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PCT 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 large intra-batch variation, poor repeatability, and unstable reagents.

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 enzyme-linked immunosorbent assay uses horseradish peroxidase or alkaline phosphatase to label antibodies, and catalyzes the color change of the substrate. Low, currently mainly used in infectious disease screening and other projects that require low detection sensitivity; immunochromatography has the advantages of simple operation and fast detection speed, but also has low sensitivity, unstable reagents, and poor repeatability , the disadvantage of being difficult to quantify
Chemiluminescent immunoassay has the advantages of simple operation, fast detection speed, and high-throughput detection, but it also has the disadvantages of heterogeneous reaction, long detection time, and large intra-assay variability

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] The anti-PCT 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:

[0030] 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.

[0031] 2) Add glutaraldehyde at a final concentration of 1.25% in a fume hood.

[0032] 3) Reaction in a water bath at 37°C for 2 hours.

[0033] 4) Dialyze with desalting column Sephadex G-25 or 0.05mol / L pH9.5 CB to remove excess glutaraldehyde.

[0034] 5) Take 0.1 mg anti-PCT antibody, prepare 1 mg / ml antibody with 0.05 mol / L pH9.5 CB, and mix the activated YFPN protein with the antibody.

[0035] 6) React overnight at 4°C.

[0036] 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

[0040] The anti-PCT 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:

[0041] 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.

[0042] 2) Add glutaraldehyde at a final concentration of 1.25% in a fume hood.

[0043] 3) Reaction in a water bath at 37°C for 2 hours.

[0044] 4) Dialyze with desalting column Sephadex G-25 or 0.05mol / L pH9.5 CB to remove excess glutaraldehyde.

[0045] 5) Take 0.1 mg anti-PCT antibody, prepare 1 mg / ml antibody with 0.05 mol / L pH9.5 CB, and mix activated YFPC protein and antibody.

[0046] 6) React overnight at 4°C.

[0047]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.

[0048] 8) ...

Embodiment 3

[0051] The main components of the kit:

[0052] 1) N-terminal fragment of fluorescent protein coupled with anti-PCT antibody;

[0053] 2) Anti-PCT antibody-coupled fluorescent protein C-terminal fragment.

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Abstract

The invention provides a PCT diagnosis kit based on a bimolecular fluorescence complementation technology, wherein the kit comprises an anti-PCT antibody conjugated fluorescent protein N-terminal fragment and an anti-PCT antibody conjugated fluorescent protein C-terminal fragment. The invention further discloses a preparation method of the PCT diagnosis kit, wherein the preparation method comprises: preparation of an anti-PCT antibody conjugated fluorescent protein N-terminal fragment, and preparation of an anti-PCT antibody conjugated fluorescent protein 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, rapid detection, no cleaning, good precision and the like, is conveniently suitable for clinical detection, can increase the accuracy of the diagnosis of sepsis in the monitoring of infections, 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 PCT in a human body, belonging to the field of disease diagnosis and detection. Background technique [0002] In recent years, the pathological and physiological processes of systemic infection have been better understood, and intensive supportive treatment has also been significantly improved. However, systemic infection and its complications are still the leading cause of death in non-cardiac intensive care units, the United States Approximately 500,000 patients with systemic infections are diagnosed each year, and approximately 40% eventually die. Therefore, there is a need for a marker that can more effectively reflect the degree of inflammatory damage caused by infection. Procalcitonin (PCT) is a new indicator with high sensitivity and specificity. It is significantly elevated when bacterial or fungal infection i...

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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