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Kit for detecting content of Dabigatran based on thrombin chromogenic substrate method

A chromogenic substrate, dabigatran technology, applied in the field of medical testing, can solve problems such as difficult quantification, weak correlation coefficient, and many interference factors

Inactive Publication Date: 2017-10-17
SHANGHAI VASCUTECH DIAGNOSIS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are many coagulation factors involved in the whole endogenous blood coagulation pathway, so this method is subject to many interference factors. The correlation coefficient between the blood concentration of dabigatran and activated partial thromboplastin time is not strong, and it is difficult to accurately quantify

Method used

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  • Kit for detecting content of Dabigatran based on thrombin chromogenic substrate method
  • Kit for detecting content of Dabigatran based on thrombin chromogenic substrate method
  • Kit for detecting content of Dabigatran based on thrombin chromogenic substrate method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Tris buffer with a concentration of 20mmol / L, then add hydrochloric acid to adjust the pH value to 7.5; then add sodium chloride, polyethylene glycol-8000 calf serum and sodium azide, and stir to obtain the final concentrations of sodium chloride 0.2 mol / L, polyethylene glycol-80000.1%, calf serum 0.1%, sodium azide 0.5mg / ml diluent.

[0043] Dissolve thrombin in diluent to form thrombin solutions with working concentrations of 0.1U / ml, 0.2U / ml, 0.5U / ml, 1U / ml and 2U / ml.

[0044] Take the chromogenic substrate H-D-Phe-Pip-Arg-pNA·2HCl of thrombin and dissolve it in the diluent to form a working concentration of 0.05mmol / L, 0.1mmol / L, 0.3mmol / L, 0.5mmol / L and 1mmol / L of thrombin chromogenic substrate solution.

Embodiment 2

[0046] Sample testing:

[0047] Take 100 μL sample and mix with 100 μl thrombin solution, incubate at 37°C for 2 minutes, then add 200 μl thrombin chromogenic substrate solution, react at 37°C and measure absorbance at 405nm wavelength for 5 minutes. The selection of the working concentration of the thrombin solution and the chromogenic substrate solution of thrombin is shown in Table 2. Record the test results, calculate the signal intensity, and compare with the standard curve to obtain the dabigatran content.

[0048] Table 1 detects the results of dabigatran assay

[0049]

[0050] It can be seen from Table 1 that the above-mentioned schemes can accurately determine the content change of dabigatran, wherein the working concentration of thrombin is 0.5U / ml and the working concentration of the chromogenic substrate of thrombin is 0.3mmol / L. Detection works best.

Embodiment 3

[0052] Preparation of Dabigatran Standards: Prepare several different concentrations of human plasma standards with known dabigatran concentrations, the concentrations are 0ng / ml, 15ng / ml, 30ng / ml, 60ng / ml, 120ng / ml ml, 250ng / ml, 500ng / ml.

[0053] Standard curve creation:

[0054] (1) Take 100 μL standard solution and 100 μL thrombin solution, mix evenly, and incubate for 2 minutes, the incubation and reaction temperature are both 37°C.

[0055] (2) Then add 200 μL thrombin chromogenic substrate solution, mix well, and measure the absorbance at a wavelength of 405 nm for 5 minutes.

[0056] (3) According to the gradient concentration of dabigatran standard solution and the corresponding absorbance value, a linear equation is used to draw a standard curve, see figure 1 , the standard curve formula is y=-0.7441x+0.2067(R 2 = 0.9927).

[0057] Table 2 detects the linear range of the dabigatran assay kit

[0058] Dabigatran concentration (ng / ml)

[0059] It can be ...

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Abstract

The invention provides a kit for detecting the content of Dabigatran based on a thrombin chromogenic substrate method. The kit comprises thrombin, a thrombin chromogenic substrate, a Human plasma standard substance containing the Dabigatran and diluting liquid; the working concentration of the thrombin is 0.1-2 U / ml; the working concentration of the thrombin chromogenic substrate is 0.05-1 mmol / L. A detection mechanism is that the thrombin is added into sample plasma and is used for cracking the thrombin chromogenic substrate to obtain an absorbance signal. As the Dabigatran in the plasma can inhibit the thrombin, the content of the Dabigatran is negatively related to the absorbance signal within a certain range. The kit realizes the monitoring of change in the content of the Dabigatran in the plasma based on the thrombin chromogenic substrate method. The kit provided by the invention realizes the rapid and accurate detection on the content of the Dabigatran; when the Dabigatran is within 0-500 ng / ml, R is more than or equal to 0.99, so that the linear relation is better; the relative deviation of the kit is smaller than 1 percent, so that the accuracy is high.

Description

technical field [0001] The invention belongs to the technical field of medical testing, and in particular relates to a kit for detecting dabigatran content based on a thrombin chromogenic substrate method. Background technique [0002] Dabigatran etexilate is a new oral anticoagulant that exerts its anticoagulant effect by directly inhibiting thrombin (coagulation factor FIIa), and can be used to prevent venous thromboembolism after hip joint / knee replacement, or Prevention of atrial fibrillation leading to stroke, etc. Although there are many anticoagulant drugs, such as warfarin, dabigatran etexilate has obvious advantages: quick onset, predictable anticoagulant efficacy, etc., but like all anticoagulants, dabigatran etexilate will cause bleeding episodes. No specific antagonists against dabigatran etexilate have been developed yet. Clinically, once patients taking dabigatran etexilate suffer from massive bleeding, or require surgery or invasive treatment, measures to r...

Claims

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Application Information

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IPC IPC(8): G01N21/31
CPCG01N21/31
Inventor 赵铁铭
Owner SHANGHAI VASCUTECH DIAGNOSIS CO LTD
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