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Test paper strip for detecting NT-proBNP as well as preparation method and application thereof

A technology of nt-probnp and test strips, which is applied in the field of test strips for detecting NT-proBNP and its preparation, can solve problems such as difficult and accurate detection, and achieve the goals of improving sensitivity and accuracy, increasing sensitivity, and reducing detection limit Effect

Inactive Publication Date: 2018-06-29
HUBEI UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Under the conditions of low antigen concentration and extremely short reaction time, it is difficult for a single immunochromatographic technique to accurately detect low-concentration NT-proBNP

Method used

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  • Test paper strip for detecting NT-proBNP as well as preparation method and application thereof
  • Test paper strip for detecting NT-proBNP as well as preparation method and application thereof
  • Test paper strip for detecting NT-proBNP as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Screening of Antibody Pairs

[0030] (1) Antibody 5B6 was used as the conjugated antibody, and an appropriate antibody was selected to pair with 5B6 to form a double-antibody sandwich model.

[0031] (2) Antibody 5B6 was used as a labeled antibody, and antibody 15C4, antibody 13G12, antibody 24E11, antibody 15F11, antibody 29D12, antibody 18H5, and antibody 16E6 were used as T-line antibodies, respectively, to prepare immunochromatographic test strips.

[0032] (3) NT-proBNP with concentrations of 30000, 9000 and 0 ng / L were detected with the immunochromatographic test strips prepared above.

[0033] Use a fluorescence detector to detect the fluorescence intensity of the T-line and C-line, draw a column graph with different antibody pair combinations as the abscissa and T / C value as the ordinate, and compare the T / C values ​​under different antigen concentrations. see results Figure 4 , when antibody 5B6 was used to label fluorescent microspheres and antibo...

Embodiment 2

[0034] Example 2 Preparation of Antibody 5B6 Labeled Fluorescent Microspheres

[0035] Draw 100 μL of carboxy fluorescent microspheres (about 1 mg carboxy fluorescent microspheres, particle size 400 nm), centrifuge at 14400 r / min for 10 min to collect fluorescent microspheres, and then wash twice with 1 mL of 0.1 M MES buffer. Add the washed fluorescent microspheres, 1.0 mg EDC and 1.0 mg NHS to 5 mL MES buffer, and centrifuge at room temperature for 45 min at 60 r / min. Collect the fluorescent microspheres after centrifugation, wash twice with 1mL 10mM PB buffer at pH 7.4, reconstitute with 500μL PB buffer, add 7.04μL antibody 5B6 (7.1mg / mL), and react overnight at 4°C . Centrifuge at 14400r / min for 8min to collect the fluorescent microspheres reacted overnight (fluorescent microspheres coupled with antibody 5B6), add the microspheres and 125μL BSA to an equal volume of 10mM pH7.4 PB buffer at room temperature for 1h, and again at 14400r / min The microspheres were collected b...

Embodiment 3

[0036] Example 3 Preparation of Biotinylated Antibody

[0037] (1) Take 100 μg of antibody 15F11 and add NHS-Biotin at a molar ratio of antibody / activated biotin of 1:100, with a total volume of 100 μL;

[0038] (2) Cultivate at room temperature for 4 hours;

[0039] (3) Dilute to 5 times the volume of the reaction solution with PB buffer, dialyze at 4°C for 72 hours, and take it out for later use.

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Abstract

The invention discloses a test paper strip for detecting NT-proBNP as well as a preparation method and application thereof, and belongs to the field of biological detection. The test paper strip mainly consists of a sample pad, a combination pad, a nitrocellulose membrane and an absorption pad, wherein the sample pad is assembled on a bottom plate; the nitrocellulose membrane contains a detectionline T and a control line C; the combination pad is fixedly provided with an antibody 5B6 labeled fluorescent microsphere and a biotinylated antibody 15F11; the detection line T is coated with streptavidin; the control line C is coated with goat anti mouse IgG. Through the preparation of the combination pad and the nitrocellulose membrane, the sample pad, the combination pad, the nitrocellulose membrane and the absorption pad are sequentially and mutually lapped and pasted on the bottom plate to be assembled; cutting is performed to obtain the test paper strip. The defects of low sensitivity and poor accuracy of a traditional fast detection method are overcome; the fast capture on trace NT-proBNP can be realized at shorter time; the detection limit is reduced; the detection sensitivity isgreatly improved.

Description

technical field [0001] The invention belongs to the field of biological detection, in particular to a test strip for detecting NT-proBNP, a preparation method and application thereof. Background technique [0002] NT-proBNP is an inactive N-terminal polypeptide fragment produced by endonuclease digestion of pro-brain natriuretic peptide (pro-BNP), which is mainly present in cardiomyocytes in response to ventricular wall tension or local ischemia. in the secretions. A large number of studies have shown that: NT-proBNP has a long half-life and strong stability. It can be used as the main index of heart failure diagnostic test and plays an important role in the stage of disease diagnosis and prognosis evaluation. Nowadays, the methods for quantitative detection of NT-proBNP mainly include radioimmunoassay, enzyme-linked immunosorbent assay, electrochemiluminescence immunoassay and fluorescence immunochromatography. The cost of radioimmunoassay technology is low, but there is ...

Claims

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Application Information

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IPC IPC(8): G01N33/533G01N33/543G01N33/558
Inventor 孙宏浩赵晓双
Owner HUBEI UNIV OF TECH
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