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A flow cytometry-based method for detecting the cleavage of the extracellular segment of the platelet receptor gpiba

A technology of flow cytometry and platelets, applied in the field of medical biology, can solve the problems of high background value of ELISA, high level of soluble fragments, lower reliability and stability of test results, etc., and achieve high reliability and good stability

Active Publication Date: 2019-07-05
XUZHOU MEDICAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, Western blot detection requires a certain number of cells to obtain the amount of protein required for detection, so in the case of thrombocytopenia, this method is not suitable for detection of GPIba extracellular segment enzyme digestion
In addition, cleavage of the extracellular segment of GPIba occurs continuously (even at rest), which can lead to high levels of soluble fragments in plasma, serum, or platelet supernatants, resulting in high background values ​​in ELISA
Detecting GPIba extracellular segment digestion by ELISA method will reduce the reliability and stability of the detection results

Method used

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  • A flow cytometry-based method for detecting the cleavage of the extracellular segment of the platelet receptor gpiba
  • A flow cytometry-based method for detecting the cleavage of the extracellular segment of the platelet receptor gpiba
  • A flow cytometry-based method for detecting the cleavage of the extracellular segment of the platelet receptor gpiba

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Embodiment 1

[0040] Embodiment 1: Detection of enzyme cleavage of the extracellular segment of GPIba in healthy human platelets

[0041] A method for detecting platelet receptor GPIba ecto-digestion enzymes based on flow cytometry, comprising the following steps:

[0042] (1) Sample preparation and processing

[0043] a. Collect 5ml of venous blood from healthy people and collect it in a test tube containing the anticoagulant trisodium citrate aqueous solution (1.6 grams of trisodium citrate dissolved in 50ml distilled water). The blood is mixed slowly and gently according to the volume ratio of 1:9;

[0044] b. At room temperature, centrifuge at 120×g for 20 minutes, use a plastic straw to gently absorb the platelet-rich plasma (PRP) in the upper layer, do not absorb the PRP near the middle layer, so as not to absorb white blood cells and cause pollution;

[0045] c. Add the aspirated PRP to 5 EP tubes respectively, and the number of platelets in each EP tube is 1×10 7 Add 1×TS buffer ...

Embodiment 2

[0061] Embodiment 2: Detection of enzyme cleavage of GPIba extracellular segment of healthy human platelets treated with NEM

[0062] N-ethylmaleimide (NEM), a reagent that can artificially induce the cleavage of the extracellular segment of GPIba, was used to treat platelets to induce cleavage of GPIba.

[0063] from Figure 7 It can be seen that the abscissa is the binding of the intracellular antibody, and the ordinate is the binding of the extracellular antibody. The 4 boxes in the figure represent the binding of each antibody respectively. The lower left quadrant represents the negative area, which represents the percentage of platelets that have neither the binding of the extracellular fragment antibody nor the binding of the intracellular fragment antibody. The upper left quadrant represents the percentage of platelets bound only by antibodies to the extracellular domain. The lower right quadrant represents platelets bound by intracellular segment antibodies. The up...

Embodiment 3

[0066] Example 3: Detection of enzyme cleavage of extracellular segment of platelet GPIba in patients with thrombocytopenia

[0067] Platelets from 5 patients with thrombocytopenia were collected, and the method of the present invention was used to detect the enzyme cleavage of the extracellular segment of GPIba. from Figure 9 As can be seen in the figure, the intact state of GPIba was significantly decreased in patients with thrombocytopenia, suggesting increased cleavage of the extracellular segment. In addition, through correlation analysis, it was found that the degree of cleavage of the extracellular segment of GPIba in patients with thrombocytopenia was not dependent on the change of platelet number (R2=0.021, P=0.815), which further verified the flow cytometry-based detection method of the present invention. The method of cleavage of the extracellular segment of GPIba does not depend on the change of platelet number.

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Abstract

The invention discloses a method for detecting the enzyme cleavage of the extracellular segment of platelet receptor GPIba based on flow cytometry. The specific steps are: (1) sample preparation and processing; (2) flow cytometry detection; (3) Weasel Software data analysis. The present invention uses two different fluorescently labeled antibodies, respectively anti-GPIba extracellular segment antibody and anti-GPIba intracellular segment antibody, and analyzes the relative value of the combination of the extracellular segment antibody and the intracellular segment antibody by flow cytometry, namely It can reflect the degree of cleavage of the extracellular segment of GPIba. The detection method of the invention does not depend on the change of the number of platelets, is suitable for the situation of reduced number of platelets, and has high reliability and good stability.

Description

technical field [0001] The invention belongs to the technical field of medical biology, and in particular relates to a flow cytometry-based method for detecting enzyme cleavage of the extracellular segment of platelet receptor GPIba. Background technique [0002] The GPIb-IX-V complex is composed of GPIba, GPIbb, GPIX and GPV ( figure 1 ). GPIba is combined with GPIbb through disulfide bonds, and then connected with GPIX and GPV through non-covalent bonds. GPIba is the major ligand-binding subunit in the GPIb-IX-V complex, consisting of 626 amino acid residues. GPIba is a membrane-expressed molecule consisting of an extracellular segment, a transmembrane region and an intracellular segment. As the most important adhesion molecule of platelets, GPIba exerts a variety of biological functions by binding to corresponding ligands. [0003] After platelet activation or some kind of stimulation, the extracellular segment of GPIba will undergo enzyme cleavage, which is mainly me...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N15/14G01N1/28G01N1/30G01N33/58
CPCG01N1/28G01N1/30G01N15/14G01N33/582
Inventor 乔建林徐开林曾令宇鞠文齐昆明
Owner XUZHOU MEDICAL UNIV
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