheme of thromboplastin, i.e. rabbit brain powder is put into an extraction reagent containing barium sulfate, triton X-100 and sodium chloride and oscillated for 15 minutes at 45 ℃, then centrifuged to obtain thromboplastin, and then the thromboplastin, a stabilizer and a protective agent form a PT reagent; compared with the former, the extraction time of the patent technology is shorter, but in the actual detection process, the plasma to be detected needs to be incubated for 3min in advance, then the reaction detection with the PT reagent can be carried out, and the incubation time is too long; the same problems exist in Chinese patent 200510030621.5 and Chinese patents 201510769631.4 and 201510769634.8, which all adopt rabbit brain powder as a source of thromboplastin, but due to process defects in the preparation process, the dosage of the rabbit brain powder in the prepared PT reagent is higher and is about 60-80 mg/mL, plasma to be detected needs to be incubated in water bath at 37 ℃ for 3min in advance, the consumed time is longer, and the detection efficiency is influenced.
Disclosure of Invention
In view of the above, the present invention provides a thromboplastin and an extraction method thereof, so that the extraction method can obtain more thromboplastin in a shorter time, and the obtained thromboplastin (tissue factor) can significantly shorten the incubation time of the plasma to be detected during detection, and simultaneously ensure higher accuracy and repeatability of the detection.
It is another object of the present invention to provide a PT reagent containing the thromboplastin, which has all the advantages of the thromboplastin.
In order to achieve the above purpose, the invention provides the following technical scheme:
an extraction method of thromboplastin comprises adding buffer solution with low metal chelating ability and nonionic surfactant into rabbit brain powder, oscillating for extraction, and centrifuging to obtain supernatant as thromboplastin;
wherein the buffer solution with low metal chelating capacity is one or more than two of Tris (hydroxymethyl) aminomethane buffer solution (Tris-HCl), piperazine-1, 4-diethylsulfonate buffer solution (PIPES), N-3- (hydroxymethyl) methyl-2-aminoethanesulfonic acid buffer solution (TES) and Bis (2-hydroxyethylamino) Tris (hydroxymethyl) methane buffer solution (Bis-Tris);
the nonionic surfactant is one or more than two of Triton X-405(Tx-405), Tween 20(Tw-20), polyoxyethylene polyoxypropylene block copolymer (F68) and Span (Span 60).
Aiming at the defect that the existing thromboplastin needs to be incubated for 3min in advance during detection, the method selects a specific buffer solution and a nonionic surfactant for extraction when the rabbit brain powder is used for extraction, and can shorten the incubation time when the obtained thromboplastin is used for detecting the plasma to be detected. Meanwhile, the extraction process is good, the time is short, and the extraction amount of thromboplastin is large.
Preferably, the concentration of the buffer solution with low metal chelating capacity is 10-100mmol/L, and the volume percentage concentration of the nonionic surfactant is 0.05-0.5%. In a specific embodiment of the invention, the buffer with low metal chelating ability has a concentration of 10mmol/L, 20mmol/L, 30mmol/L, 50mmol/L, 75mmol/L or 100mmol/L, and the volume percent concentration of the nonionic surfactant is 0.05%, 0.1%, 0.2% or 0.5%.
Preferably, the concentration of the rabbit brain powder is 20-40 mg/mL. In a specific embodiment, the concentration of the rabbit brain powder is 20 mg/mL.
Preferably, the shaking extraction is carried out in a shaking table at 37 ℃ for 30min, and the centrifugation is carried out at 8000r/min for 3 min.
In the process of detecting the thrombin dosage form PT extracted and obtained by the invention, the blood plasma to be detected can be detected only by incubating for 1min, and has no obvious difference with the PT value incubated for 3 min. However, the PT values of the existing patents 200510030621.5, 201510769631.4 and 201510769634.8 incubated for 1min and 3min respectively have a large difference, which indicates that the existing technology needs a long plasma incubation time, so that the stability and correctness of the detection result can be ensured.
Based on the technical effects, the invention provides the thromboplastin obtained by any one of the extraction methods and the application thereof in preparing a PT reagent.
Meanwhile, the invention also provides a PT reagent which comprises the thromboplastin, a compound containing calcium ions, an antioxidant and a stabilizer.
