Separation and purification method of blood coagulation factor xiii and its application

A coagulation factor, separation and purification technology, applied in separation methods, peptide preparation methods, chemical instruments and methods, etc., can solve the problems of consuming a large amount of human blood, low extraction efficiency, low yield, etc., to avoid the reduction of protein activity, Improve extraction efficiency and high affinity

Active Publication Date: 2021-10-19
宝锐生物科技泰州有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, the existing methods for purifying blood coagulation factors still use the classic precipitation method, which has low extraction efficiency, low yield, and low product purity, which greatly affects the application effect of FXIII.
At the same time, the FXIII used in the existing viscoelastic coagulation analysis is all extracted from human blood, and the content of FXIII in human blood is extremely low, so a large amount of human blood is required to obtain a sufficient amount of FXIII, and human blood is a scarce resources, for clinical use only
In addition, the use of human blood products also has a high biosafety risk

Method used

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  • Separation and purification method of blood coagulation factor xiii and its application
  • Separation and purification method of blood coagulation factor xiii and its application
  • Separation and purification method of blood coagulation factor xiii and its application

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preparation example Construction

[0043] As a preferred embodiment, the immunized animals of the immune response are BalB / C mice. BalB / C mice can rapidly induce plasmacytoma after injection of mineral oil, and this strain is widely used in the production of hybridoma and monoclonal antibody. The use of BALB / c mice in this protocol improves the efficiency and cost of monoclonal antibody production. In a preferred embodiment of the present invention, the preparation method of the active component containing blood coagulation factor XIII comprises the following steps:

[0044] Firstly, mammalian plasma is separated and subjected to membrane filtration to obtain component A; then component A is prepared as cryoprecipitate and then resuspended, and the supernatant is collected by solid-liquid separation to obtain the active component containing coagulation factor XIII.

[0045] As a preferred embodiment, the above-mentioned active components containing coagulation factor XIII are extracted by using mammalian blood...

Embodiment 1

[0072] Embodiment 1 prepares the active component containing coagulation factor XIII as antigen from pig blood

[0073] The preparation method of the active component containing blood coagulation factor XIII comprises the following steps:

[0074] (a) Weigh 20 kg of fresh citrated anticoagulated porcine whole blood, centrifuge at 8,000 rpm at 4°C for 20 min, collect the upper layer of plasma, and obtain component 1;

[0075] (b), component 1 is filtered with a 0.45um microporous membrane to obtain component 2;

[0076] (c) Put component 2 in a freezer at -40°C overnight to freeze all the plasma, then take it out and put it in a freezer at 4°C to slowly thaw, during the thawing process, a large amount of cryoprecipitate will be precipitated. Centrifuge at 10,000 g for 30 min in a GL-21M centrifuge to collect cryoprecipitate at 4°C to obtain fraction 3;

[0077] (d) Resuspend the cryoprecipitate of component 3 with 1000mL 8% cold ethanol solution, centrifuge at -3°C to collect...

Embodiment 2

[0083] Example 2 Preparation of Mouse Monoclonal Antibody

[0084] (1) Immunization of Balb / C mice

[0085] Take 5 Balb / C mice, immunize for the first time on the first day, mix the antigen + the same amount of complete Freund's adjuvant, fully emulsify, 100μg / mouse, and inject subcutaneously. The total amount of injection is 200 μl. The second immunization was carried out on the 14th day, and the antigen + equal amount of incomplete Freund's adjuvant was mixed, fully emulsified, 100 μg per mouse, and injected subcutaneously.

[0086] One week later, measure the potency by ELISA method. If the titer does not meet the fusion requirements, immunize once every 14 days according to the second immunization method, and measure the titer one week later. Three days after the last booster immunization, the animals were sacrificed by removing their eyeballs and bleeding, and the blood was stored for later use.

[0087] (2) Fusion cell preparation

[0088] Aseptically remove the sple...

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Abstract

The invention provides an immunoaffinity chromatography column filler, an immunoaffinity chromatography column, a method for separating and purifying blood coagulation factor XIII and applications thereof, and relates to the technical field of preparation of functional blood coagulation factors. The immunoaffinity chromatography column filler is mainly obtained by coupling an anticoagulation factor XIII monoclonal antibody with an affinity chromatography medium. According to the principle of high-specificity selection of antigen and antibody, during the purification process of coagulation factor XIII on the immunoaffinity chromatography column containing this filler, the monoclonal antibody against coagulation factor XIII can specifically adsorb coagulation factor XIII, thus effectively The purity and extraction efficiency of the blood coagulation factor XIII are improved, and the purity of the prepared blood coagulation factor XIII is as high as 95%, which can be widely used in the preparation of viscoelastic coagulation analysis and detection reagents.

Description

technical field [0001] The invention relates to the technical field of preparation of functional blood coagulation factors, in particular to a method for separating and purifying blood coagulation factor XIII and its application. Background technique [0002] Viscoelastic coagulation analysis and detection is to simulate physiological hemostasis in vivo by placing trace whole blood samples in vitro, comprehensively and dynamically observe the whole process of coagulation and fibrinolysis, and can quickly and completely detect the whole process from the beginning of coagulation to the formation and stability of blood clots. Real-time monitoring of changes in blood coagulation status, and providing data on the properties and dynamics of coagulation and fibrinolysis, so as to analyze the function and interaction of various components of the coagulation system. In the process of viscoelastic coagulation analysis and detection, coagulation factor XIII (FXIII) is needed as an acti...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): B01D15/20C12N9/10C07K1/22
CPCB01D15/20C12N9/1044C12Y203/02013
Inventor 严俊刘涛师丽佳
Owner 宝锐生物科技泰州有限公司
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