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Mouse anti-human CD61 monoclonal antibody hybridoma cell line, monoclonal antibody and preparation method and application thereof and flow cytometry detection reagent

A hybridoma cell line and monoclonal antibody technology, applied in anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, biochemical equipment and methods, biological testing, etc.

Active Publication Date: 2017-08-18
HENAN UNIV OF CHINESE MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there is no published literature on mouse anti-human CD61 monoclonal antibody hybridoma cell lines in the prior art

Method used

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  • Mouse anti-human CD61 monoclonal antibody hybridoma cell line, monoclonal antibody and preparation method and application thereof and flow cytometry detection reagent
  • Mouse anti-human CD61 monoclonal antibody hybridoma cell line, monoclonal antibody and preparation method and application thereof and flow cytometry detection reagent
  • Mouse anti-human CD61 monoclonal antibody hybridoma cell line, monoclonal antibody and preparation method and application thereof and flow cytometry detection reagent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] The preparation of hybridoma cell line IID5G8 comprises the following steps:

[0034] 1. Immunity

[0035] 1. Preparation of immunogen

[0036] (1) Take 3mL fresh buffy coat retrieved from Henan Red Cross Blood Center, add 7mL 1×PBS to dilute and mix;

[0037] (2) Take a 15mL centrifuge tube, add 4mL of lymphocyte separation medium, and slowly and steadily add 10mL of diluted buffy coat to the surface of the lymphocyte separation medium;

[0038] (3) Horizontal low-temperature centrifuge 500×g, speed up to 1 gear, speed down to 0 gear, 18°C, centrifuge for 20 minutes;

[0039] (4) Collect the ring-shaped milky white layer and the upper layer into a new centrifuge tube;

[0040] (5) Add 1×PBS to 14mL, centrifuge at 500×g, 18°C ​​for 20min, and discard the supernatant;

[0041] (6) Repeat washing once with step (5);

[0042] (7) Add an appropriate amount of 1×PBS to resuspend, stain with 0.4% trypan blue solution, count under a microscope, and adjust to the required ...

Embodiment 2

[0101] The preparation of mouse anti-human CD61 monoclonal antibody (mouse peritoneal induction method), comprises the following steps:

[0102] 1. Ascites preparation

[0103] (1) Select old female Balb / c mice, and inject liquid paraffin into the abdominal cavity of mice after filtration sterilization, 500 μL / mouse, for standby;

[0104] (2) Transfer the positive monoclonal cell line IID5G8 into a cell culture dish for expanded culture, and intraperitoneally inject hybridoma cells 7 days later, 0.8×10 6 A total of 4 mice were inoculated, and the injection dose was 500 μL / mouse;

[0105] (3) After one week, observe the occurrence of ascites in the mice. When the abdomen of the mice is obviously protruding and the skin is tight, puncture the skin of the abdomen of the mice with a 20mL sterile needle, and collect the ascites into a centrifuge tube;

[0106] (4) Centrifuge the ascites at 3000r / min for 5min, suck out the transparent liquid in the middle, aliquot and store at -80...

Embodiment 3

[0119] FITC-labeled CD61 monoclonal antibody, its preparation steps are as follows:

[0120] (1) Take the CD61 monoclonal antibody (1mg / mL) to be cross-linked for AKTA purification;

[0121] (2) Dissolve FITC in DMSO (Note: FITC used for each cross-linking should be freshly prepared and kept away from light) at a concentration of 1 mg / mL;

[0122] (3) Slowly add FITC into the antibody solution according to the ratio of P:F (CD61 ​​monoclonal antibody: FITC) of 1 mg: 150 μg, shake gently while adding, make it mix with the antibody evenly, and react at 4°C in the dark for 8 hours;

[0123] (4) Add 5mol / L of NH 4 Cl to a final concentration of 50mmol / L, stop the reaction at 4°C for 2 hours;

[0124] (5) Dialyze the cross-linked product in PBS more than four times until the dialysate is clear;

[0125] (6) Identification of cross-linked substances

[0126] Protein concentration (mg / mL) = (A280-0.31×A495) / 1.4;

[0127] F / P ratio: 3.1×A495 / (A280-0.31×A495), the value should be ...

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Abstract

The invention discloses a mouse anti-human CD61 monoclonal antibody hybridoma cell line, a monoclonal antibody and a preparation method and application thereof and a flow cytometry detection reagent, and belongs to the technical field of biological engineering. According to the mouse anti-human CD61 monoclonal antibody hybridoma cell line, human peripheral blood leukocytes are used as an immunogen for immunizing Balb / c mice; a cell fusion technology is adopted and hybridoma cells are screened by utilizing an immunocytochemistry and a flow cytometry indirect immunofluorescent method; an antibody generated by monoclonal hybridoma cells is subjected to specific identification by applying an immunoprecipitation-mass-spectrometric method; Western Blot proves that one hybridoma cell line IID5G8 capable of stably secreting a mouse anti-human CD61 monoclonal antibody is obtained and a preservation number is CGMCC (China General Microbiological Culture Collection Center) No. 13300. The monoclonal antibody is prepared by adopting an animal in-vivo induction method; a heavy chain of the antibody is identified to be IgG1 and a light chain is identified to be kappa type; after the antibody is purified, an FITC (Fluorescein Isothiocyanate) label can be used for detecting antigen expression of human peripheral blood platelets CD61 by utilizing flow cytometry direct-labeling immunofluorescence.

Description

technical field [0001] The present invention relates to a mouse anti-human CD61 monoclonal antibody hybridoma cell line, and also relates to the mouse anti-human CD61 monoclonal antibody and FITC-labeled monoclonal antibody secreted by the hybridoma cell line, and the production of the above-mentioned monoclonal antibody The preparation method and application, and also relates to the flow detection reagent containing the above-mentioned monoclonal antibody, belong to the technical field of bioengineering. Background technique [0002] CD61, also known as CD61A, GPIIb / IIIa or integrin β3 chain, has a molecular weight of 110kDa and is expressed on platelets, megakaryocytes and macrophages. CD61 belongs to integrin β subunit, its ligands and related molecular formulas are fibronectin, fibrinogen, plasminogen, prothrombin, TSP, vitronectin, vWF, tenascin, MMP-2, laminin protein and osteopontin. [0003] CD41 / CD61 are receptors for vitronectin, fibrinogen, fibrin and vWF that a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/28G01N33/68G01N33/577
CPCC07K16/2848
Inventor 张振强张钟允王爱萍宋军营曾华辉孙向东袁永闫敏刘文弟
Owner HENAN UNIV OF CHINESE MEDICINE
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