Among them, in the PT reagent, preferably, the antioxidant is 2, 6-di-tert-butyl-4-methylphenol and/or polyethylene glycol-20000, and the stabilizer is one or more of serine, bovine serum albumin and sucrose.
Preferably, the compound containing calcium ions is calcium chloride.
In a particular embodiment of the invention, the PT reagent may be selected as follows:
the thromboplastin with the volume percentage concentration of 20 percent is 0.025mol/L CaCl25wt% serine, 0.6wt% polyethylene glycol-20000, 0.1 wt% BSA, 0.01wt% BHT, 1.2wt% sucrose.
According to the technical scheme, in the process of extracting the thromboplastin by taking the rabbit brain powder as the raw material, the specific buffer solution and the nonionic surfactant are selected for extraction, the extracted thromboplastin is short in time and large in extraction amount, and only blood plasma to be detected needs to be incubated for 1min when PT is detected.
Detailed Description
The embodiment of the invention discloses thromboplastin, an extraction method thereof and a PT reagent, and a person skilled in the art can appropriately improve process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to those skilled in the art are deemed to be included within the invention. While the thromboplastin and its extraction method have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that the techniques of the present invention may be practiced and applied by modifying or appropriately modifying or combining the products and methods described herein without departing from the spirit, scope, and spirit of the invention.
The thromboplastin, its extraction method and PT reagent provided by the present invention are further described below.
Example 1: the extraction method of the invention is adopted to extract the thromboplastin
Adding buffer solution with low metal chelating ability and nonionic surfactant into rabbit brain powder, mixing well, and incubating in a constant temperature water bath at 37 deg.C for 30 min. And finally, centrifuging the mixed solution at 8000r/min for 3min, and taking supernatant fluid to obtain the extracted thromboplastin.
Wherein, the concentration of the rabbit brain powder is 20mg/mL, the concentration of the buffer solution with low metal chelating capacity is 20mmol/L, and the volume percentage concentration of the nonionic surfactant is 0.1%;
the buffer solution with low metal chelating capacity is selected from Tris (hydroxymethyl) aminomethane buffer solution (Tris-HCl), piperazine-1, 4-diethylsulfonate buffer solution (PIPES), N-3- (hydroxymethyl) methyl-2-aminoethanesulfonic acid buffer solution (TES) or Bis (2-hydroxyethylamino) Tris (hydroxymethyl) methane buffer solution (Bis-Tris);
the nonionic surfactant is selected from Triton X-405(Tx-405), Tween 20(Tw-20), polyoxyethylene polyoxypropylene block copolymer (F68) or Span (Span 60).
Example 2: PT reagent of the present invention
20% by volume of the thromboplastin according to the invention (example 1, Tris-HCl as buffer with low metal chelating capacity and F68 as non-ionic surfactant), 0.025mol/L CaCl25wt% serine, 0.6wt% polyethylene glycol-20000, 0.1 wt% BSA, 0.01wt% BHT, 1.2wt% sucrose.
Example 3: time consuming extraction of thromboplastin with different buffers and surfactants
On the basis of example 1, different buffers and surfactants were selected for thromboplastin extraction and the respective time consumption was counted, and the results are shown in table 1.
TABLE 1 extraction time results (min)
| Tris-HCL | PIPES | TES | Bis-Tris | |
| Tx-405 | 40 | 30 | 40 | 50 |
| Tw-20 | 30 | 50 | 40 | 40 |
| F68 | 30 | 40 | 30 | 30 |
| Span 60 | 30 | 30 | 40 | 40 |
As can be seen from Table 1, the buffer and the nonionic surfactant selected in the present invention take relatively little time in the extraction process of the rabbit brain powder. Among them, the more preferable combination form is a combination which takes 30 min.
Example 4: extraction amount of thromboplastin by combining different buffers and surfactants
The extraction of thromboplastin was carried out according to the method described in example 1, with the difference in the choice of buffer and non-ionic surfactant at the dose of 1g of rabbit brain powder, corresponding to thromboplastin extraction amounts shown in table 2:
TABLE 2 thromboplastin extraction amount control Table (unit: mg)
As can be seen from Table 2, the final thromboplastin extraction amount is higher under various combinations of the buffer solution and the nonionic surfactant selected by the invention under the same rabbit brain powder dosage. The extraction method of the invention is shown to obtain higher thromboplastin extraction amount under the condition of relatively less usage of rabbit brain powder. In each combination, the highest thromboplastin extraction was achieved in the combination of Tris-HCl as the buffer and F68 as the nonionic surfactant, which was significantly higher than the other combinations.
