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Anti-sFc [epsilon] RI [alpha] monoclonal antibody and application thereof

A monoclonal antibody and antibody technology, applied in the fields of genetic engineering and immunology, can solve problems such as poor stability, low yield, and low activity

Active Publication Date: 2019-12-17
李莉 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, when developing such anti-sFcεRIα antibodies, they often encounter bottlenecks such as decreased antibody affinity, low activity, poor stability, or low yield.

Method used

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  • Anti-sFc [epsilon] RI [alpha] monoclonal antibody and application thereof
  • Anti-sFc [epsilon] RI [alpha] monoclonal antibody and application thereof
  • Anti-sFc [epsilon] RI [alpha] monoclonal antibody and application thereof

Examples

Experimental program
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Effect test

preparation example Construction

[0104] Preparation of monoclonal antibodies

[0105] Antibodies of the present invention can be prepared by various techniques known to those skilled in the art. For example, an antigen of the invention may be administered to an animal to induce the production of monoclonal antibodies. For monoclonal antibodies, hybridoma technology can be used to prepare (see Kohler et al., Nature 256; 495, 1975; Kohler et al., Eur.J.Immunol.6:511, 1976; Kohler et al., Eur.J.Immunol. 6:292,1976; Hammerling et al., In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981) or can be prepared by recombinant DNA methods (US Patent No. 4,816,567).

[0106] Representative myeloma cells are those that fuse efficiently, support stable high-level production of antibody by selected antibody-producing cells, and are sensitive to culture medium (HAT medium matrix), including myeloma cell lines, such as murine Myeloma cell lines, including those derived from MOPC-21 and MPC-11 mouse tumors (...

Embodiment 1

[0139] Example 1 Preparation of anti-sFcεRIα monoclonal antibody

[0140] 1. Establishment and screening of hybridoma cell lines

[0141] (1) Immunized animals:

[0142] Take FcεRIα prokaryotic expression full-length protein and add an equal amount of Freund’s complete adjuvant, grind and mix thoroughly, and then subcutaneously inject multiple points to immunize BALB / c mice. Two weeks later, add Freund’s incomplete adjuvant for the second time, and then Immunize once every two weeks, 6 times in total, 50 μg each time, and the last tail vein booster immunization; a total of 5 mice were immunized, marked by punching the right ear.

[0143] (2) Cell fusion:

[0144] When the titer of anti-human FcεRIα antibody in the mouse serum was above 1:50000, the mice that were successfully immunized were selected for cell fusion. Collect the splenocytes of the mouse, and divide 1×10 8 mouse splenocytes with 1×10 7 Mix SP2 / 0 cells, centrifuge at 1200rpm for 5min, discard the supernatant...

Embodiment 2

[0167] Example 2 Anti-sFcεRIα antibody titer detection

[0168] 1. Indirect ELISA to detect the titer of monoclonal antibody B5A11

[0169] (1) Dilute FcεRIα to 5 μg / mL (concentration obtained by checkerboard method) with coating buffer, add 100 μL / well to ELISA 96-well plate, and coat overnight at 4°C;

[0170] (2) The next day, pour out the liquid in the wells, wash the plate 5 times, stay in the wells for 30 seconds each time, and pat dry;

[0171] (3) Add 1% BSA-PBS, 200 μL / well for blocking, block at 37°C for 2 hours, wash the plate, and pat dry;

[0172] (4) Add different dilutions of B5A11 monoclonal antibody to the wells, use a commercially available mouse anti-human FcεRI antibody as a positive control (1:1000, eBioscience, USA), and the same concentration of BSA as a negative control, incubate at 37°C for 1 h, wash plate, pat dry;

[0173] (5) Add 100 μL of HRP-goat anti-mouse IgG secondary antibody (1:5000, diluted in 1% BSA-PBS) to each well, incubate at 37°C fo...

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Abstract

The invention provides an anti-sFc [epsilon] RI [alpha] monoclonal antibody and application thereof. Specifically, the invention provides preparation and application of a hybridoma cell strain for generating an anti-sFc [epsilon] RI [alpha] monoclonal antibody. The anti-sFc [epsilon] RI [alpha] monoclonal antibody aims at a non-IgE binding site of sFc [epsilon] RI [alpha], and the combination of the anti-sFc [epsilon] RI [alpha] monoclonal antibody and the non-IgE binding site does not influence the combination of sFc [epsilon] RI [alpha] and IgE. The antibody can be combined with a sFc [epsilon] RI [alpha] antigen with high specificity, and has high affinity, good specificity and high antibody titer (the combination rate of the antibody diluted by 1: 50 times and sFc [epsilon] RI [alpha]reaches 96.7%). The antibody can simply, conveniently, quickly and specifically carry out accurate analysis and detection on trace sFc [epsilon] RI [alpha], and lays a good foundation for researchingthe effect of the protein on various diseases.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and immunology, and relates to anti-sFcεRIα monoclonal antibody and application thereof. Background technique [0002] When allergens first enter the body, B cells are stimulated to produce allergen-specific IgE antibodies. FcεRI is a high affinity receptor for IgE (IgE high affinity receptor, FcεRI), which is a tetramer composed of α, β and two γ subunits, mainly expressed on the surface of mast cells and basophils, involved in IgE mediated allergic diseases. Its α subunit is divided into an extracellular region, a transmembrane region and an intracellular region. The extracellular region is a high-affinity binding site for the ligand IgE, and the binding force between the two is greater than the antigen-antibody binding force. The combination of IgE and FcεRI on the surface of mast cells can make the cells in a sensitized state. When the same allergen enters the body again, the sen...

Claims

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Application Information

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IPC IPC(8): C07K16/28C12N5/20G01N33/68G01N33/577C12R1/91
CPCC07K16/28G01N33/6872G01N33/577C07K2317/35G01N2333/705G01N33/68A61K39/395C12R2001/91C12N1/00
Inventor 李莉林堃
Owner 李莉
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