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Anti-human FcepsilonRIalpha subunit monoclonal antibody and application thereof

A technology for cloning antibodies and single subunits, applied in the field of hybridoma cell lines, can solve the problems of low specificity and poor immune effect of anti-human FcεRIα subunit monoclonal antibodies, and achieve high basic research value and clinical application value , Uniform cell size and good growth

Inactive Publication Date: 2014-12-17
李莉 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The technical problem to be solved by the present invention is to provide a highly specific anti-human FcεRIα subunit monoclonal antibody that can be mass-produced for the problems of low specificity and poor immune effect of existing anti-human FcεRIα subunit monoclonal antibodies Antibody and application thereof, and a hybridoma cell line secreting the anti-human FcεRIα subunit monoclonal antibody

Method used

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  • Anti-human FcepsilonRIalpha subunit monoclonal antibody and application thereof
  • Anti-human FcepsilonRIalpha subunit monoclonal antibody and application thereof
  • Anti-human FcepsilonRIalpha subunit monoclonal antibody and application thereof

Examples

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Effect test

Embodiment 1

[0044] Example 1 Preparation of monoclonal antibody against human FcεRIα subunit and hybridoma cell line B4C2 secreting the antibody

[0045] 1. Animal immunization

[0046] Human FcεRIα subunit recombinant protein was used to immunize 6-week-old Balb / c female mice weighing 18-20 g (purchased from Hunan Slack Jingda Experimental Animal Co., Ltd.), and human FcεRIα subunit recombinant protein was taken (the concentration of the subunit recombinant protein was 0.1μg / ml) 0.5ml was mixed with an equal volume of Freund's complete adjuvant and fully emulsified, then injected 0.5ml each subcutaneously at multiple points in the back and abdomen, with an interval of 3 weeks, and took 0.5ml human FcεRIα subunit recombinant protein (this subunit recombinant protein The concentration of the protein is 0.1 μg / ml) plus an equal volume of Freund's incomplete adjuvant, mixed and fully emulsified, and the second intraperitoneal injection of 0.5 ml per mouse. The third immunization was perform...

Embodiment 2

[0054] Example 2 Identification of anti-human FcεRIα subunit monoclonal antibody secreted by hybridoma cell line B4C2

[0055] 1. Screen positive clones of hybridoma cell lines by flow cytometry

[0056] Take CHO3D10 cells in the logarithmic growth phase (Chinese hamster ovary cells, donated by the Laboratory Department of Changzheng Hospital), wash with phosphate buffer saline (PBS), centrifuge at 1000rpm / min for 5min; discard the supernatant, and take 1% bovine serum white Protein-phosphate buffered saline (BSA-PBS, said percentage is mass percentage) adjusted cell concentration to be 2×10 5 cells / ml; take 1ml of CHO3D10 cells, add 100μl of purified anti-human FcεRIα subunit monoclonal antibody (prepared in Example 1), incubate at 4°C for 45min, wash twice with PBS, centrifuge at 1000rpm / min for 5min, discard the supernatant, add 100μl working concentration of goat anti-mouse IgG(H+L)-PE fluorescent antibody, incubate at 4°C for 45min, wash twice with PBS, centrifuge at 100...

Embodiment 3

[0064] Example 3 Application of anti-human FcεRIα subunit monoclonal antibody MAb-B4C2

[0065] Tonsil tissue slices derived from human tissues were baked in an oven at 58°C for 1 hour, and immediately placed in xylene I for 15 minutes and xylene II for 15 minutes after taking out the tissue slices from the oven; Soak in 95% ethanol and 75% ethanol solution for one minute, and then wash twice with distilled water; heat-repair the tissue slices in citric acid pH 6.0 repair solution for 15 minutes, then cool to room temperature; wash the tissue slices three times with PBS , shake off and dry the liquid around the tissue on the section, place it flat in a wet box, add 50 μl of 3% peroxidase blocker hydrogen peroxide dropwise on the tissue, and react in the dark for 20 minutes; after washing with PBS three times, Shake off and dry the liquid around the tissue on the tissue section, and add 50 μl of blocking solution (5% BSA-PBS, the percentage is mass percentage) dropwise at room ...

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Abstract

The invention discloses an anti-human FcepsilonRIalpha subunit monoclonal antibody and application thereof. A hybridoma cell strain producing the monoclonal antibody is collected in China Center for Type Culture Collection, and the collection number thereof is CCTCC NO:201211. The anti-human FcepsilonRIalpha subunit monoclonal antibody prepared by the invention can be combined with a human IgE high-affinity receptor FcepsilonRIalpha, and can be used for detecting FcepsilonRIalpha expression on the surface of a cell; and the monoclonal antibody is of great significance for prevention and treatment of mast cell and basophilic granulocyte related diseases such as allergic dermatitis, dermatomyositis, psoriasis and the like, and has great clinical application value. The hybridoma cell strain provided by the invention is favorable in cell strain growth vigor, uniform in cell body size, muddy or transparent and vigorous in division, and has a proliferative potential of infinite division.

Description

technical field [0001] The invention belongs to the field of bioengineering, and particularly relates to an anti-human FcεRIα subunit monoclonal antibody and application thereof, and a hybridoma cell strain secreting the anti-human FcεRIα subunit monoclonal antibody. Background technique [0002] IgE (immunoglobulin E) receptors are important mediators of allergic reactions. Mast cells and basophils are the main effector cells of type Ⅰ allergic diseases and some mast cell related diseases such as allergic dermatitis, dermatomyositis and psoriasis. The complex formed by the combination of the allergen and its specific IgE binds to the IgE receptors on the cell surface such as mast cells and basophils, and then micro-aggregates the IgE receptors, triggering signal transduction, including protein kinase activation, phosphatidylinositol hydrolysis and Ca 2+ Flow, etc., causing cell mediator and cytokine synthesis, increased expression of adhesion molecules, cell stretching, m...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/28G01N33/68G01N33/577A61K39/395A61P17/00A61P17/06C12R1/91
Inventor 李莉王娟
Owner 李莉
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