Unlock instant, AI-driven research and patent intelligence for your innovation.

Activity determination method of low concentration Ulinastatin

A technology for ulinastatin and activity determination, which is applied in the direction of material analysis by observing the effect on chemical indicators, and analysis by chemical reaction of materials, etc., can solve the problem of high cost of inspection reagents, inability to sample detection, and equipment requirements Advanced problems, to achieve the effect of high sensitivity, high equipment requirements and low cost

Active Publication Date: 2013-01-16
YANGZHOU AIDEA BIOTECH
View PDF3 Cites 0 Cited by
  • 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 commonly used in the Chinese Pharmacopoeia and the Japanese Pharmacopoeia. Inhibition, use a spectrophotometer to detect the amount of p-nitroaniline (PNA) generated per unit time to detect the activity of ulinastatin. This method has high accuracy, but the equipment requirements are also high. The spectrophotometer used needs to be able to measure the kinetic energy. The concentration of ulinastatin determined by the current method is about 50U / ml, and it is unable to 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • 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 0.2 M 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 0.0005...

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 0.2 M 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 in a 37°C water bath for 60 minutes, add 0.6ml of 20% trichloroacetic acid stop solution to each tube, mix imme...

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 0.2 M triethanolamine buffer solution to the tube, and add 2.8ml of the triethanolamine buffer solution to the other two tubes;

[0057] 2) to tube 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 stop ...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

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 the plasma and is released in processes involving inflammatory reactions. It is an anti-inflammatory molecule associated with the human body during the inflammatory process (MJ Pugia et al, Bikunin (urinary trypsin inhibitor): structure , 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...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/78
Inventor 苗丕渠许冬至许珍
Owner YANGZHOU AIDEA BIOTECH