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Determination method of hirudin antithrombin activity

An antithrombin and determination method technology, applied in the field of medical testing, can solve the problems of unsatisfactory repeatability and accuracy, standard curve error, large environmental impact, etc., and achieves low equipment requirements, good accuracy, and low detection concentration. Effect

Inactive Publication Date: 2017-06-16
GUANGXI TEACHERS EDUCATION UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These methods can be divided into: (1) titration method: adopt fibrinogen or other reagents to be used as the indicator of titration and test hirudin binding activity, this method is simple, time-saving, economical and convenient, widely used, but repeatability and accuracy The degree is not ideal, and it is greatly affected by the environment
(2) Spectroscopic method: the change of light scattering after coagulation or the addition of radiotracing thrombin substrate was used to evaluate the activity of hirudin, and the change of light scattering was used after coagulation or adding radio-labeled thrombin substrate for Assessing the activity of hirudin, this method is accurate and stable, but requires the use of an expensive professional dispersive fluorescence instrument, and the establishment of a standard curve may cause errors
(3) Immunoassay method: Hirudin activity is measured by immunoprecipitation and western blotting. This method is accurate, stable, and has certain reproducibility, but some drugs need to be imported and are expensive

Method used

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  • Determination method of hirudin antithrombin activity
  • Determination method of hirudin antithrombin activity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Add 11.5 μL, 10.5 μL, 1.5 μL, and 1.5 μL of binding buffer to the four tubes arranged from left to right, and then add 0 μL, 1 μL, 10 μL, and 10 μL of hirudin solution to the four tubes. Add 2 μL of thrombin solution respectively, after mixing, place the four tubes in a water bath at 37°C for 5 min, and add 1.5 μL of cross-linking agent BS to the first three tubes from left to right 3 solution, add 1.5 μL of cross-linking buffer to the fourth tube, after 30 min of cross-linking reaction, add 1 μL of stop buffer solution to the four tubes, react at room temperature for 15 min to terminate the cross-linking, and then add 4 μL of 5 % SDS-PAGE protein loading buffer, after mixing, take 10 μL sample from each tube for SDS-PAGE analysis and optical density scanning, and use 0.1 μg and 1.0 μg bovine serum albumin (BSA) as a control, the gel Electrophoresis was stained with Coomassie brilliant blue to obtain figure 1 The electropherogram, wherein, the described binding buffer ...

Embodiment 2

[0033] Step 1: Add 10.5 μL, 9.5 μL, 8.5 μL, 7.5 μL, 6.5 μL, 5.5 μL, 4.5 μL and 3.5 μL of binding buffer to the tubes marked 1-8, then add 1 μL and 2 μL of hirudin solution in sequence , 3 μL, 4 μL, 5 μL, 6 μL, 7 μL and 8 μL, add 2 μL thrombin solution to 8 tubes respectively, after mixing, place them in a water bath at 37°C for 5 minutes, the binding buffer is 0.9 mass fraction % sodium chloride solution; the concentration of the hirudin solution is 0.2 mg / mL, and the thrombin solution is 4.8 mg / mL with a mass fraction of 0.9% sodium chloride solution as the solvent. 3 NIH / μL solution;

[0034] Step 2: Add 1.5 μL cross-linking agent BS to 8 tubes respectively 3 Solution, cross-linking reaction 30min at room temperature, the cross-linking agent BS 3 The solution consists of 12.5mmol BS 3 and 20mmol of sodium phosphate dissolved in 1.5 μL of a 0.9% solution of sodium chloride;

[0035] Step 3: Add 1 μL of stop buffer solution to 8 tubes respectively, and react for 15 minutes...

Embodiment 3

[0041] On the basis of Example 2, the samples in the 8 tubes were analyzed by SDS-PAGE, and 0.1 μg and 1 μg of bovine serum albumin were used as controls for scanning densitometric detection.

[0042] The masses of the corresponding hirudin-thrombin compounds in the 8 tubes detected by optical density scanning were 0.45 μg, 0.91 μg, 1.36 μg, 1.82 μg, 2.27 μg, 2.4 μg, 2.4 μg and 2.4 μg, marked by 6 The quality of the hirudin-thrombin compound in the tube of -8 can draw the quality of effective thrombin to be 2.0 μ g (that is, 5 / 6 of 2.4 μ g), so the mass fraction of effective thrombin in the thrombin solution is 41.7 %(2.0 / 4.8×100%), since the hirudin in the tube labeled 1-5 is completely reacted, the activity of hirudin antithrombin in the tube labeled 1-5 is thus 5625ATU / mg . Antithrombin activity was calculated as follows:

[0043] The mass of hirudin-thrombin compound is: 0.45 μg;

[0044] The mass of effective thrombin is: 0.45×5 / 6=0.375μg;

[0045] The mass of hirudin...

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Abstract

The invention discloses a method of determining anti-thrombin activity of hirudin. The method includes: allowing hirudin having certain mass and containing unknown activity to react with sufficient thrombin, allowing cross-linking reaction under the action of cross-linking agent BS3 to obtain a first mixture system containing the hirudin, the thrombin and a hirudin-thrombin compound after completion of cross-linking, performing optical density scanning after electrophoresis, acquiring mass of the hirudin-thrombin compound according to protein band density of the hirudin-thrombin compound on the basis of a control having known protein band density, combining the hirudin and the thrombin according to a mass ratio: 1:5 so as to obtain the hirudin-thrombin compound so as to acquire mass of effective thrombin participating in reaction, and performing calculating according to the mass fraction of the effective thrombin in the thrombin and the mass of the hirudin so as to obtain an activity value of the hirudin, having the certain mass, in inhibiting the thrombin, namely an anti-thrombin activity value of the hirudin. The method is simple to operate, good in accuracy and low in investment and has low equipment requirements.

Description

technical field [0001] The invention relates to the field of medical testing, in particular to a method for measuring the antithrombin activity of hirudin. Background technique [0002] Due to the increase in heart and cerebrovascular diseases, hirudin has attracted more and more attention as an effective antithrombotic drug exhibiting great potential in inhibiting thrombin. Natural hirudin can be obtained from farmed leeches (Hirudo medicinalis) from the saliva. In order to meet the growing demand, research on recombinant hirudin has been carried out since 1986. [0003] Hirudin is an acidic polypeptide with 64-69 amino acids, a molecular weight of 7 kilodaltons, and an isoelectric point of 3.8. The traits of hirudin are similar to tadpoles, and it has two functional domains: the n-terminus is the head and the c-terminus is the tail, and there are abundant negatively charged residues at the 49th-65th amino acid of the c-terminus. Thrombin has three functional domains: th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N27/447
Inventor 杨剑熊文朋刘艳芳修立辉
Owner GUANGXI TEACHERS EDUCATION UNIV
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