KIM-1 detection kit based on bimolecular fluorescence complementary technology as well as preparation and use methods

A KIM-1, detection kit technology, applied in the field of bimolecular fluorescence complementation, can solve the problems of long operation time, poor detection effect, low detection sensitivity, 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

This method has the characteristics of simple operation and long reagent stability period, but the detection sensitivity of this method is low, and the operation time is long. At present, it is ma

Method used

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  • KIM-1 detection kit based on bimolecular fluorescence complementary technology as well as preparation and use methods
  • KIM-1 detection kit based on bimolecular fluorescence complementary technology as well as preparation and use methods
  • KIM-1 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-KIM-1 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:

[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-KIM-1 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 reaction...

Embodiment 2

[0038] The anti-KIM-1 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-KIM-1 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.

[0...

Embodiment 3

[0049] The main components of the kit:

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

[0051] 2) Anti-KIM-1 antibody-coupled fluorescent protein C-terminal fragment.

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Abstract

The invention provides a KIM-1 diagnostic kit based on a bimolecular fluorescence complementary technology. The kit comprises an anti-KIM-1 antibody coupled fluorescin N-terminal fragment and an anti-KIM-1 antibody coupled fluorescin C-terminal fragment. The invention further discloses a preparation method of the KIM-1 diagnostic kit based on the bimolecular fluorescence complementary technology.The method comprises the steps of preparing the anti-KIM-1 antibody coupled fluorescin N-terminal fragment and preparing the anti-KIM-1 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 convenient in clinical detection use and can be used for improving the accuracy of acute renal injury diagnosis when the kit is applied to acute renal 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 the content of KIM-1 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 incr...

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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