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Cys C detection kit based on bimolecular fluorescence complementary technology as well as preparation and use methods

A detection kit, bi-molecular technology, applied in biological testing, fluorescence/phosphorescence, analytical materials, etc., can solve the problems of high laboratory requirements, long operation time of enzyme immunoassay, poor stability of latex reagents, etc.

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

Among them, the one-way immunodiffusion method is generally used for qualitative detection; the enzyme immunoassay method takes a long time to operate and is cumbersome, and has higher requirements for the experimenter; although the time-resolved fluorescence immunoassay based on immune chromatography has high sensitivity, its CV is relatively low. Large, there is no feasible solution at present; radioimmunoassay is gradually eliminated because of radioactive pollution; particle-enhanced immunoturbidimetric method, which is widely used at present, has matured technology, is easy to operate, and has few interference factors, but liquid latex The stability of the reagent is not good, and the accuracy of the test result is not good

Method used

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  • Cys C detection kit based on bimolecular fluorescence complementary technology as well as preparation and use methods
  • Cys C detection kit based on bimolecular fluorescence complementary technology as well as preparation and use methods
  • Cys C detection kit based on bimolecular fluorescence complementary technology as well as preparation and use methods

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] The anti-Cys C 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 0.1 mg anti-Cys C antibody, prepare 1 mg / ml antibody with 0.05 mol / L pH9.5 CB, and mix the activated YFPN protein and 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 react...

Embodiment 2

[0038] The anti-Cys C antibody is coupled to the C-terminal fragment of fluorescent protein, 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-Cys C antibody, prepare 1 mg / ml antibody with 0.05 mol / L pH9.5 CB, and mix 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.

[0...

Embodiment 3

[0049] The main components of the kit:

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

[0051] 2) Anti-Cys C antibody-coupled fluorescent protein C-terminal fragment.

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Abstract

The invention provides a Cys C diagnostic kit based on a bimolecular fluorescence complementary technology. The kit comprises an anti-Cys C antibody coupled fluorescin N-terminal fragment and an anti-Cys C antibody coupled fluorescin C-terminal fragment. The invention further discloses a preparation method of the Cys C diagnostic kit based on the bimolecular fluorescence complementary technology.The method comprises the steps of preparing the anti-Cys C antibody coupled fluorescin N-terminal fragment and preparing the anti-Cys C antibody coupled fluorescin C-terminal fragment. Finally, the invention further discloses a use method of the kit. The kit provided by the invention has the advantages of simplicity in operation, wide linear range, good specificity, free from cleaning, high accuracy and the like, is convenient in clinical detection use and can be used for reflecting symptoms of kidney diseases in time and accurately reflecting disease situation changes when the kit is appliedto kidney disease monitoring, thereby being extensively popular to the market and 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 Cys C in a human body, belonging to the field of disease diagnosis and detection. Background technique [0002] Cystatin C (cysteine ​​proteinase inhibitor C, Cys C) is a cysteine ​​protease inhibitor, also known as γ-microprotein and γ-microglobulin, with a molecular weight of 13.3KD. It is a low molecular weight, Basic non-glycosylated protein, composed of 122 amino acid residues, can be produced by all nucleated cells in the body with a constant production rate, and widely exists in nucleated cells and body fluids of various tissues. Circulating cystatin C is cleared only by glomerular filtration, is an endogenous marker of changes in glomerular filtration rate, and is reabsorbed in the proximal convoluted tubule, but is completely eliminated after reabsorption It is metabolized and decomposed and does not return t...

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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