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

A detection kit and bimolecular technology, applied in biological testing, fluorescence/phosphorescence, analytical materials, etc., can solve the problems of reducing the precision of reagents and the accuracy of detection results that cannot meet the requirements

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

Although the detection speed of colloidal gold immunochromatography is fast, the accuracy of the detection results is increasingly unable to meet the requirements
The electrochemiluminescence method improves the sensitivity and accuracy of detection, but like the magnetic particle chemiluminescence method, it is a heterogeneous reaction, and the operation process needs to be cleaned, which reduces the precision of the reagent.

Method used

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  • CTnT detection kit based on bimolecular fluorescence complementary technology as well as preparation and use methods
  • CTnT detection kit based on bimolecular fluorescence complementary technology as well as preparation and use methods
  • CTnT 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-cTnT antibody is coupled to the N-terminal fragment of the 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-cTnT antibody, prepare 1 mg / ml antibody with 0.05 mol / L pH9.5 CB, and mix activated YFPN protein with 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 reaction.

...

Embodiment 2

[0037] The anti-cTnT 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:

[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-cTnT 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 reaction.

...

Embodiment 3

[0048] The main components of the kit:

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

[0050] 2) C-terminal fragment of fluorescent protein coupled with anti-cTnT antibody.

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Abstract

The invention provides a cTnT diagnostic kit based on a bimolecular fluorescence complementary technology. The kit comprises an anti-cTnT antibody coupled fluorescin N-terminal fragment and an anti-cTnT antibody coupled fluorescin C-terminal fragment. The invention further discloses a preparation method of the cTnT diagnostic kit based on the bimolecular fluorescence complementary technology. Themethod comprises the steps of preparing the anti-cTnT antibody coupled fluorescin N-terminal fragment and preparing the anti-cTnT 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 rapidness in operation, good specificity, good precision, high accuracy and the like, is convenientin clinical detection use and can be used for improving the accuracy of acute myocardial injury diagnosis when the kit is applied to myocardial injury 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 cTnT content in human body, belonging to the field of disease diagnosis and detection. Background technique [0002] Troponin T (TnT) has a molecular weight of 37KD and is a tropomyosin-binding subunit. There are three subtypes: skeletal muscle troponin T (sTnT), including fast skeletal muscle and slow skeletal muscle, and cardiac muscle. The majority of cardiac troponin T (cTnT) exists on the filaments in the form of C-T-I complexes, and 6%-8% exists in the free form in the myocardial cytoplasm. Because the gene encoding of cTnT and skeletal muscle TnT is different, there is no expression of cTnT in skeletal muscle. cTnT is 40% dissimilar to the two skeletal muscle subtypes. cTnT molecules are stable, hydrophilic, and have good reactivity to specific antigenic determinants. [0003] Cardiomyocyte damage is inevitable in patients...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N21/64
CPCG01N21/6486G01N33/68G01N2800/324
Inventor 徐林
Owner NANJING TZONE BIOLOGICAL SCI & TECH
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