Anti-human Fc epsilon RI alpha subunit monoclonal antibody and application thereof

Abstract

The invention discloses an anti-human Fc epsilon RI alpha subunit monoclonal antibody and application thereof. A hybridoma cell strain capable of producing the monoclonal antibody is preserved in China Center for Type Culture Collection and has the preservation number of CCTCC NO. C201326. The anti-human Fc epsilon RI alpha monoclonal antibody prepared by the invention can be bound with high-affinity receptor Fc epsilon RI alpha of human IgE and can be applied in detecting the expression of cell surface Fc epsilon RI alpha. The monoclonal antibody has important significance and relatively large clinical value for prevention and treatment of mast cell and basophilic granulocyte-related diseases, such as allergic dermatitis, dermatomyositis and psoriasis. The hybridoma cell strain disclosed by the invention grows well, and is bright and transparent, the cell body is uniform, the division is strong and the hybridoma cell strain has indefinitely divided proliferative capability.

Description

technical field

[0001] The invention belongs to the field of bioengineering, and particularly relates to an anti-human FcεR I α subunit monoclonal antibody and its application, and a hybridoma cell strain secreting the anti-human FcεR I α 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...

Examples

Embodiment 1

[0044] Example 1 Preparation of monoclonal antibody against human FcεR I α subunit and hybridoma cell line B5B4 secreting the antibody

[0045] 1. Animal immunization

[0046] Immunize 6-week-old Balb / c female mice weighing 18-20 g with human FcεR Ⅰ α subunit recombinant protein (purchased from Hunan Slake Jingda Experimental Animal Co., Ltd.): take 0.5ml human FcεR Ⅰ α subunit recombinant protein (the recombinant Protein concentration (0.1 μg / ml) was mixed with an equal volume of Freund's complete adjuvant and fully emulsified, and 0.5 ml of each mouse was subcutaneously injected into the back and abdomen at multiple points, with an interval of 3 weeks, and 0.5 ml of human FcεR Ⅰ α subunit recombinant protein ( The concentration of the recombinant protein was 0.1 μg / ml) and an equal volume of Freund's incomplete adjuvant was mixed and fully emulsified, and the second intraperitoneal injection of 0.5 ml was performed for each mouse. The third immunization was performed after ...

Embodiment 2

[0054] Example 2 Identification of anti-human FcεR Ⅰ α subunit monoclonal antibody secreted by hybridoma cell line B5B4

[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 1000 rpm / min for 5 min; discard the supernatant, and replace with 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; add 100 μl of purified anti-human FcεR Ⅰ α subunit monoclonal antibody (prepared in Example 1) to 1 ml of CHO3D10 cells, incubate at 4°C for 45 min, wash twice with PBS, centrifuge at 1000 rpm / min for 5 min , discard the supernatant, add 100 μl working concentration of goat anti-mouse IgG (H+L)-PE fluorescent antibody, incubate at 4°C for 45 min, wash twice with PB...

Embodiment 3

[0064] Example 3 Application of anti-human FcεR Ⅰ α subunit monoclonal antibody MAb-B5B4

[0065]Tonsil tissue slices derived from human tissues were baked in an oven at 58°C for 1 h, and immediately placed in xylene I for 15 min and xylene II for 15 min after taking out the tissue slices from the oven; Soak in water ethanol, 95% ethanol, and 75% ethanol solution for one minute, and then wash twice with distilled water; microwave heat repair for 15 min in citric acid repair solution with pH6.0, and then cool to room temperature; slice the tissue After washing with PBS three times, shake off and dry the liquid around the tissue on the tissue section, place it flat in a wet box, add 50 μl of 3% peroxidase blocking agent hydrogen peroxide on the tissue, and react in the dark for 20 min After washing with PBS three times, shake off and dry the liquid around the tissue on the section, and add 50 μl of blocking solution (5% BSA-PBS, the percentage is mass percentage) dropwise at roo...

technical field

[0001] The invention belongs to the field of bioengineering, and particularly relates to an anti-human FcεR I α subunit monoclonal antibody and its application, and a hybridoma cell strain secreting the anti-human FcεR I α 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...

Examples

Embodiment 1

[0044] Example 1 Preparation of monoclonal antibody against human FcεR I α subunit and hybridoma cell line B5B4 secreting the antibody

[0045] 1. Animal immunization

[0046] Immunize 6-week-old Balb / c female mice weighing 18-20 g with human FcεR Ⅰ α subunit recombinant protein (purchased from Hunan Slake Jingda Experimental Animal Co., Ltd.): take 0.5ml human FcεR Ⅰ α subunit recombinant protein (the recombinant Protein concentration (0.1 μg / ml) was mixed with an equal volume of Freund's complete adjuvant and fully emulsified, and 0.5 ml of each mouse was subcutaneously injected into the back and abdomen at multiple points, with an interval of 3 weeks, and 0.5 ml of human FcεR Ⅰ α subunit recombinant protein ( The concentration of the recombinant protein was 0.1 μg / ml) and an equal volume of Freund's incomplete adjuvant was mixed and fully emulsified, and the second intraperitoneal injection of 0.5 ml was performed for each mouse. The third immunization was performed after ...

Embodiment 2

[0054] Example 2 Identification of anti-human FcεR Ⅰ α subunit monoclonal antibody secreted by hybridoma cell line B5B4

[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 1000 rpm / min for 5 min; discard the supernatant, and replace with 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; add 100 μl of purified anti-human FcεR Ⅰ α subunit monoclonal antibody (prepared in Example 1) to 1 ml of CHO3D10 cells, incubate at 4°C for 45 min, wash twice with PBS, centrifuge at 1000 rpm / min for 5 min , discard the supernatant, add 100 μl working concentration of goat anti-mouse IgG (H+L)-PE fluorescent antibody, incubate at 4°C for 45 min, wash twice with PB...

Embodiment 3

[0064] Example 3 Application of anti-human FcεR Ⅰ α subunit monoclonal antibody MAb-B5B4

[0065]Tonsil tissue slices derived from human tissues were baked in an oven at 58°C for 1 h, and immediately placed in xylene I for 15 min and xylene II for 15 min after taking out the tissue slices from the oven; Soak in water ethanol, 95% ethanol, and 75% ethanol solution for one minute, and then wash twice with distilled water; microwave heat repair for 15 min in citric acid repair solution with pH6.0, and then cool to room temperature; slice the tissue After washing with PBS three times, shake off and dry the liquid around the tissue on the tissue section, place it flat in a wet box, add 50 μl of 3% peroxidase blocking agent hydrogen peroxide on the tissue, and react in the dark for 20 min After washing with PBS three times, shake off and dry the liquid around the tissue on the section, and add 50 μl of blocking solution (5% BSA-PBS, the percentage is mass percentage) dropwise at roo...

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