Method for detecting anti-AQP4 antibody based on quantum dot polystyrene microsphere

A technology of polystyrene microspheres and quantum dots, applied in the fields of bioanalytical chemistry and nanobiology, can solve the problems of low protein expression affecting detection results, difficulty in realizing large-scale commercial production, and difficulty in establishing quality control standards, etc., to achieve Rapid detection of standardization and automation, easy standardization and automation, wide excitation spectral range effects

Inactive Publication Date: 2019-12-27
SOUTHERN MEDICAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0004] At present, the commonly used detection methods for clinically detecting AQP4 mainly include the following two types: (1) Indirect immunofluorescence assay (IIF) based on mouse brain tissue sections with high expression of AQP4 protein. Although this method has high sensitivity, However, the operation is complex, the production of tissue slices is highly demanding, and quality control is difficult, and it requires inspectors to have a wealth of neurophysiological knowledge to accurately determine the test results
(2) A cell-based assay (CBA) based on cells successfully expressing AQP4 protein. This method has relatively high specificity. The low expression level affects the interpretation of the test results, and due to the inconsistent transfection efficiency, it is difficult to establish a unified quality control standard, making it difficult to achieve large-scale commercial production

Method used

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  • Method for detecting anti-AQP4 antibody based on quantum dot polystyrene microsphere
  • Method for detecting anti-AQP4 antibody based on quantum dot polystyrene microsphere
  • Method for detecting anti-AQP4 antibody based on quantum dot polystyrene microsphere

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Embodiment 1

[0054] A method for detecting anti-AQP4 antibodies based on quantum dot polystyrene microspheres, such as Figure 1 to Figure 3 shown, including the following steps:

[0055] (1) Preparation of quantum dot polystyrene QPs microspheres;

[0056] (2) obtaining the functional fragment target gene of AQP4 protein for expression;

[0057] (3) Utilize EDC-NHS to activate the carboxyl groups on the surface of the QPs microspheres, and couple the carboxyl groups on the surface of the activated QPs microspheres with the amino groups of the AQP4 protein to make functionalized QPs microspheres;

[0058] (4) Flow cytometric detection by oil-soluble CdSe quantum dots with a fluorescence emission peak of 600nm to 650nm and FITC-labeled goat anti-human IgG red-green dual fluorescence localization system, and the detection results of the functionalized QPs microspheres to read.

[0059] Specifically, step (1) adopts chemical osmosis method to prepare QPs microspheres, and the specific proc...

Embodiment 2

[0098] A method for detecting anti-AQP4 antibodies based on quantum dot polystyrene microspheres, other features are the same as in Example 1, the difference is that the specific detection steps of the detection sample are as follows:

[0099] (1) Take 10 μL serum sample, dilute it with PBS solution according to the ratio of serum: PBS solution = 1:10, fully mix the diluted serum with 80 μg of QPs-AQP4 complex, and incubate at 37°C for one hour.

[0100] (2) Wash the incubated product 3 times with PBS solution, 5 minutes each time, and spin dry.

[0101] (3) Dilute FITC-labeled goat anti-human IgG with PBS solution according to the ratio of FITC-labeled goat anti-human IgG: PBS solution = 1:50, and add 100 μL of diluted FITC-labeled goat anti-human IgG to every 100 μL serum mixture sample , and incubate the mixture at 37°C for one hour in the dark.

[0102] (4) Wash the incubated functionalized QPs microspheres-anti-AQP4 antibody-goat anti-human IgG immune complex three times...

Embodiment 3

[0107] A method for detecting anti-AQP4 antibodies based on quantum dot polystyrene microspheres, comprising the following steps:

[0108] (1) Preparation of quantum dot polystyrene QPs microspheres;

[0109] (2) obtaining the functional fragment target gene of AQP4 protein for expression;

[0110] (3) Utilize EDC-NHS to activate the carboxyl groups on the surface of the QPs microspheres, and couple the carboxyl groups on the surface of the activated QPs microspheres with the amino groups of the AQP4 protein to make functionalized QPs microspheres;

[0111] (4) The red-green dual fluorescence localization system of oil-soluble CdSe quantum dots with a fluorescence emission peak of 600nm and FITC-labeled goat anti-human IgG was used for flow cytometry detection, and the detection results of the functionalized QPs microspheres were interpreted.

[0112] Specifically, step (1) adopts chemical osmosis method to prepare QPs microspheres, and the specific process includes:

[0113...

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Abstract

The invention relates to a method for detecting an anti-AQP4 antibody based on a quantum dot polystyrene microsphere. A novel immune method combining a quantum dot nanoprobe with a flow cytometry technology is used for detecting contained anti-AQP4 antibodies and breaks through the bottleneck of the traditional way that an organic fluorescent dye is taken as a detection tool. Meanwhile, the advantages of the flow cytometry technology that detection is rapid and standarization and automation can be easily realized are utilized, and the defects of an existing AQP4 antibody detection technology are made up. AQP4 antibodies can be simply, rapidly and repeatably detected.

Description

technical field [0001] The invention relates to the fields of bioanalytical chemistry and nano-biotechnology, in particular to a method for detecting anti-AQP4 antibodies based on quantum dot polystyrene microspheres. Background technique [0002] Human autoantibodies are important serological markers of autoimmune diseases, and their rapid detection has important application value for the diagnosis, treatment and prognosis evaluation of autoimmune diseases. With the improvement of the sensitivity and specificity of autoantibody detection methods, the diagnostic criteria of related autoimmune diseases are also updated and adjusted accordingly. [0003] Neuromyelitis optica (NMO) is an acute or subacute inflammatory demyelinating lesion involving the optic nerve and spinal cord simultaneously or sequentially. In 2004, Lennon et al first found NMO-IgG antibodies in the serum of NMO patients, that is, anti-aquaporin 4 (AQP4) antibodies. AQP4 is the main aquaporin in the centr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543G01N33/533G01N21/64G01N15/14
CPCG01N33/54346G01N33/54313G01N33/533G01N21/6428G01N15/14
Inventor 刘天才李鹏陈振华
Owner SOUTHERN MEDICAL UNIVERSITY
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