Example 5: comparison of clotting times for different PT reagents
On the basis of example 1, different buffers and surfactants are selected to extract thromboplastin, different PT reagents are formed under the requirement of example 2, each PT reagent is used for carrying out a blood coagulation time test, the test environment is 37 ℃, the plasma is incubated for 1 minute, and quality Control plasma (normal quality Control plasma Control N: 10.5-14.1 s) purchased from Simens is used for the test, and the test results are as follows:
TABLE 3 comparison of clotting time and CV value of different PT reagents (unit: second)
As can be seen from the above table, the blood coagulation time of each PT reagent prepared from the thromboplastin extracted above was substantially the same as the blood coagulation time of the PT reagent prepared from the thromboplastin extracted above; while the CV values tested were within the normal range. More importantly, the plasma incubation time is shorter than that required in the prior art, requiring only 1 min.
Example 6: effect of Tris-HCl concentration on Final PT reagent detection
On the basis of example 1, the immobilized surfactant was F68 (0.1%), thromboplastin was extracted using various concentrations of Tris-HCl and tested by composing PT reagent in the manner of example 2, the buffer concentration is shown in Table 4;
TABLE 4 buffer concentration
During testing, 0.1mL of plasma to be tested (normal quality Control plasma Control N: 10.5-14.1 s; abnormal quality Control plasma Control P: 16.3-24.5 s) is taken, incubated for 1min and 3min at 37 ℃, 0.2mL of pre-heated PT reagent at 37 ℃ is added, and the solidification time is recorded, namely the PT value. The results of the test on a brochure semi-automatic hemagglutination instrument CA-52 (n is 10 times, and the PT value is s) are shown in a table 5 for different incubation times of each group, and the results of the CV value for 1min are shown in a table 6.
TABLE 5 PT test results for different incubation times in each group
| Tris-HCl concentration (mmol/L) | 0 | 10 | 20 | 50 | 75 | 100 | 120 |
| 1min/Control N | 17.8 | 13.3 | 12.3 | 14.0 | 15.7 | 13.1 | 13.3 |
| 3min/Control N | 14.9 | 12.1 | 12.2 | 13.2 | 14.5 | 12.6 | 14.2 |
| 1min/Control P | 33.7 | 23.5 | 22.7 | 25.1 | 26.5 | 26.5 | 23.7 |
| 3min/Control P | 27.8 | 22.2 | 22.3 | 24.6 | 22.3 | 24.3 | 24.3 |
Table 6 CV value test results of each group
As can be seen from the tables, the results of the PT reagent of the present invention detected at 1min and 3min are not obvious, but the blank control is obvious. In each concentration range, the detection difference is the smallest when the concentration of Tris-HCl is 20 mmol/L.
Example 7: effect of F68 concentration on Final PT reagent detection
On the basis of example 1, the immobilization buffer was Tris-HCl, at a concentration of 20mmol/L, and thromboplastin was extracted using different concentrations of F68 and tested by composing PT reagent in the manner of example 2, with the concentration of F68 shown in Table 7.
TABLE 7F68 concentrations
During testing, 0.1mL of plasma to be tested (normal quality Control plasma Control N: 10.5-14.1 s; abnormal quality Control plasma Control P: 16.3-24.5 s) is taken, incubated for 1min and 3min at 37 ℃, 0.2mL of pre-heated PT reagent at 37 ℃ is added, and the solidification time is recorded, namely the PT value. The results of the test on a brochure semi-automatic hemagglutination instrument CA-52 (n is 10 times, and the PT value is s) are shown in Table 8, and the results of the CV value at 1min of incubation are shown in Table 9.
