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MPO detection kit based on bimolecular fluorescence complementation technology, preparation method and use method thereof

A detection kit and bimolecular technology, applied in the field of bimolecular fluorescence complementation, can solve the problems of reducing detection repeatability and the like

Inactive Publication Date: 2018-08-10
NANJING TZONE BIOLOGICAL SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The magnetic particle chemiluminescence method is improved from the enzyme-linked immunosorbent assay method. Compared with it, it has the characteristics of simple operation and fast detection speed. However, the magnetic particle chemiluminescence method is a heterogeneous reaction, and the operation process needs to be cleaned, which reduces the detection efficiency. Repeatability

Method used

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  • MPO detection kit based on bimolecular fluorescence complementation technology, preparation method and use method thereof
  • MPO detection kit based on bimolecular fluorescence complementation technology, preparation method and use method thereof
  • MPO detection kit based on bimolecular fluorescence complementation technology, preparation method and use method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] The anti-MPO 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:

[0029] 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.

[0030] 2) Add glutaraldehyde at a final concentration of 1.25% in a fume hood.

[0031] 3) Reaction in a water bath at 37°C for 2 hours.

[0032] 4) Dialyze with desalting column Sephadex G-25 or 0.05mol / LpH9.5CB to remove excess glutaraldehyde.

[0033] 5) Take 0.1 mg anti-MPO antibody, prepare 1 mg / ml antibody with 0.05 mol / L pH9.5CB, and mix the activated YFPN protein and antibody.

[0034] 6) React overnight at 4°C.

[0035] 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 reaction.

[0036]...

Embodiment 2

[0039] The anti-MPO 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:

[0040] 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.

[0041] 2) Add glutaraldehyde at a final concentration of 1.25% in a fume hood.

[0042] 3) Reaction in a water bath at 37°C for 2 hours.

[0043] 4) Dialyze with desalting column Sephadex G-25 or 0.05mol / L pH9.5CB to remove excess glutaraldehyde.

[0044] 5) Take 0.1 mg anti-MPO antibody, prepare 1 mg / ml antibody with 0.05 mol / L pH9.5CB, and mix the activated YFPC protein and antibody.

[0045] 6) React overnight at 4°C.

[0046] 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 reaction.

[0047] ...

Embodiment 3

[0050] The main components of the kit:

[0051] 1) N-terminal fragment of fluorescent protein coupled with anti-MPO antibody;

[0052] 2) Anti-MPO antibody-coupled fluorescent protein C-terminal fragment.

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Abstract

The invention provides a MPO detection kit based on a bimolecular fluorescence complementation technology, a preparation method and a use method thereof, wherein the kit comprises an anti-MPO antibodyconjugated fluorescent protein N-terminal fragment and an anti-MPO antibody conjugated fluorescent protein C-terminal fragment. The invention further discloses a preparation method of the MPO diagnosis kit, wherein the preparation method comprises: preparation of an anti-MPO antibody conjugated fluorescent protein N-terminal fragment, and preparation of an anti-MPO antibody conjugated fluorescentprotein C-terminal fragment. The invention further discloses a use method the kit. According to the present invention, the kit has advantages of simple operation, good specificity, no cleaning, highaccuracy and the like, is conveniently suitable for clinical detection, can increase the accuracy of the diagnosis of cardiovascular diseases in the monitoring of cardiovascular diseases, and has 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 MPO in a human body, belonging to the field of disease diagnosis and detection. Background technique [0002] Myeloperoxidase (MPO), also known as peroxidase, is a heme protease containing a heme prosthetic group secreted by neutrophils, monocytes, and macrophages in certain tissues. A member of the peroxidase superfamily. In mature granulocytes, MPO is the most abundant glycoprotein, accounting for about 5% of the total protein content in peripheral blood polymorphonuclear neutrophils (PMNs), and 95% of MPO in blood comes from PMNs. [0003] The relative molecular mass of MPO is 150×10 3 , is a dimer composed of two subunits, and each subunit is composed of a heavy chain (α chain, with a relative molecular mass of about 60×10 3 ) and a light chain (β chain, relative molecular mass about 15×10 3 ) constitutes. The...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/533G01N33/544
CPCG01N33/533G01N33/544G01N33/6893
Inventor 徐林
Owner NANJING TZONE BIOLOGICAL SCI & TECH
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