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Heparin content detection method

A detection method and heparin technology, applied in the field of biological detection, can solve the problems of accurate, reliable and simple detection results, and the inability to use automatic detection instruments.

Inactive Publication Date: 2014-09-17
SHANGHAI VASCUTECH DIAGNOSIS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Chinese patent CN 103063593 A discloses a method for determining the content of heparin in human antithrombin III concentrate. Although the content of heparin combined with antithrombin III is measured, it uses microwells with manual loading. Plate method, cannot be used on automatic detection equipment
[0011] To sum up, there is no detection method in the existing products that is simple, accurate and reliable, and can be applied to the detection of heparin content on automatic detection instruments

Method used

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Examples

Experimental program
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Embodiment 1

[0091] The kit 1 provided by the present invention is prepared, comprising reagent 1 and reagent 2, and is used for determining the heparin content in blood.

[0092] Specifically: Reagent 1 activates the chromogenic substrate of factor X (FXa), and its specific preparation method is as follows: taking 4-Nz-D-Arg-Gly-Arg-p-nitroanilide 2HCl (S-2782) dissolved in a concentration of 20mM Tris buffer, then add hydrochloric acid to adjust the pH value to 7.4; then add sodium chloride and polyethylene glycol-8000, stir to obtain reagent 1, and its final concentrations are: 4-Nz-D-Arg-Gly -Arg-p-nitroanilide 2HCl 0.5 mM, NaCl 0.15 M, PEG-8000 1%.

[0093] Reagent 2 is activating factor X (FXa), and its specific preparation method is as follows: taking activating factor X (FXa) and dissolving it in a Tris buffer with a concentration of 20 mM, then adding hydrochloric acid to adjust the pH value to 7.4; then adding sodium chloride and polyethylene Diol-8000, and after stirring, reage...

Embodiment 2

[0103] The difference between this example and Example 1 is only that the heparin contained in the standard and sample described in Example 1 is Unfractionated heparin (UFH), while the heparin contained in the standard and sample described in this example is Low molecular weight heparin (low-molecular-weight heparin, LMWH). Other operations and proportions are the same as those in Example 1.

[0104] According to the gradient concentration of heparin standard solution and the corresponding absorbance value, a linear equation is used to draw a standard curve, please see the appendix figure 2 , its standard curve formula is y=-1.0344x+1.3977(R 2 =0.9867).

[0105] The specificity, sensitivity and linear range of the obtained kit are shown in the following experiments.

Embodiment 3

[0107] The kit 3 provided by the present invention is prepared, including Reagent 1, Reagent 2 and Reagent 3, and is used to measure the heparin content in blood. At this time, the measured heparin content needs to consider the endogenous antithrombin expression level of the sample.

[0108] Wherein, the preparation of reagent 1 and reagent 2 and the standard curve are the same as those in Example 1.

[0109] Reagent 3 is antithrombin, and its specific preparation method is as follows: dissolving antithrombin in a Tris buffer solution with a concentration of 20 mM, and then adding hydrochloric acid to adjust the pH value to 7.4. Then add sodium chloride and polyethylene glycol-8000 to obtain reagent 3, and its final concentrations are: antithrombin 0.2~0.5U / mL, sodium chloride 0.1~0.3M, polyethylene glycol 0.5~1% .

[0110] According to the gradient concentration of heparin standard solution and the corresponding absorbance value, a linear equation is used to draw a standard ...

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Abstract

The invention discloses a heparin content detection method. The heparin content detection method comprises the following steps of by a developing substrate method, mixing a sample to be detected and a FXa activity detection reagent, carrying out incubation, detecting signal intensity of the developing substrate, calculating signal intensity and comparing the signal intensity and a standard curve to obtain heparin content of the sample to be detected, wherein the developing substrate is a developing substrate of an activation factor X(FXa) and is selected from Benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide.HCl, CH3O-CO-D-CHA-Gly-Arg-p-nitroanilide.AcOH, Acetyl-D-Arg-Gly-Arg-p-nitroanilide.2HCl, and 4-Nz-D-Arg-Gly-Arg-p-nitroanilide.2HCl. The heparin content detection method can be used for detecting common unclassified heparin, low-molecular weight heparin and fondaparinux, has good detection stability and good repeatability, can accurately show heparin content, has high sensitivity and accuracy, can fast find the optimal dosage scope of heparin and is free of multitime detection on patients. The heparin content detection method can be used for automation apparatuses such as a hemagglutination analyzer or a biochemical analyzer, realizes automation, is conducive to clinical popularization use and has the characteristics of simple operation, high sensitivity and good repeatability.

Description

technical field [0001] The invention belongs to the technical field of biological detection and relates to a detection method for heparin content. Background technique [0002] Heparin, a glycosaminoglycan, is a widely used anticoagulant drug. Heparin is a mucopolysaccharide sulfate composed of D-glucosamine, L-iduronic acid, N-acetylglucosamine and glucuronic acid alternately. The molecular weight ranges from 5 to 30KDa, of which sulfate accounts for about 40%. The anticoagulant function of heparin is mainly realized through its combination with antithrombin. The arginase active site of antithrombin III (AT-III) can combine with serine-containing thrombin and the serinase active sites of coagulation factors XIIa, XIa, Xa, and IXa to form antithrombin without coagulation activity III-coagulation factor complex to achieve anticoagulant effect. Heparin has a large number of negative charges and can bind to the positively charged lysine on antithrombin III. The unique pentos...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/31G01N21/78
Inventor 赵铁铭
Owner SHANGHAI VASCUTECH DIAGNOSIS CO LTD
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