TABLE 8 test results
| Concentration of F68 (v/v) | Blank space | 0.02％ | 0.05％ | 0.1％ | 0.2％ | 0.5％ |
| 1min/Control N | 18.6 | 13.4 | 12.1 | 14.1 | 15.6 | 14.1 |
| 3min/Control N | 15.7 | 12.9 | 12.2 | 12.7 | 14.3 | 14.2 |
| 1min/Control P | 35.6 | 26.7 | 22.7 | 22.5 | 24.7 | 23.7 |
| 3min/Control P | 29.3 | 25.3 | 21.9 | 22.6 | 22.6 | 23.9 |
TABLE 9 CV value test results of each group
As can be seen from the tables, the results of the PT reagent of the present invention detected at 1min and 3min are not obvious, but the blank control is obvious. In each concentration range, the detection difference was the smallest at a F68 concentration of 0.1%.
Example 8: comparison with existing PT reagent
1. Grouping
Test groups: PT reagent of example 2;
control group 1: patent 200510030621.5 PT reagent prepared in example 1;
control group 2: patent 201510769631.4 PT reagent in example 1;
control group 3: patent 201510769634.8 PT reagent of example 1 (i.e. rabbit brain powder extract reagent);
2. method of producing a composite material
During testing, 0.1mL of plasma to be tested (normal quality Control plasma Control N: 10.5-14.1 s; abnormal quality Control plasma Control P: 16.3-24.5 s) is taken, incubated for 1min and 3min at 37 ℃, 0.2mL of pre-heated PT reagent at 37 ℃ is added, and the solidification time is recorded, namely the PT value. The results are shown in table 10 for each group of different incubation times PT tested on a brochure semi-automatic hemagglutinator CA-52 (n 10 times, PT values in s).
TABLE 10 test results
It is obvious from table 10 that the detection results of PT reagents in the prior patents meet the standard of quality control plasma only after 3min incubation, and exceed the standard requirements of quality control plasma after 1min incubation; the detection results are similar no matter the incubation time is 1min or 3min, and the requirements of quality control of blood plasma are met.
The foregoing is only for the purpose of understanding the method of the present invention and the core concept thereof, and it will be understood by those skilled in the art that various changes and modifications may be made without departing from the principle of the invention, and the invention also falls within the scope of the appended claims.
Claims (3)
1. An extraction method of thromboplastin is characterized in that a buffer solution with low metal chelating capacity and a nonionic surfactant are added into rabbit brain powder of 20-40mg/mL, oscillation extraction is carried out, then centrifugation is carried out, and the obtained supernatant is thromboplastin;
wherein the buffer solution with low metal chelating capacity is one of a trihydroxymethyl aminomethane buffer solution, a piperazine-1, 4-diethylsulfonic acid buffer solution, an N-3- (hydroxymethyl) methyl-2-aminoethanesulfonic acid buffer solution and a bis (2-hydroxyethylamino) tris (hydroxymethyl) methane buffer solution; the concentration of the buffer solution with low metal chelating capacity is 10-100 mmol/L;
the non-ionic surfactant is one of Triton X-405, Tween 20, polyoxyethylene polyoxypropylene block copolymer and span; the volume percentage concentration of the nonionic surfactant is 0.05-0.5%.
2. The extraction method according to claim 1, wherein the shaking extraction is carried out in a shaker at 37 ℃ for 30min to 50 min.
3. A PT reagent comprising 20% by volume of the thromboplastin prepared by the extraction method of claim 1 or 2, 0.025mol/L CaCl25wt% serine, 0.6wt% polyethylene glycol-20000, 0.1 wt% BSA, 0.01wt% BHT and 1.2wt% sucrose.
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| CN107988140B (en) * | 2018-01-25 | 2021-02-02 | 鲁东大学 | Abalone blood cell anticoagulant protective agent and preparation method thereof |
| CN108508220A (en) * | 2018-03-23 | 2018-09-07 | 天津药物研究院新药评价有限公司 | A kind of PT reagents are for measuring the active purposes of FXa in blood plasma |
| CN110887970B (en) * | 2019-11-29 | 2023-10-31 | 北京赛科希德科技股份有限公司 | Extraction buffer solution, rabbit brain extraction solution, PT detection reagent and PT detection kit |
| CN111638374B (en) * | 2020-06-08 | 2022-10-18 | 深圳市国赛生物技术有限公司 | In-vitro diagnostic kit for determining prothrombin time |
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