A kind of thromboplastin and its extraction method and pt reagent

An extraction method, thromboplastin technology, applied in biochemical equipment and methods, enzymes, hydrolytic enzymes, etc., can solve the problems of difficult process control, affecting detection efficiency, complex process, etc., shorten incubation time, improve detection efficiency, Very time consuming and short effect

Active Publication Date: 2020-02-04
SINOCARE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

TF is prepared by genetic engineering, the process is complicated, and the process is difficult to control. In the later stage, phosphatidylethanolamine, phosphatidylcholine and phosphatidylserine are added for esterification
[0004] The existing easy preparation method is usually extracted from rabbit brain powder, such as Chinese patent 201410093404.X, which discloses a technical scheme for extracting rabbit brain tissue factor (thrombin) from rabbit brain, but from rabbit brain powder to In the process of rabbit brain tissue factor, this patented technology needs to incubate rabbit brain powder in normal saline at 37°C for 60 minutes, the extraction time is long and the extraction efficiency is low; Box, which involves the technical scheme of thromboplastin, that is, rabbit brain powder is shaken at 45°C for 15 minutes in an extraction reagent containing barium sulfate, Triton X-100, and sodium chloride, and then centrifuged to obtain thromboplastin, and then mixed with Stabilizer and protective agent constitute PT reagent; compared with the former, the extraction time of this patented technology is shorter, but in the actual detection process, the plasma to be tested needs to be incubated for 3 minutes in advance before it can react with PT reagent for detection, and the incubation time is too long; Chinese patents 201510769631.4 and 201510769634.8 have the same problem as Chinese patent 200510030621.5. Although these patents all use rabbit brain powder as the source of thromboplastin, due to process defects in the preparation process, the prepared PT reagent is the product of rabbit brain powder. The dosage is relatively high, about 60-80mg / mL, and the plasma to be tested needs to be pre-incubated in a 37°C water bath for 3 minutes, which takes a long time and affects the detection efficiency

Method used

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  • A kind of thromboplastin and its extraction method and pt reagent
  • A kind of thromboplastin and its extraction method and pt reagent
  • A kind of thromboplastin and its extraction method and pt reagent

Examples

Experimental program
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Effect test

Embodiment 1

[0025] Example 1: Using the extraction method of the present invention to extract thromboplastin

[0026] Add a buffer with low metal chelating ability and a non-ionic surfactant to the rabbit brain powder, and after the mixture is uniformly placed in a constant temperature water bath at 37° C., incubate for 30 min. Finally, the mixed solution is centrifuged at 8000r / min for 3min, and the supernatant is removed, which is the extracted thromboplastin.

[0027] Among them, the concentration of rabbit brain powder is 20mg / mL, the concentration of buffer with low metal chelating ability is 20mmol / L, and the volume percentage concentration of non-ionic surfactant is 0.1%;

[0028] The buffer with low metal chelating ability is selected from tris buffer (Tris-HCl), piperazine-1,4-diethanesulfonic acid buffer (PIPES), N-3-(hydroxymethyl) Methyl-2-aminoethanesulfonic acid buffer (TES) or bis(2-hydroxyethylamino) tris(hydroxymethyl)methane buffer (Bis-Tris);

[0029] The nonionic surfactant i...

Embodiment 2

[0030] Example 2: PT reagent of the present invention

[0031] The thromboplastin of the present invention with a volume percentage concentration of 20% (Example 1, the low metal chelating capacity buffer is Tris-HCl, and the non-ionic surfactant is F68), 0.025mol / L CaCl 2 , 5wt% serine, 0.6wt% polyethylene glycol-20000, 0.1wt% BSA, 0.01wt% BHT, 1.2wt% sucrose.

Embodiment 3

[0032] Example 3: Time-consuming extraction of thromboplastin with different buffers and surfactants

[0033] On the basis of Example 1, different buffers and surfactants were selected to extract thromboplastin, and the respective time-consuming statistics were calculated. The results are shown in Table 1.

[0034] Table 1 Extraction time results (min)

[0035] Tris-HCL PIPES TES Bis-Tris Tx-40540304050 Tw-2030504040 F6830403030 Span 6030304040

[0036] It can be seen from Table 1 that the various combinations of buffer and nonionic surfactants selected in the present invention take relatively less time in the extraction process of rabbit brain powder. Among them, a more preferable combination is a combination that takes 30 minutes.

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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. According to the extraction method, a buffering solution low in metal chelating capacity and a non-ionic surface active agent are added into rabbit brain powder, and oscillating extraction is performed; and then centrifuging is performed, and obtained supernate is the thromboplastin. According to the extraction method, in the process of extracting the thromboplastin with the rabbit brain powder as raw materials, the specific buffering solution and the non-ionic surface active agent are selected for performing extraction, the extracted thromboplastin consumes short time, and the extraction amount is large; and when PT is detected, it is only needed to incubate to-be-detected blood plasma for 1 min, and compared with long incubation time of an existing PT reagent, the thromboplastin can improve the detection efficiency.

Description

Technical field [0001] The invention relates to the technical field of biochemical detection, in particular to a thromboplastin and its extraction method and PT reagent. Background technique [0002] Prothrombin time (prothrombin time, PT) is a screening test to check exogenous coagulation factors. It is widely used in the examination of various factors before surgery and various exogenous coagulation pathways, quantitative detection of coagulation factors and oral administration Testing of anticoagulant therapy. If the content of fibrinogen (factor I), prothrombin (factor II), factor V, VII, and X decreases, the PT time will have sensitivity changes. [0003] The prothrombin time (PT) reagent is mainly composed of thromboplastin, a mixture of phospholipids, and calcium ions. The most important component in the reagent is thromboplastin, that is, coagulation factor III, usually called tissue factor (TF). The process of preparing TF by genetic engineering is complicated and diffi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/64G01N33/86
CPCC12N9/6424G01N33/86
Inventor 周杨美慧彭希望宋耀平
Owner SINOCARE
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