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Human anti-thrombin III heparin combination ratio detection method

A technology of antithrombin and detection method, which is applied in the field of biomedical detection and analysis, can solve the problems of high price, cumbersome and difficult operation, and low accuracy of calculation results, and achieve high precision and accuracy of results, simple sample processing, and elimination of The effect of human interference

Active Publication Date: 2014-04-23
SHANDONG TAIBANG BIOLOGICAL PROD CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The detection method provided by the European Pharmacopoeia is cross-linked immunoelectrophoresis detection method, which has the following disadvantages: ① It is necessary to prepare or purchase rabbit anti-human antithrombin III antiserum, which is expensive, time-consuming, and cumbersome and difficult to operate; ② Electrophoresis takes a long time , about 20 hours; ③The temperature during electrophoresis is required to be controlled at 4°C; ④It is difficult to stain and decolorize after electrophoresis; ⑤The agarose electrophoresis gel is fragile and difficult to operate; Obviously, the accuracy of calculation results is low; ⑦Without related kit products, it is difficult to achieve unification of standards

Method used

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  • Human anti-thrombin III heparin combination ratio detection method

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Experimental program
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Effect test

Embodiment １

[0047] 1. Reagents and samples are shown in Table 1:

[0048] Table 1

[0049]

[0050] 2. Solution preparation:

[0051] Phosphate buffer: Weigh 12.0 g of sodium dihydrogen phosphate, dissolve it in 950 ml of purified water, adjust the pH to 7.4 with 0.2 mol / L sodium hydroxide solution, dilute to 1 L with purified water, and mix well.

[0052] Mobile phase: Take 495ml of the above-mentioned phosphate buffer solution, add 5ml of isopropanol, mix well and ultrasonicate for 30min for degassing.

[0053] Heparin solution: dilute heparin sodium injection to 5000IU / ml with the above mobile phase.

[0054]Inactivated human antithrombin III: add Bicine solution (50mmol / L Bicine (N-di(hydroxyethyl)glycine), 100mmol / L NaHCO3, pH 8.3) to human antithrombin III standard ; Then add 10~30mmol / L trinitrobenzenesulfonic acid, and treat at 25°C for 30min; add 20 times the volume of Tris buffer (50mmol / LTris–HCl, 100mmol / L NaCl, pH7.4) and mix well. After concentration by ultrafiltratio...

Embodiment 2

[0078] 1. Reagents and samples are shown in Table 5:

[0079] table 5

[0080]

[0081] 2. Solution preparation:

[0082] Phosphate buffer: Weigh 20.0 g of sodium dihydrogen phosphate, dissolve it in 950 ml of purified water, adjust the pH to 6.4 with 0.2 mol / L sodium hydroxide solution, dilute to 1 L with purified water, and mix well.

[0083] Mobile phase: Take 495ml of the above-mentioned phosphate buffer solution, add 5ml of isopropanol, mix well and ultrasonicate for 30min for degassing.

[0084] Heparin solution: Dilute heparin sodium injection to 2000IU / ml with the above mobile phase.

[0085] Inactivated human antithrombin III: add Bicine solution (50mmol / L Bicine (N-bis(hydroxyethyl)glycine), 100mmol / L NaHCO) to human antithrombin III standard 3 , pH 8.3); then add 10~30mmol / L trinitrobenzenesulfonic acid, and treat at 25°C for 30min; add 20 times the volume of Tris buffer (50mmol / LTris–HCl, 100mmol / L NaCl, pH7.4) Mix well. After concentration by ultrafiltrati...

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Abstract

The invention discloses a human anti-thrombin III heparin combination ratio detection method. The detection method comprises the steps of using an efficient size exclusion chromatography column and a sodium salt buffer solution with mobile phases comprising isopropanol and phosphoric acid at pH of 6.0-7.5 to calculate a combination ratio between the human anti-thrombin III and the heparin through a ratio of an area of characteristic peak and a corresponding standard curve; adding inactivated human anti-thrombin III into a standard product of the human anti-thrombin III according to a ratio, and manufacturing a standard curve according to change of the ratio of the area of characteristic peak. According to the method, the human factors are excluded; the accuracy and the precision of results are high, the experiment operation steps are simple, the detection time can be shortened, the experiment cost is reduced and the working efficiency of detectors is greatly improved.

Description

technical field [0001] The invention relates to the technical field of detection and analysis of biomedicine, in particular to a method for detecting the binding ratio of human antithrombin III heparin. Background technique [0002] Human antithrombin III (Human Antithrombin III, AT-III) is secreted by liver cells and vascular endothelial cells. It is a single-chain α2 glycoprotein containing 10% sugar, with a relative molecular weight of 58-64 kD and an isoelectric point of 4.8~ 5.3, composed of 432 amino acid residues, the content of antithrombin III in normal human plasma is 0.12~0.3g / L. It regulates coagulation activity by inhibiting the activity of thrombin, FIXa, FXa, FXIa, FXⅡa and other activated coagulation factors, and maintains the normal physiological balance of blood. Human antithrombin III product is a plasma protein drug purified from healthy human plasma. It has high purity and maintains its natural physiological activity. It is mainly used for the treatment...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02
Inventor 周安朱孟沼陈晨张翠萍席智赢菅长永马山
Owner SHANDONG TAIBANG BIOLOGICAL PROD CO LTD
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