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Determination method for heparin activity

A determination method and heparin technology, applied in the measurement of color/spectral characteristics, etc., can solve the problems of large deviation and unsuitable for the detection of low-activity heparin samples, and achieve the effects of high accuracy, simple operation and improved detection efficiency.

Active Publication Date: 2013-04-24
BLOOD TRASFUSION INST CHINESE ACAD OF MEDICAL SCI +1
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  • Abstract
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Problems solved by technology

The 32nd edition of the United States Pharmacopeia (United States Pharmacopeia 32, USP32) adopts the chromogenic substrate method for the determination of heparin activity, and utilizes the fact that the heparin-antithrombin complex can inhibit the chromogenic reaction of activated coagulation factor X hydrolyzing the chromogenic substrate. Basic principle, although the method reduces the detection time and simplifies the operating procedure, due to the limitation of its reaction system and time, it is necessary to use a logarithmic equation to fit the standard curve, and after the calculation of the logarithmic equation, the detection result and the actual deviation Larger, less suitable for detection of low activity heparin samples

Method used

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  • Determination method for heparin activity

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Embodiment 1

[0032] Example 1: Determination of Heparin Activity in Standards

[0033] (1) The heparin standard (1000IU / ml) produced by the European Directorate for Quality Medicines (EDQM) was used as the test product, and the test product was mixed with 0.02mol / L Tris buffer (pH8.4). Dilute 20,000 times, 50,000 times and 100,000 times respectively as standard substances A, B, and C to be tested.

[0034] (2) Take another batch of EDQM heparin standard and dilute it with 0.02mol / L Tris buffer (pH8.4) to a gradient concentration of 0.1 IU / ml, 0.075 IU / ml, 0.05 IU / ml, 0.025 IU / ml ml, 0.0125 IU / ml, 0.00625 IU / ml, and 0 IU / ml were used as solutions for standard curve formulation.

[0035] (3) Take 20 μl of the standard curve formulation solution and standard substances A, B, and C to be tested in different wells of the microplate;

[0036] (4) Add 20 μl of 1 IU / ml antithrombin-Ⅲ standard solution to each well, mix well, and incubate at 37°C for 3 minutes;

[0037] (5) Add 20 μl of 10 nka...

Embodiment 2

[0051] Example 2: Determination of Heparin Activity in Human Plasma

[0052] (1) Take the isolated human fresh sodium citrate anticoagulated plasma as the test sample, and use the plasma and the 10-fold diluted plasma as the test samples A and B respectively;

[0053] (2) Dilute EDQM heparin standard with 0.02mol / L Tris buffer (pH8.4) to 0.1 IU / ml, 0.075 IU / ml, 0.05 IU / ml, 0.025 IU / ml, 0.0125 IU / ml, 0.00625 IU / ml , 0 IU / ml gradient concentration, make a standard curve to formulate the solution.

[0054] (3) Take 40 μl of the standard curve formulation solution and samples A and B to be tested in different wells of the microplate;

[0055] (4) Add 40 μl of 1 IU / ml antithrombin solution to each well, mix well, and incubate at 37°C for 3 minutes;

[0056] (5) Add 40 μl of 10 nkat / ml bovine FXa solution to each well, mix well, and incubate at 37°C for 1.5 minutes;

[0057] (6) Add 40 μl of 2.5 mmol / L S-2765 solution to each well, mix well, and incubate at 37°C for 3 minutes; ...

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Abstract

The invention discloses a determination method for heparin activity. The method comprises three steps of treatment of standard substances and samples, establishment of a standard curve and sample measurement, and calculation of heparin activity contents in the samples, wherein reaction systems and time are as follows: (1) 10-40 microliters standard curve establishment solution and samples with 0-0.1 IU / ml heparin activity concentration are mixed with 10-40 microliters antithrombase with 1 IU / ml concentration, and are incubated for 3 minutes at the temperature of 37 DEG C; (2) 10-40 microliters activated bovine blood coagulation factor X with 10 nkat / ml concentration is added for mixing, and is incubated for 1.5 minutes at the temperature of 37 DEG C; (3) 10-40 ml color development substrate S-2765 with 2.5 mmol / L concentration is added for mixing, and is incubated for 3 minutes at the temperature of 37 DEG C; and (4) 10-40 microliters acetic acid with 50% of volume concentration is added for evenly mixing, and then the reaction is stopped. The method has the advantages of lower reagent dosage, high detection efficiency and high precision of measurement results, and is more suitable for detecting the samples with lower heparin activity.

Description

technical field [0001] The invention belongs to the technical field of biomedical detection, and in particular relates to a method for measuring heparin activity. Background technique [0002] Heparin is a polymer composed of two polysaccharides connected alternately. It has a strong anticoagulant effect both in vivo and in vitro. It is widely used clinically as an anticoagulant drug, mainly for the treatment of thrombotic diseases. Treatment and anticoagulant treatment of cardiovascular surgery, hemodialysis, extracorporeal circulation and other processes. [0003] As a drug, the precise detection of heparin's activity and function is of great significance for the quality control of the production process and the dynamic monitoring of patients in clinical treatment. As a biologically active substance, its detection cannot be done by chemical or physical methods alone, but must be combined with biological detection methods. At present, coagulation method and chromogenic sub...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/31
Inventor 曹海军叶生亮胡吉军袁靖李长清陈云华刘欣晏杨刚
Owner BLOOD TRASFUSION INST CHINESE ACAD OF MEDICAL SCI
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