NT-proBNP detection kit based on bimolecular fluorescence complementary technology as well as preparation and use methods

A detection kit, nt-probnp technology, applied in biological testing, fluorescence/phosphorescence, analytical materials, etc., can solve problems such as reducing detection repeatability

Inactive Publication Date: 2018-06-29
NANJING TZONE BIOLOGICAL SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The CMIA method is improved from the ELISA method. Compared with the ELISA method, it has the characteristics of simple operation and fast detection speed. However, the CMIA method is a heterogeneous reaction, and the operation process needs to be cleaned, which reduces the repeatability of the detection.

Method used

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  • NT-proBNP detection kit based on bimolecular fluorescence complementary technology as well as preparation and use methods
  • NT-proBNP detection kit based on bimolecular fluorescence complementary technology as well as preparation and use methods
  • NT-proBNP detection kit based on bimolecular fluorescence complementary technology as well as preparation and use methods

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] The anti-NT-proBNP antibody is coupled to the N-terminal fragment of fluorescent protein. Taking the yellow fluorescent protein (YFP) 1-154 amino acid fragment YFPN as an example, the specific implementation process is as follows:

[0027] 1) Add 0.1mg of YFPN protein into a centrifuge tube, and prepare with 0.05mol / L pH9.5 carbonate buffer (CB) to dilute the YFPN protein to 1mg / ml.

[0028] 2) Add glutaraldehyde at a final concentration of 1.25% in a fume hood.

[0029] 3) Reaction in a water bath at 37°C for 2 hours.

[0030] 4) Dialyze with desalting column Sephadex G-25 or 0.05mol / L pH9.5 CB to remove excess glutaraldehyde.

[0031] 5) Take 0.1 mg anti-NT-proBNP antibody, prepare 1 mg / ml antibody with 0.05 mol / L pH9.5 CB, and mix the activated YFPN protein and antibody.

[0032] 6) React overnight at 4°C.

[0033] 7) Blocking: add 50 μl of 0.2 mol / L lysine solution, and block for 2 hours at room temperature to block residual aldehyde groups and terminate the reac...

Embodiment 2

[0037] The anti-NT-proBNP antibody is coupled to the fluorescent protein C-terminal fragment, taking the yellow fluorescent protein (YFP) 155-238 amino acid fragment YFPC as an example, the specific implementation process is as follows:

[0038] 1) Add 0.1mg of YFPC protein into a centrifuge tube and prepare with 0.05mol / L pH9.5 carbonate buffer (CB) to dilute the YFPC protein to 1mg / ml.

[0039] 2) Add glutaraldehyde at a final concentration of 1.25% in a fume hood.

[0040] 3) Reaction in a water bath at 37°C for 2 hours.

[0041] 4) Dialyze with desalting column Sephadex G-25 or 0.05mol / L pH9.5 CB to remove excess glutaraldehyde.

[0042] 5) Take 0.1 mg anti-NT-proBNP antibody, prepare 1 mg / ml antibody with 0.05 mol / L pH9.5 CB, and mix the activated YFPC protein and antibody.

[0043] 6) React overnight at 4°C.

[0044] 7) Blocking: add 50 μl of 0.2 mol / L lysine solution, and block for 2 hours at room temperature to block residual aldehyde groups and terminate the reacti...

Embodiment 3

[0048] The main components of the kit:

[0049] 1) N-terminal fragment of fluorescent protein coupled with anti-NT-proBNP antibody;

[0050] 2) Anti-NT-proBNP antibody-coupled fluorescent protein C-terminal fragment.

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Abstract

The invention provides an NT-proBNP diagnostic kit based on a bimolecular fluorescence complementary technology. The kit comprises an anti-NT-proBNP antibody coupled fluorescin N-terminal fragment andan anti-NT-proBNP antibody coupled fluorescin C-terminal fragment. The invention further discloses a preparation method of the NT-proBNP diagnostic kit based on the bimolecular fluorescence complementary technology. The method comprises the steps of preparing the anti-NT-proBNP antibody coupled fluorescin N-terminal fragment and preparing the anti-NT-proBNP antibody coupled fluorescin C-terminalfragment. Finally, the invention further discloses a use method of the kit. The kit provided by the invention has the advantages of good specificity, convenience in operation, rapidness in detection,wide linear range, free from cleaning, high accuracy and the like, is convenient in clinical detection use and can be used for improving the accuracy of heart failure diagnosis when the kit is appliedto heart failure monitoring, thereby having a great market value.

Description

technical field [0001] The invention relates to a bimolecular fluorescence complementary technology used for in vitro immunodiagnosis to detect the content of NT-proBNP in a human body, belonging to the field of disease diagnosis and detection. Background technique [0002] In 1988, a polypeptide with strong natriuretic, diuretic, vasodilator and hypotensive effects was first isolated from pig brain by Japanese scholar Tetsuji Sudoh, named Brain Natriuretic Peptide (BNP) , BNP mainly exists in the granules of the ventricular septum, and its secretion increases when the volume of the ventricle expands and the pressure load increases. NT-proBNP and BNP belong to the same family of natriuretic peptides, and their biological sources are the same, but their biological effects and clinical significance are not completely the same. When the pressure load of ventricular myocytes increases, pro-brain natriuretic peptide of 134 amino acid residues is synthesized, and then the signal ...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N21/64
CPCG01N21/6486G01N33/6893
Inventor 徐林
Owner NANJING TZONE BIOLOGICAL SCI & TECH
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