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A method for detecting mmp-9 by using fluorescent zymogram imprinting technique

A technology of MMP-9 and technical detection, applied in the biological field, can solve the problems such as difficult to meet the sensitivity, achieve the effect of avoiding different background coloring and improving detection sensitivity

Active Publication Date: 2019-06-21
SHAANXI MYBIOTECH CO LTD
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Problems solved by technology

Gelatin zymography is difficult to meet the sensitivity required for clinical detection

Method used

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  • A method for detecting mmp-9 by using fluorescent zymogram imprinting technique
  • A method for detecting mmp-9 by using fluorescent zymogram imprinting technique
  • A method for detecting mmp-9 by using fluorescent zymogram imprinting technique

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Embodiment Construction

[0026] The present invention will be further described in detail below in conjunction with specific embodiments, which are explanations of the present invention rather than limitations.

[0027] On the premise of retaining the advantages of the gelatin zymography method, the present invention adds a simple serum concentration step to increase the enzyme concentration in the sample by 5 times. At the same time, a fluorescent-labeled substrate is used to replace the traditional gelatin, and the protein is transferred to the surface of the nitrocellulose membrane to react with the substrate using the capillary blotting method. There is no background coloring caused by a large amount of protein in the sample that is common in other zymography detection methods. The detection sensitivity can be significantly improved, and accurate quantitative analysis can be performed. Aiming at the low concentration of MMP-9 in serum, this method adds a simple concentration step on the basis of m...

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Abstract

The invention belongs to the technical field of biotechnologies and discloses a method for detecting MMP-9 by a fluorescence zymography imprinting technology. The method includes: 1) subjecting a to-be-detected serum sample to incubation centrifuge according to a gel protein concentration method to increase enzyme concentration of the serum sample by five times; 2) adopting a fluorescence zymography imprinting method for distinguishing various enzymes and proteins in the concentrated to-be-detected serum sample by electrophoresis according to molecular weights; 3) adopting a capillary imprinting method for transferring the proteins distinguished at the step 2) to the surface of a nitrocellulose membrane, and degrading a substrate pre-cured on the surface of the nitrocellulose membrane; 4) after degradation of the substrate, separating a fluorescence group from a quenching group to emit fluorescence under a specific excitation wavelength, and detecting fluorescence intensity to judge enzyme activity of MMP9 in the to-be-detected serum sample. On the premise that advantages of gelatin zymography are kept, serum is concentrated, and detection sensitivity is improved; by adoption of the fluorescence zymography imprinting method, experimental results are low in background, and high sensitivity and quantifiability of the results are realized.

Description

technical field [0001] The invention belongs to the technical field of biotechnology, and relates to a method for detecting enzyme activity of a low-concentration MMP-9 sample, in particular to a method for detecting MMP-9 by using a fluorescence zymogram imprinting technique. Background technique [0002] Matrix Metalloproteinase (MMP) is a group of Ca 2+ / Zn 2+ The main function of dependent neutral protease is to degrade and remodel the extracellular matrix (extracellular matrix, ECM) of various tissues in the whole body, and more than 20 kinds have been found. Among them, MMP2 and MMP-9 (gelatinase B) are important members of the matrix metalloproteinase family. In some pathological processes, the abnormal activation of these two proteases will degrade the main components of the blood-brain barrier basement membrane, laminin and Fibronectin, etc., destroy the blood-brain barrier, causing the permeability of the blood-brain barrier to increase or open. [0003] In rece...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/64G01N33/573
CPCG01N21/6428G01N33/573G01N2021/6432G01N2333/96491
Inventor 闫亚平秦李娜李科
Owner SHAANXI MYBIOTECH CO LTD
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