FXa activity detection reagent, preparation method and application of FXa activity detection reagent

A technology of activity detection and detection kit, applied in the field of biological detection, which can solve the problems of no detection method, accurate, reliable and simple detection results

Inactive Publication Date: 2014-09-24
SHANGHAI VASCUTECH DIAGNOSIS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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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  • FXa activity detection reagent, preparation method and application of FXa activity detection reagent
  • FXa activity detection reagent, preparation method and application of FXa activity detection reagent
  • FXa activity detection reagent, preparation method and application of FXa activity detection reagent

Examples

Experimental program
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Effect test

Embodiment 1

[0091] Prepare kit 1 provided by the present invention, comprising reagent 1 and reagent 2, 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: take 4-Nz-D-Arg-Gly-Arg-p-nitroanilide 2HCl (S-2782) and dissolve it at a concentration of 20mM Tris buffer solution, then add hydrochloric acid to adjust the pH value to 7.4; then add sodium chloride and polyethylene glycol-8000, and stir to prepare reagent 1, the final concentrations are: 4-Nz-D-Arg-Gly -Arg-p-nitroanilide 2HCl 0.5 mM, sodium chloride 0.15 M, polyethylene glycol-8000 1%.

[0093] Reagent 2 is activated factor X (FXa), and its specific preparation method is: take activated factor X (FXa) and dissolve it in Tris buffer solution with a concentration of 20mM, then add hydrochloric acid to adjust the pH value to 7.4; add sodium chloride and polyethylene Diol-8000 was stirred to prepare reagent 2. The ...

Embodiment 2

[0103] The difference between this example and Example 1 is that the heparin contained in the standard and sample described in Example 1 is unfractionated heparin (Unfractionated heparin, UFH), while the heparin contained in the standard and sample described in this example is Low-molecular-weight heparin (LMWH). Other operations and proportioning are consistent with embodiment 1.

[0104] According to the gradient concentration of heparin standard solution and the corresponding absorbance value, use linear equation to draw the standard curve, please refer to the attached figure 2 , the 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] Preparation of kit three provided by the present invention, including reagent 1, reagent 2 and reagent 3, is used to measure the heparin content in the 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 in Example 1.

[0109] Reagent 3 is antithrombin, and its specific preparation method is: take antithrombin and dissolve it in Tris buffer solution with a concentration of 20 mM, and then add hydrochloric acid to adjust the pH value to 7.4. Then add sodium chloride and polyethylene glycol-8000 to obtain reagent 3, the final concentrations of which 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, use linear equation to draw the standard curve, p...

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Abstract

The invention discloses a detection method for heparin content. The detection method comprises the steps of mixing a sample to be tested with an FXa activity detection reagent by a developing substrate method to obtain a mixture, incubating the mixture, and detecting the signal intensity of a developing substrate; and finally calculating signal intensity and comparing the calculated signal intensity with a standard curve to obtain the heparin content of the sample to be tested, wherein the developing substrate is a developing substrate of an activated factor X (FXa) and is selected from any one of the following substrates of 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 detection method disclosed by the invention can be used for detecting general non-graded heparin, low-molecular-weight heparin and fondaparinux; furthermore, according to the detection method, the detection stability and the repetitiveness are high, the heparin content can be reflected accurately, the sensitivity and the accuracy are extremely high, and the optimal dosage range of the heparin can be found out quickly, so that a patient does not need to be detected for multiple times. Moreover, the detection method can be also used on an automatic instrument such as a coagulation analyzer or a biochemical analyzer, so that automation is realized, and clinical popularization and application can be assisted; the detection method has the characteristics of simplicity in operation, high sensitivity and high repetitiveness.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and relates to a FXa activity detection reagent and its preparation method and application. 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 ...

Claims

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Application Information

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