Preparation method for prothrombin time determination reagent

A prothrombin time and reagent technology, which is applied in biological testing, material testing and other directions, can solve the problems of complex preparation process, difficult process control, low sensitivity, etc., and achieves stable process, good stability and sensitivity, and good reliability. Actionable effect

Inactive Publication Date: 2012-07-11
苏州良辰生物医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The tissue factor obtained by this method based on genetic engineering used in PT reagents at present is mainly human tissue factor and rabbit tissue factor, but this PT reagent and its preparation method also have defects, or poor stability, or insufficient sensitivity. High, or the preparation process is complex and difficult to control, etc.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] The preparation method of the PT reagent of the present embodiment, comprises the steps:

[0021] (1) Using genetic engineering to express sheep tissue factor in Escherichia coli to prepare sheep tissue factor with a purity of more than 90%, and dissolve the sheep tissue factor in an appropriate amount of PBS buffer to form a 54ug / ml tissue factor solution;

[0022] (2) Take 0.3g each of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine, and pour them into a 2L glass beaker to prepare PBS with a mass content of 1% triton, a pH value of 7.4, and a concentration of 20mmol Add 800ml of buffer solution into the above beaker, and gently stir for 90 minutes at 15°C~25°C to obtain a uniformly dispersed phospholipid solution;

[0023] (3) Take 5ml of the tissue factor solution prepared in step (1) (containing about 270ug of sheep tissue factor) and add it to the phospholipid solution obtained in step (2), and then dilute to 900ml with PBS buffer, and...

Embodiment 2

[0027] The preparation method of the PT reagent of the present embodiment, comprises the steps:

[0028] (1) Express sheep tissue factor in Escherichia coli by genetic engineering, prepare sheep tissue factor with a purity of more than 90%, and dissolve the sheep tissue factor in an appropriate amount of PBS buffer to form a 50ug / ml tissue factor solution;

[0029] (2) Take 0.3g each of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine, and pour them into a 2L glass beaker to prepare PBS with a mass content of 1% triton, a pH value of 7.4, and a concentration of 20mmol Add 800ml of buffer solution into the above beaker and gently stir for 60 minutes at 15°C~25°C to obtain a uniformly dispersed phospholipid solution;

[0030] (3) Take 5ml of the tissue factor solution prepared in step (1) (containing about 250ug of sheep tissue factor) and add it to the phospholipid solution obtained in step (2), and then dilute to 900ml with PBS buffer, and continue...

Embodiment 3

[0034] The preparation method of the PT reagent of the present embodiment, comprises the steps:

[0035] (1) Using genetic engineering to express sheep tissue factor in Escherichia coli to prepare sheep tissue factor with a purity of more than 90%, and dissolve the sheep tissue factor in an appropriate amount of PBS buffer to form a 60ug / ml tissue factor solution;

[0036] (2) Take 0.3g each of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine, and pour them into a 2L glass beaker to prepare PBS with a mass content of 1% triton, a pH value of 7.4, and a concentration of 20mmol Add 800ml of buffer solution into the above beaker, and stir gently at 15°C~25°C for 120 minutes to obtain a uniformly dispersed phospholipid solution;

[0037] (3) Take 5ml of the tissue factor solution prepared in step (1) (containing about 300ug of sheep tissue factor) and add it to the phospholipid solution obtained in step (2), and then dilute to 900ml with PBS buffer, an...

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Abstract

The invention relates to a preparation method for prothrombin time determination reagent (PT reagent), comprising the following steps. Cow or sheep tissue factor with the purity of more than 90% is prepared by the genetic engineering method, phospholipid is dispersed in phosphate buffer solution containing surfactant, and then the tissue factor and the phospholipid are mixed and sufficiently combined, and finally, C18 resin is adopted to adsorb and separate the surfactant, and the PT reagent product is obtained after freeze-drying. The preparation method is stable in technique and strong in operability, and further, the obtained PT reagent has better stability, sensitiveness and fewer impurities, and the quality can be easily ensured.

Description

technical field [0001] The invention relates to a prothrombin time test in the field of clinical blood diagnosis, in particular to a preparation method of a reagent for measuring the prothrombin time. Background technique [0002] Coagulation tests are performed clinically for many purposes, such as determining the bleeding tendency of patients undergoing surgery, and monitoring patients receiving anticoagulant therapy to prevent blood clotting. The "prothrombin time (PT)" test is one of the coagulation tests. The PT test is performed by adding thromboplastin to the blood sample required for the PT test, thereby activating the extrinsic coagulation pathway. Thromboplastin, also known as tissue factor (TF), is a membrane-associated protein. After forming a VIIa / TF complex with factor VIIa, it will activate a series of specific enzymes involved in the coagulation pathway, resulting in thrombin and fibrous Protein formation, activation of platelets, and eventually blood clot ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/86
Inventor 王小良
Owner 苏州良辰生物医药科技有限公司
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