NGAL detection kit based on bimolecular fluorescence complementary technology, preparation and use methods

A detection kit and bimolecular technology, applied in the field of bimolecular fluorescence complementation, can solve the problems of complex reagents, easy inactivation of enzymes, and low sensitivity

Inactive Publication Date: 2018-07-06
NANJING TZONE BIOLOGICAL SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The reaction specificity of latex turbidimetry is not good, and the reagents required are relatively complicated
The enzymes of enzyme-linked immunosorbent assay are easy to inactivate, resulting in low sensitivity, and the spatial structure of the labeled substance is easily affected by the macromolecular labeling of the enzyme, which limits the im...

Method used

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  • NGAL detection kit based on bimolecular fluorescence complementary technology, preparation and use methods
  • NGAL detection kit based on bimolecular fluorescence complementary technology, preparation and use methods
  • NGAL detection kit based on bimolecular fluorescence complementary technology, preparation and use methods

Examples

Experimental program
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Effect test

Embodiment 1

[0027] The anti-NGAL antibody is coupled to the N-terminal fragment of fluorescent protein, taking the fragment YFPN of 1-154 amino acids of yellow fluorescent protein (YFP) as an example, the specific implementation process is as follows:

[0028] 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.

[0029] 2) Add glutaraldehyde at a final concentration of 1.25% in a fume hood.

[0030] 3) Reaction in a water bath at 37°C for 2 hours.

[0031] 4) Dialyze with desalting column Sephadex G-25 or 0.05mol / L pH9.5 CB to remove excess glutaraldehyde.

[0032] 5) Take 0.1mg anti-NGAL antibody, prepare 1mg / ml antibody with 0.05mol / L pH9.5 CB, and mix activated YFPN protein with antibody.

[0033] 6) React overnight at 4°C.

[0034] 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.

...

Embodiment 2

[0038] The anti-NGAL 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:

[0039] 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.

[0040] 2) Add glutaraldehyde at a final concentration of 1.25% in a fume hood.

[0041] 3) Reaction in a water bath at 37°C for 2 hours.

[0042] 4) Dialyze with desalting column Sephadex G-25 or 0.05mol / L pH9.5 CB to remove excess glutaraldehyde.

[0043] 5) Take 0.1 mg anti-NGAL antibody, prepare 1 mg / ml antibody with 0.05 mol / L pH9.5 CB, and mix the activated YFPC protein and antibody.

[0044] 6) React overnight at 4°C.

[0045] 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.

[0046...

Embodiment 3

[0049] The main components of the kit:

[0050] 1) N-terminal fragment of fluorescent protein coupled with anti-NGAL antibody;

[0051] 2) Anti-NGAL antibody conjugated fluorescent protein C-terminal fragment.

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Abstract

The invention provides a NGAL diagnostic kit based on bimolecular fluorescence complementary technology. The kit comprises an anti-NGAL antibody conjugation fluorescent protein N-terminal fragment andan anti-NGAL antibody coupled fluorescent protein C-terminal fragment. The present invention also discloses a preparation method of the NGAL diagnostic kit based on the bimolecular fluorescence complementary technology. The preparation method comprises preparation of the anti-NGAL antibody coupled fluorescent protein N-terminal fragment and preparation of the anti-NGAL antibody coupled fluorescent protein C-terminal fragment. The present invention also finally discloses a use method of the kit. The kit has the advantages of fast detection, simple operation, good specificity, no cleaning, highprecision, and the like, is convenient for clinical detection, can be used for monitoring of acute kidney injury, has great significance for preventing the acute kidney injury, 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 NGAL in a human body, belonging to the field of disease diagnosis and detection. Background technique [0002] Acute kidney injury (acute kidney injury, AKI) is common and serious clinically. In the United States, community-acquired AKI accounts for 1% of the total inpatients, and the incidence of AKI in inpatients ranges from 0.15% to 7.5%, which is much higher than that in the community. The incidence of AKI in the ICU ward is as high as 5% to 20%. There is no large-scale epidemiological survey in my country, but the incidence rates reported in various places are relatively high. For example, Peking University Hospital reported that the incidence rate of AKI in patients after coronary artery bypass grafting was 27.94%. Although the level of clinical treatment continues to improve, the incidence of AKI is still incre...

Claims

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Application Information

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