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Method for detecting human neutrophil elastase based on nucleic acid aptamer

A neutrophil and elastase technology, which is applied in the field of protein detection, can solve the problems of low sensitivity of nucleic acid probe replacement method and cannot meet high-sensitivity detection, and achieve the effects of improved detection sensitivity, easy operation and improved sensitivity

Inactive Publication Date: 2013-02-13
SHANXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The reported fluorescence polarization method or fluorescent dye-labeled nucleic acid probe displacement method using nucleic acid aptamers to detect HNE has low sensitivity and cannot meet the requirements of highly sensitive detection (Jayasena, S.D.Clin.Chem.1999, 45, 1628-1650 ; He, J.L.; Wu, Z.-S.; Zhang S.-B.; Shen, G.-L.; Yu, R.-Q. Talanta 2010, 80, 1264–1268.)

Method used

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  • Method for detecting human neutrophil elastase based on nucleic acid aptamer
  • Method for detecting human neutrophil elastase based on nucleic acid aptamer
  • Method for detecting human neutrophil elastase based on nucleic acid aptamer

Examples

Experimental program
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Effect test

Embodiment 1

[0036] Example 1: Detection of neutrophil elastase using chromogenic substrate Meo-Suc-AAPV-pNA and nucleic acid aptamer-modified ELISA plate

[0037] Immobilization of aptamers on ELISA plates: aptamers labeled with biotin have the following sequence: 5'-biotin-TAG CGA TAC TGC GTG GGT TGG GGC GGG TAG GGC CAG CAG TCTCGT-3 '. Add 100 microliters of streptavidin (10 μg / mL, 0.1 M Na 2 CO 3 , pH 9.0) were incubated overnight at 4°C. After washing, use blocking solution (20mM Tris-HCl, 140mM NaCl, 2mg / mL BSA, 0.1%Tween 20, pH 7.5) to incubate at 37°C for 30 minutes to block the unbound sites on the microwell plate. Add 100 μl of biotin-labeled aptamer (200 nM aptamer, 20 mM Tris-HCl, 1 M NaCl, 0.1% Tween 20, pH 7.5) to each well, react at 37 ° C for 1 hour, and wash for 3 Next, store at 4°C for later use.

[0038] Detection of neutrophil elastase: Neutrophil elastase (HNE) was captured with a reaction solution (100mM Tris-HCl, 150mM NaCl, 2mM MgCl 2 , 6mM KCl, 0.1%Tween 20, p...

Embodiment 2

[0039] Example 2: Detection of neutrophil elastase using fluorescent substrate Meo-Suc-AAPV-AMC and nucleic acid aptamer-modified microtiter plate

[0040] Immobilization of aptamers on ELISA plates: aptamers with biotin have the following sequence: 5'-biotin-TAG CGA TAC TGC GTG GGT TGG GGC GGG TAG GGC CAG CAG TCTCGT-3' . Add 100 microliters of streptavidin (10 μg / mL streptavidin, 0.1M Na 2 CO 3 , pH 9.0) were incubated overnight at 4°C. After washing, use blocking solution (20mM Tris-HCl, 140mM NaCl, 2mg / mL BSA, 0.1%Tween 20, pH 7.5) to incubate at 37°C for 30 minutes to block the unbound sites on the microwell plate. Add 100 μl of biotin-labeled aptamer (200 nM aptamer, 20 mM Tris-HCl, 1 M NaCl, 0.1% Tween20, pH 7.5) to each well, react at 37 ° C for 1 hour, wash 3 times, Store at 4°C for later use.

[0041] Detection of neutrophil elastase: Neutrophil elastase (HNE) was captured in reaction buffer (100mM Tris-HCl, 150mM NaCl, 2mM MgCl 2 , 6mM KCl+0.1%Tween 20, pH 7.0)...

Embodiment 3

[0042] Example 3: Specificity investigation of the method for detecting human neutrophil elastase using chromogenic substrate Meo-Suc-AAPV-pNA and nucleic acid aptamer-modified ELISA plate.

[0043] The detection was carried out according to the detection steps in Example 1, and the detected protein concentrations were as follows: human neutrophil elastase (HNE), porcine pancreatic elastase (PPE), chymotrypsin, human coagulation Enzyme (human α-thrombin), proteinase 3 (proteinase 3), and cathepsin G (cathepsin G) concentrations were 0.5 nM. Detection of the coexistence of human neutrophil elastase (0.5nM) and other proteins, including lysozyme, human transferrin, hemoglobin (Hb), human albumin (human serum albumin, HSA) and immunoglobulin G (IgG), the concentration of the coexisting protein was 50nM. Only human neutrophil elastase was specifically detected, and other proteins under investigation did not produce any interference ( Figure 4 ).

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Abstract

The invention provides a method for detecting human neutrophil elastase based on nucleic acid aptamer and a kit for detecting human neutrophil elastase based on nucleic acid aptamer. According to the method, the nucleic acid aptamer is modified on an ELISA Plate, used as a recognition reagent, and selectively captures neutrophil elastase molecules; the substrate is catalyzed by the neutrophil elastase to generate products; and by measuring the change of absorbance or fluorescence intensity, the high-sensitivity high-specificity detection of the human neutrophil elastase is realized. The method is easy to implement and high in specificity and sensitivity, and high throughput detection is easy to implement.

Description

technical field [0001] The invention relates to protein detection technology, in particular to a method and a kit for detecting human neutrophil elastase based on nucleic acid aptamers. Background technique [0002] Human neutrophil elastase (HNE) is an important protease released by neutrophils, which undertakes a variety of biological functions. HNE is closely related to the occurrence and development of many human diseases, such as emphysema, cystic Acute pulmonary fibrosis, acute lung injury and acute respiratory distress syndrome, chronic intestinal diseases, myocarditis, arthritis, etc. In addition, HNE also plays an important role in the body's immune defense, inflammatory response and its regulation. HNE is an important biomarker and a measurement index for the diagnosis of various diseases. Establishing a highly sensitive and specific HNE detection method is of great significance in the study of the physiological and pathological functions of HNE, and the diagnosi...

Claims

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Application Information

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IPC IPC(8): G01N33/558G01N33/531G01N21/31
Inventor 赵强程琳
Owner SHANXI UNIV
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