Reagent for detecting anti-platelet surface receptor-specific autoantibodies and preparation method and application thereof

A technology of surface receptors and autoantibodies, applied in biological testing, measuring devices, material inspection products, etc., can solve problems such as low sensitivity and cumbersome operation, and achieve high sensitivity, increased sensitivity, and high specificity

Inactive Publication Date: 2018-02-23
SUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The invention discloses a reagent for detecting anti-platelet surface receptor-specific autoantibodies and a preparation method thereof, which is used for detecting free platelet surface receptor-specific autoantibodies in serum or plasma, has high specificity and sensitivity, and It is easy to operate and effectively solves the shortcomings of the existing double-antibody sandwich detection method, such as low sensitivity and cumbersome operation

Method used

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  • Reagent for detecting anti-platelet surface receptor-specific autoantibodies and preparation method and application thereof
  • Reagent for detecting anti-platelet surface receptor-specific autoantibodies and preparation method and application thereof
  • Reagent for detecting anti-platelet surface receptor-specific autoantibodies and preparation method and application thereof

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Effect test

Embodiment 1

[0036] Example 1 Preparation of reagents for detecting free anti-platelet surface receptor-specific autoantibodies

[0037] Acquisition of DNA Fragments

[0038] Obtain human platelet membrane protein GP Ibα subunit from the NCBI database, sequence accession number: NM_000173.6; human platelet glycoprotein GP Ibβ subunit, sequence accession number: NM_000407.4; human platelet glycoprotein GP IX, sequence accession number: NM_000174.4; human platelet integrin GP IIb subunit, sequence accession number: NM_000419.4; human platelet integrin GP IIIa subunit, sequence accession number: NM_000212.2. Submit the gene company for synthesis, and then connect it to the vector pcDNA3.3 to construct pcDNA-GP Ibα, pcDNA-GP Ibβ, pcDNA-GP IX, pcDNA-GPIIb and pcDNA-GPIIIa recombinant plasmids, and the base sequence is confirmed to be accurate after enzyme digestion and sequencing Correct; the sequencing sequences of pcDNA-GP Ibα, pcDNA-GP Ibβ, pcDNA-GP IX, pcDNA-GP IIb and pcDNA-GPIIIa recombi...

Embodiment 2

[0060] Example 2 Detection of surface-specific autoantibodies on free platelets in serum

[0061] Take out 50 μl of fixed and stable CHO-IbIX, CHO-IIbIIIa and CHO-123 cells, add 50 μl of pre-diluted serum samples (diluted 200 times with PBS), and incubate at 37°C for 1 hour; add PBS, 800rpm / min Centrifuge for 5min; discard the supernatant, add FITC-goat anti-human IgG (fluorescein-labeled goat anti-human immunoglobulin polyclonal antibody), incubate at 37°C for 0.5h; add PBS, centrifuge at 800rpm / min for 5min; discard the supernatant.

[0062] Use the fluorescence method as follows: add PBS to resuspend cells, take 10 μl of cell suspension (containing 1×10 4 1 cell), add 10 μl of DAPI-containing mounting medium, drop on the slide, and add a cover slip. Direct observation under a fluorescence microscope, if the cell membrane shows green fluorescence, it is positive, otherwise it is negative.

[0063] Figure 4 Serum samples from clinically diagnosed ITP patients (the levels ...

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Abstract

The invention discloses a reagent for detecting anti-platelet surface receptor-specific autoantibodies and a preparation method and application thereof. The reagent has high specificity and sensitivity. The method comprises expressing a human platelet-specific receptor membrane glycoprotein GP Ib-IX and/or GP IIb/IIIa receptor through CHO cells, carrying out co-incubation on the specific membraneglycoprotein receptor expression-positive CHO cells and serum to be detected and adding a fluorescein-labeled anti-human immunoglobulin polyclonal antibody (secondary antibody) for bonding. If there is an anti-platelet receptor-specific antibody in serum or plasma, the CHO-membrane glycoprotein specific antibody-fluorescein-labeled anti-human immunoglobulin polyclonal antibody complex structure isformed, has high CHO cell fluorescence intensity and can be used for flow cytometry or fluorescence microscope examination. The method is easy to operate, has enough specific membrane glycoprotein expression positive CHO cell sources, high specificity and sensitivity and high diagnosis efficiency and can be used for basic research and clinical examination.

Description

technical field [0001] The invention belongs to the technical field of detecting anti-platelet surface receptor-specific autoantibodies, in particular to a reagent for detecting anti-platelet surface receptor-specific autoantibodies, a preparation method and application thereof, and has high specificity and sensitivity. Background technique [0002] Bleeding disorders are common and frequently-occurring diseases in clinical practice. If they are not diagnosed quickly and intervened in time, they may lead to severe pain, organ damage and even death. Worldwide, only about 30% of patients with bleeding disorders are diagnosed. Immune thrombocytopenic (Immune thrombocytopenic, ITP) is an immune-mediated thrombocytopenic syndrome and one of the most common clinical bleeding disorders, which can occur in all age groups and is more common in women. It can be divided into two types: acute and chronic. Chronic is common in adults, and acute is more common in children. The clinical ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/533G01N33/68
CPCG01N33/533G01N33/6893G01N2800/222
Inventor 戴克胜
Owner SUZHOU UNIV
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