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Activity determination method of low concentration Ulinastatin

A technology for ulinastatin and activity determination, applied in the direction of color/spectral characteristic measurement, etc., can solve the problems of high cost of inspection reagents, inability to detect samples, and high equipment requirements, and achieve high sensitivity, high equipment requirements, and low cost.

Active Publication Date: 2012-02-15
YANGZHOU AIDEA BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The latter two methods have higher sensitivity, but require more expensive equipment, and the cost of inspection reagents is high and difficult to obtain
The chromogenic substrate method is a commonly used method in the Chinese Pharmacopoeia and the Japanese Pharmacopoeia. Inhibition, using a spectrophotometer to detect the amount of p-nitroaniline (PNA) per unit time to detect the activity of ulinastatin, this method has high accuracy, but the equipment requirements are also high, and the used spectrophotometer needs to be able to measure power The concentration of ulinastatin measured by this current method is about 50U / ml, which cannot detect low concentration ulinastatin (1-20U / ml) samples, such as: the content of ulinastatin in urine Only at 3-9U / ml, activity detection of ulinastatin as low as 1IU / ml is required during process research

Method used

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  • Activity determination method of low concentration Ulinastatin
  • Activity determination method of low concentration Ulinastatin
  • Activity determination method of low concentration Ulinastatin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Take a sample during the purification process of ulinastatin, the concentration of which was determined to be 61U / ml according to the method described in the Chinese Pharmacopoeia 2010 edition, and accurately add 5 times the volume of water to dilute the sample to obtain a low-concentration ulinastatin sample. Carry out ulinastatin concentration determination according to the method of the present invention:

[0025] A low-concentration ulinastatin activity assay method, comprising the steps:

[0026] 1) Take 3 test tubes, tube A 0 , Tube A s , Tube A T , and to A 0 Add 3ml of 0.2M triethanolamine buffer solution to the tube, and add 2.8ml of the triethanolamine buffer solution to the other two tubes;

[0027] 2) to tube A s Add 0.2ml of 10U / ml ulinastatin reference substance to tube A T Add 0.2ml of the sample solution to be tested, mix well, add 0.2ml of 0.01mg / ml trypsin solution to each tube (the final concentration of trypsin in the reaction system is 0.00058...

Embodiment 2

[0039] The sample of 61U / ml in embodiment 1 is diluted 61 times, detects according to the method of the present invention:

[0040] A low-concentration ulinastatin activity assay method, comprising the steps:

[0041] 1) Take 3 test tubes, tube A 0 , Tube A s , Tube A T , and to A 0 Add 3ml of 0.2M triethanolamine buffer solution to the tube, and add 2.8ml of the triethanolamine buffer solution to the other two tubes;

[0042] 2) to tube A s Add 0.2ml of 1U / ml ulinastatin reference substance to tube A T Add 0.2ml of the sample solution to be tested, mix well, add 0.2ml of 0.001mg / ml trypsin solution to each tube (the final concentration of trypsin in the reaction system is 0.000058mg / ml), mix immediately and place in a 37°C water bath keep warm for 10 minutes;

[0043] 3) Add 0.2ml of 20mg / ml BAPNA substrate solution to each tube, mix immediately and keep warm for 60 minutes in a 37°C water bath, add 0.6ml of 20% trichloroacetic acid stop solution to each tube, mix immedi...

Embodiment 3

[0054] The sample of 61U / ml in embodiment 1 is diluted 3 times, detects according to the inventive method:

[0055] A low-concentration ulinastatin activity assay method, comprising the steps:

[0056] 1) Take 3 test tubes, tube A 0 , Tube A s , Tube A T , and to A 0 Add 3ml of 0.2M triethanolamine buffer solution to the tube, and add 2.8ml of the triethanolamine buffer solution to the other two tubes;

[0057] 2) to pipe A s Add 0.2ml of 20U / ml ulinastatin reference substance into tube A T Add 0.2ml of the sample solution to be tested, mix well, add 0.2ml of 0.1mg / ml trypsin solution to each tube (the final concentration of trypsin in the reaction system is 0.0058mg / ml), mix immediately and place in a 37°C water bath keep warm for 10 minutes;

[0058] 3) Add 0.2ml of 20mg / ml BAPNA substrate solution to each tube, mix immediately and keep warm in a 37°C water bath for 10 minutes, add 0.6ml of 20% trichloroacetic acid stop solution to each tube, mix immediately to termin...

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Abstract

The invention discloses an activity determination method of low concentration Ulinastatin. The determination method comprises steps of: using 1-20 U / ml Ulinastatin with a known concentration as a reference substance according to a Ulinastatin concentration of a sample to be detected; adding a trypsin solution respectively and insulating; detecting a remaining activity of trypsin after inhibition by Ulinastatin to detect the Ulinastatin content in the sample, wherein the remaining activity of trypsin is determined by a spectrophotometer at 405 nm luminous absorption using BAPNA (benzoyl DL-arginine p nitroaniline) as a substrate. According to the method, a detection sensitivity of the Ulinastatin sample raises to 1 U / ml, so the method can be directly used in urine sample determination to facilitate determination of patient condition, can determine low concentration samples in various links of Ulinastatin production and processing and can cooperate with quality control and technology research of Ulinastatin production effectively to increase product quality and yield.

Description

technical field [0001] The invention relates to a method for measuring the activity of ulinastatin, in particular to a method for measuring the activity of low-concentration ulinastatin. Background technique [0002] Ulinastatin (English name: Ulinastatin, abbreviated UTI, urinary trypsin inhibitor) is a broad-spectrum hydrolase inhibitor, in the human body in the form of two precursor molecules inter-α-inhibitor (IαI) and pre -α-inhibitor (PαI) exists in plasma and is released in processes involving inflammatory reactions. , biological relevance, and measurement, Adv Clin Chem. 2007; 44: 223-45). Inflammatory processes of various origins promote the release of serine proteases from neutrophils, macrophages, mast cells, lymphocytes, and endothelial cells that contribute to the accelerated clearance of pathogens. However, if these hydrolytic enzymes spread from the local area, it may cause damage to normal cells. To limit the action of these hydrolases, granulocyte elastas...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/31
Inventor 苗丕渠许冬至许珍
Owner YANGZHOU AIDEA BIOTECH