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Method for measuring content of heparin in human antithrombase-III concentrate

An antithrombin and concentrate technology, applied in the field of biological detection, can solve the problems of large interference of antithrombin-III and low measurement accuracy, and achieve the effects of high sensitivity, small reagent dosage and high accuracy

Active Publication Date: 2013-04-24
BLOOD TRASFUSION INST CHINESE ACAD OF MEDICAL SCI +1
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0006] Purpose of the invention: The purpose of the present invention is to propose a new heparin in antithrombin-III concentrate in view of the technical problems such as large interference of the sample antithrombin-Ⅲ and low measurement accuracy in the above-mentioned measurement technology. Determination method of content

Method used

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  • Method for measuring content of heparin in human antithrombase-III concentrate
  • Method for measuring content of heparin in human antithrombase-III concentrate
  • Method for measuring content of heparin in human antithrombase-III concentrate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Determination of the content of heparin in antithrombin-Ⅲ solution (heat treatment at different temperatures)

[0029] 1. Processing of samples to be tested:

[0030] (1) Preparation of samples to be tested: take human antithrombin-Ⅲ and heparin standard products, prepare three kinds of human antithrombin-Ⅲ solutions containing different concentrations of heparin, and the concentrations of human antithrombin-Ⅲ are 1IU / ml, the heparin content is 0.02, 0.04 and 0.05IU / ml respectively;

[0031] (2) Divide the three prepared samples into four groups: control group, 60°C heat treatment group, 70°C heat treatment group and 80°C heat treatment group. The solution in the control group was not treated, and the solutions in the three heat treatment groups were incubated at 60°C, 70°C and 80°C for 30 minutes.

[0032] 2. Preparation of standard curve and determination of samples:

[0033] (1) Dilute the heparin standard with known potency to 0.0125, 0.025, 0.05, 0.1...

Embodiment 2

[0051] Example 2: Determination of the content of heparin in antithrombin-Ⅲ solution (using different heat treatment time)

[0052] 1. Processing of samples to be tested:

[0053] (1) Preparation of samples to be tested: take human antithrombin-Ⅲ and heparin standard products, and prepare human antithrombin-Ⅲ solution containing heparin, in which the concentration of human antithrombin-Ⅲ is 1IU / ml. Heparin content is 0.05IU / ml;

[0054] (2) Divide the prepared sample to be tested into two parts, and divide them into three groups A, B and C respectively. C) Incubate under three different temperature conditions for 20 min; the three groups in the second copy were incubated under three different temperature conditions of 60°C (A), 70°C (B) and 80°C (C) for 50 min.

[0055] 2. Preparation of standard curve and determination of samples:

[0056] (1) Dilute the heparin standard with known potency to 0, 0.025, 0.05, 0.1IU / ml with Tris buffer (pH8.4) as the standard solution;

...

Embodiment 3

[0074] Example 3: Determination of Heparin Content in Antithrombin-Ⅲ Concentrate

[0075] 1. Processing of samples to be tested:

[0076] Preparation of the sample to be tested: take the human antithrombin-Ⅲ concentrate purified by heparin affinity chromatography, its concentration is determined according to the method of the 32nd edition of the United States Pharmacopoeia, and accurately diluted to 1IU / ml, the diluted sample Divide into three parts and incubate for 30 min at three different temperature conditions of 60°C, 70°C and 80°C respectively.

[0077] 2. Preparation of standard curve and determination of samples:

[0078] (1) Dilute the heparin standard with known potency to 0, 0.0125, 0.025, 0.05, 0.1IU / ml with Tris buffer (pH8.4) as the standard solution;

[0079] (2) Take 40 μl of heparin standard solution and three kinds of sample solutions to be tested in different wells of the microplate;

[0080] (3) Add an equal volume of 1IU / ml antithrombin-Ⅲ standard sol...

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Abstract

The invention discloses a method for measuring the content of heparin in a human antithrombase-III concentrate. The method is characterized by comprising the steps of treating a sample to be measured, making a standard curve and measuring the sample, wherein the step of treating the sample to be measured includes diluting and heating, and a micro color developing substrate method is adopted in the steps of making the standard curve and measuring the sample. The method has the benefits as follows: the micro color developing substrate method is utilized to perform the measurement operation and has the advantages of high sensitivity, simplicity and time saving for operation, low usage amount of required detection reagent and the like; the sample to be measured-the antithrombase-III concentrate is heated for 20-50 min at the temperature of 60-80 DEG C, so that antithrombase-III components with great influence on results in the sample to be measured can be inactivated, but the activity of the heparin is not affected, and the accuracy of the measurement results is improved; and the stability and the accuracy are high as the results are calculated by drawing the standard curve.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a method for determining the content of heparin in human antithrombin-Ⅲ concentrate. Background technique [0002] Human antithrombin-Ⅲ (antithrombin-Ⅲ, AT-Ⅲ) concentrate is a plasma protein product separated and purified from human plasma. It is mainly used to treat antithrombin-Ⅲ deficiency and surgery caused by various reasons. caused by thrombus etc. At present, the purification and preparation process of antithrombin-Ⅲ uses heparin affinity chromatography as the main step. During the purification process, the heparin in the chromatographic packing material will fall off. The heparin content was detected. The European Pharmacopoeia 7.0 (EP7.0) and the United States Pharmacopoeia 32nd edition (United States Pharmacopeia 32, USP32) require antithrombin-Ⅲ concentrate as per international unit (IU) of antithrombin-Ⅲ The content of heparin in the medici...

Claims

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Application Information

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