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Thromboplastin, extraction method thereof and PT reagent

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

Active Publication Date: 2017-04-26
SINOCARE
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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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  • Thromboplastin, extraction method thereof and PT reagent
  • Thromboplastin, extraction method thereof and PT reagent
  • Thromboplastin, extraction method thereof and PT reagent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1: Adopt extraction method of the present invention to extract thromboplastin

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

[0027] Wherein, the concentration of rabbit brain powder is 20mg / mL, the concentration of buffer solution with low metal chelating ability is 20mmol / L, and the concentration of nonionic surfactant is 0.1% by volume;

[0028] The buffer with low metal chelating ability is selected from Tris hydroxymethylaminomethane 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 nonion...

Embodiment 2

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

[0031] The volume percent concentration is 20% thromboplastin of the present invention (embodiment 1, the buffer solution of low metal chelating ability is Tris-HCl, and nonionic 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 by different buffers and surfactants

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

[0034] Table 1 Extraction time results (min)

[0035] 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

[0036] As can be seen from Table 1, various combinations of buffer and nonionic surfactants used in the present invention take relatively little time in the process of extracting rabbit brain powder. Among them, the more preferred combination form is the 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, its extraction method and PT reagent. Background technique [0002] Prothrombin time (PT) is a screening test for checking exogenous coagulation factors. Detection of anticoagulant therapy. If the content of fibrinogen (factor I), prothrombin (factor II), factor V, VII, and X decreases, the sensitivity of PT time will change. [0003] Prothrombin time (PT) reagent is mainly composed of three parts: thromboplastin, phospholipid mixture and calcium ion. The most important component in the reagent is thromboplastin, namely blood coagulation factor III, usually called tissue factor (tissue factor, TF). TF is prepared by genetic engineering, the process is complicated, and the process is difficult to control. Phosphatidylethanolamine, phosphatidylcholine and phosphatidylserine are added later for esterification. [0004] The existing easy preparatio...

Claims

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Application Information

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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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