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Method for detecting enzymatic activity of phospholipid-depending factor X activator

A blood coagulation factor and detection method technology, which is applied in the directions of biochemical equipment and methods, microbiological determination/inspection, color/spectral characteristic measurement, etc., can solve problems such as inability to accurately reflect the characteristics of enzyme activity, and achieve easy control and process Simple, efficient effect

Inactive Publication Date: 2012-11-28
ZHAOKE PHARMA HEFEI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method cannot rule out that there may be a nonlinear relationship between the time of the early stage of activation and the amount of Xa produced, so it cannot accurately reflect the characteristics of the activity of the enzyme

Method used

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  • Method for detecting enzymatic activity of phospholipid-depending factor X activator
  • Method for detecting enzymatic activity of phospholipid-depending factor X activator
  • Method for detecting enzymatic activity of phospholipid-depending factor X activator

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] ①Use 0.02M Tris-HCl buffer (pH 7.4) to prepare the FXA chromogenic substrate S-2765 into a solution with a concentration of 3mM; accurately measure 0.8ml of the prepared 3mM S-2765 solution and add 0.6ml of 0.075M CaCl 2 solution, mix well and set aside;

[0039] ②Take one piece of Human Factor X (100μg), dilute it with 0.02M Tris-HCl buffer (pH 7.4) to a solution with a concentration of 2.5μM, mix well and set aside;

[0040] ③ Add 10 mg of human serum albumin to 10 ml of 0.02M Tris-HCl buffer (pH 7.4) to prepare a serum albumin dilution with a concentration of 1 mg / ml;

[0041] ④Use the prepared 1mg / ml serum albumin diluent and 0.02M Tris-HCl buffer solution (pH 7.4) to dilute the FXA monomer standard substance into 7 different concentrations of standard substance mother solutions, the concentrations of which are: 1.4, 1.2, 1.0, 0.8, 0.6, 0.4, 0.2 Ku / ml;

[0042] ⑤ Randomly select the snake venom hemocoagulase injection preparation produced by Zhaoke Pharmaceutical ...

Embodiment 2

[0047] Take 10 samples of FXA to be tested with concentrations of 0.34, 0.355, 1.904, 0.421, 0.602, 0.206, 0.477, 0.662, 0.683 and 1.767mg / ml prepared by Zhaoke Pharmaceutical (Hefei) Co., Ltd., respectively, with 0.02M Tris Dilute the batch of test samples with HCl buffer solution and 1 mg / ml serum albumin diluent (the diluted concentration of the batch of test samples is 35 ug / ml). In the microtiter plate, the standard curve reaction system is the same as in Example 1, with 3 duplicate wells each. The reaction system of the sample to be tested was 23 μl of the chromogenic substrate S-2765 solution, 30 μl of the Human Factor X solution, and 7 μl of the sample solution to be tested in each well, and mixed immediately. Put the prepared microplate plate into the microplate reader preheated for 4 minutes to 37 ° C for 15 minutes, and measure the absorbance A405 of the enzymatic hydrolysis product p-nitroaniline (PNA) in each hole at a wavelength of 405 nm in parallel to make For...

Embodiment 3

[0051] Take 20 batches of samples of snake venom hemagglutinin intermediates produced by Zhaoke Pharmaceutical (Hefei) Co., Ltd., the batch numbers are 20080403, 20080404, 20080405, 20080406, 20080407, 20080501, 20080602, 20080605, 20090104, 20090105, 2040203 , 20090402, 20090501, 20090601, 20090702, 20090703, 20091001, 20100401 and 20100501, the specifications are 200, 100 and 400Ku / ml respectively, and the batch is diluted with 0.02M Tris-HCl buffer and 1mg / serum albumin diluent to be tested Sample (the concentration of the batch of samples to be tested after dilution is 0.8Ku / ml). In the microtiter plate, the standard curve reaction system is the same as in Example 1, and each concentration has 3 duplicate wells. The reaction system of the sample to be tested was 23 μl of the chromogenic substrate S-2765 solution, 30 μl of the Human Factor X solution, and 7 μl of the sample solution to be tested in each well, and mixed immediately. Put the prepared microplate plate into th...

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Abstract

The invention discloses a method for quickly and specifically detecting the enzymatic activity of a phospholipid-depending factor X activator. A chromogenic substrate assay is adopted, a microplate reader is used, a linear reaction curve is obtained in a certain reaction time and a certain-activity standard substance range, a linear relation between a light absorption value of a product and the concentration of the phospholipid-depending factor X activator is established, and the corresponding enzymatic activity of a sample is calculated. In addition, the recovery rate and accuracy of the standard curve are carefully researched. The method is high in accuracy, simple in steps and convenient to impellent in a laboratory, and is suitable for quality control of X activator raw materials and hemocoagulase preparations.

Description

technical field [0001] The invention relates to the fields of medicines and biological products, in particular to a method for measuring phospholipid-dependent blood coagulation factor X activator (FXA) enzyme activity by a chromogenic substrate method. Background technique [0002] Snake venom hemagglutinin is extracted from viper venom and purified by modern biotechnology. It is an enzyme preparation mainly for hemostasis, containing two components that can promote blood coagulation, namely batroxobin and phospholipid-dependent coagulation factor X activator (FXA for short). Among them, FXA is a highly specific enzyme with proteolytic properties, which is widely distributed in the snake venoms of rattlesnakes and vipers. Most of the snake venom FXA is purified from viper and Macrovipera lebetina, which are called RVV-X and VLFXA respectively, and RVV-X has the strongest activity. These two FXAs are composed of a heavy chain α and two light chains β and γ of the C-type le...

Claims

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Application Information

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IPC IPC(8): G01N21/31C12Q1/37
Inventor 戴向荣刘娟娟方丽杨中强李小羿张国辉
Owner ZHAOKE PHARMA HEFEI
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