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New application of nuclear fast red staining in anti-double-stranded DNA detection method

A detection method, nuclear fast red technology, applied in the field of immunoassay, can solve the problems such as difficult to distinguish the fluorescence model of the moving matrix, blurred structure, difficult to distinguish the flagellar matrix, etc.

Pending Publication Date: 2020-10-09
TIANJIN BAODI HOSPITAL
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Problems solved by technology

[0004] The problems existing in the current detection method are: under the fluorescent microscope, the nucleus, kinetosome and flagellar matrix in Chrymena luliflora are difficult to distinguish, the structure is blurred, and the fluorescence characteristics can appear. The three organelles can be negative and positive at the same time, or one of them In the case of one or two positives, etc., the nuclei and kinetosomes are basically similar in size, and it is difficult to distinguish the fluorescence model of the kinetosome

Method used

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  • New application of nuclear fast red staining in anti-double-stranded DNA detection method

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Experimental program
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Embodiment 1

[0012] 1. Reagent Anti-double-stranded DNA antibody IgG detection kit (containing PBS Tween buffer, biological thin film, fluorescent yellow isothiocyanate [FITC]-labeled fluorescent secondary antibody, mounting agent) was provided by Omen Medical Diagnostics (China ) Limited offer.

[0013] 2. For the mounting agent containing nuclear fast red, add nuclear fast red 0.2g / L to the mounting medium (glycerol) of the kit and dissolve it for 24 hours before use.

[0014] 3. Instrument: EURO Star 3Plus fluorescence microscope reagent

Embodiment 2

[0016] Detection steps:

[0017] Experimental preparation: PBS Tween buffer: dissolve 1 sachet of phosphate in 1 liter of distilled water, add 2 mL of Tween 20 and mix well;

[0018] Sample preparation: The serum sample to be tested was diluted 1:10 with PBS Tween buffer. For example: add 11.1 μl serum sample to 100 μl PBS Tween buffer and mix thoroughly;

[0019] Adding samples: Place the sample adding plate on the foam plate, and add 11 μL of diluted serum sample to each reaction area of ​​the sample adding plate, avoiding the generation of air bubbles. Start incubation after adding all the samples to be tested (up to 200 samples each time);

[0020] Incubation: Place the biological slide with the biological thin side down, and cover it in the groove of the sample plate, and the reaction will start immediately. It should be ensured that each sample is in contact with the biochip and that the samples are not in contact with each other. Incubate at room temperature for 30 ...

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Abstract

The invention discloses a new application of nuclear fast red staining in an anti-double-stranded DNA detection method. The invention belongs to the technical field of immunoassay. According to a technical scheme adopted by the invention, crithidia luciliae is adopted as an antigen matrix, slide-sealing staining is conducted by using a glycerin nucleus fast red staining solution, and indirect immunofluorescence detection of an anti-dsDNA antibody is carried out, cell nucleuses in the crithidia luciliae body are orange red or yellow; wherein the part connected with the flagellum is a flagellummatrix and is dyed into light orange or light yellow; the part having a volume slightly smaller than that of the cell nucleus and greater than the flagellum matrix and located between two cell organelles is a movable matrix, and if green fluorescence appears on the movable matrix, the anti-dsDNA antibody is positive. The method has the technical characteristic that the three cell organelles including the cell nucleus, the movable matrix and the flagellum matrix can be clearly distinguished under a fluorescence microscope.

Description

technical field [0001] The invention belongs to the technical field of immune analysis; or it is a method for testing materials by using visible light to produce color changes in the results of test reactions, in particular a method for measuring anti-double-stranded DNA using nuclear fast red staining and immunofluorescence detection. Background technique [0002] The routine methods for detecting anti-double-stranded DNA (dsDNA) antibodies in the laboratory include ELISA, indirect immunofluorescence detection (CLIFF) and immunoblotting (IBT), but the American College of Rheumatology (ACR) revised it in 1997. The detection method of anti-dsDNA antibody is not limited in the diagnostic criteria of SLE classification. Clinical laboratories often use indirect immunofluorescence to detect dsDNA. This method uses Chrysalis luciliae as the antigen matrix to detect anti-double-stranded DNA antibodies in serum. Chrithidia luciliae belongs to the protozoan phylum moss The genus Sho...

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Application Information

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IPC IPC(8): G01N33/569G01N33/53
CPCG01N33/56905G01N33/5308
Inventor 李立和李志永齐秀会
Owner TIANJIN BAODI HOSPITAL
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