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Method for detecting superoxide dismutase through quantum dot labeled direct competitive fluorescence immunoassay

A technology of superoxide and fluorescence immunity, which is applied in the field of immunoassay, can solve the problems of time-consuming and cumbersome operation, and achieve the effect of simple operation, strong fluorescence intensity and long fluorescence stability time

Pending Publication Date: 2021-05-14
COFCO GROUP +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In this method, the antibody is coated on the microplate, and proteins and enzymes are added for specific binding, then the enzyme-labeled antibody (ie, the detection antibody) is added, and finally the substrate is added for color development. After a certain period of time, a microplate reader is used to detect a certain Calculate the concentration of the drug to be tested in the sample based on the absorbance value at a specific wavelength, which is cumbersome and time-consuming

Method used

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  • Method for detecting superoxide dismutase through quantum dot labeled direct competitive fluorescence immunoassay
  • Method for detecting superoxide dismutase through quantum dot labeled direct competitive fluorescence immunoassay
  • Method for detecting superoxide dismutase through quantum dot labeled direct competitive fluorescence immunoassay

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Embodiment 1

[0082] The method for the direct competitive fluorescent immunodetection superoxide dismutase of quantum dot label of the present invention comprises the following steps:

[0083] (1) Activation of quantum dots

[0084] Measure 100 μL CdSe / ZnS quantum dots, add 100 μL 3mg / mL N-hydroxysuccinimide (NHS) phosphate solution, 100 μL 2mg / mL 1-ethyl-(3-dimethylaminopropyl)-carbodiethylene Dissolve in amine hydrochloride (EDC) phosphate solution and 700μL 25mM pH 6.0 phosphate buffer, after ultrasonic dispersion, react in a constant temperature incubator at 37°C and 250rmp for 30min, centrifuge at 10000rpm for 10min, remove the supernatant, and obtain the activated subsequent quantum dots.

[0085] (2) Coupling of activated quantum dots and superoxide dismutase

[0086] Use 0.01M pH7.4 phosphate buffer as solvent to prepare 1 mg / mL superoxide dismutase phosphate solution, take 1mL superoxide dismutase phosphate solution to redissolve the activated quantum dots, Ultrasonic dispersio...

Embodiment 2

[0100] Except for the following step (4), the same operation as in Example 1 was used to obtain the fluorescence intensity and prepare a standard curve. The linear correlation coefficient of the standard curve obtained in this study was 0.921.

[0101] (4) Antibody coating: use pH=7.4, 0.01M phosphate buffer to dilute the superoxide dismutase polyclonal antibody at 1:500, and add 100 μL of the obtained antibody dilution to each well of the microtiter plate , after overnight coating in a refrigerator at 4°C, take it out, wash three times with 0.01M pH7.4 phosphate buffer containing 0.5% Tween-20 at room temperature and spin dry to remove excess antibody, then add to each well 300 μL of 1% BSA blocking solution was used to block the blank site at 37° C. for 1.5 h to obtain a coated polyclonal antibody against superoxide dismutase.

Embodiment 3

[0103] Except for the following step (5), the same operation as in Example 1 was used to obtain the fluorescence intensity and prepare a standard curve. The linear correlation coefficient of the standard curve obtained in this study was 0.969.

[0104] (5) Formation of luminescent immune complexes: Add 50 μL standard solution containing superoxide dismutase and 50 μL Dilution of the QDs-SOD complex. In this step, superoxide dismutase will compete with QDs-SOD for binding to the solid-phase antibody coated on the microtiter plate, and the competition reaction lasts at 37°C for 0.5h. Wash three times with 0.01M pH7.4 phosphate buffer containing 0.5% Tween-20 to remove non-specific adsorption and form antibody-antigen luminescent immune complex.

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Abstract

The invention relates to a method for detecting superoxide dismutase by quantum dot labeled direct competitive fluorescence immunoassay, which comprises the following steps of: labeling superoxide dismutase by using CdSe / ZnS quantum dots to form a superoxide dismutase-quantum dot fluorescence probe (QDs-SOD) compound; detecting the concentration of the superoxide dismutase in the sample liquid to be detected by directly competitively combining the QDs-SOD compound and the superoxide dismutase in the sample liquid to be detected with the coated superoxide dismutase polyclonal antibody. The method disclosed by the invention has the characteristics of high sensitivity, high specificity, simplicity in operation and the like, and can be used for rapidly detecting the superoxide dismutase.

Description

technical field [0001] The invention belongs to the technical field of immunodetection methods, in particular, the invention relates to a method for detecting superoxide dismutase by quantum dot-labeled direct competitive fluorescence immunoassay and a superoxide dismutase detection kit. Background technique [0002] Superoxide dismutase (Superoxide Dismutase, SOD), widely present in the cells and tissues of animals and plants, is one of the key enzymes in the biological defense system. SOD can eliminate active oxygen and other harmful substances produced by animals and plants during their metabolism, maintain their normal life activities, enhance plant defense capabilities, and delay aging. During the growth of animals and plants and during the storage of agricultural and sideline products such as tomatoes, the content of SOD is constantly changing due to the influence of cell metabolism. Therefore, the content of superoxide dismutase can indirectly reflect the changes of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/573G01N33/532G01N21/64
CPCG01N33/573G01N33/532G01N21/6428G01N2333/90283
Inventor 翟晨李梦瑶王书雅杨悠悠谢云峰
Owner COFCO GROUP
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