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Anti-human interleukin-6 receptor [beta]-chain monoclonal antibody, preparation method and application thereof

A monoclonal antibody and interleukin technology, applied in the direction of anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, anti-animal/human immunoglobulin, antibody, etc. , elevated levels of high-density lipoprotein and low-density lipoprotein, and increased risk of cardiovascular disease

Active Publication Date: 2015-11-11
SHANGHAI INST OF IMMUNOLOGY +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in-depth studies have found that tocilizumab acts on the classical signal and the trans signal at the same time, completely blocking the activity of interleukin-6, which also brings a series of side effects, such as the occurrence of infection, the level of alanine aminotransferase in the blood increases, Elevated levels of total blood cholesterol, high-density lipoprotein and low-density lipoprotein, and increased risk of cardiovascular disease have hindered its widespread clinical application

Method used

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  • Anti-human interleukin-6 receptor [beta]-chain monoclonal antibody, preparation method and application thereof
  • Anti-human interleukin-6 receptor [beta]-chain monoclonal antibody, preparation method and application thereof
  • Anti-human interleukin-6 receptor [beta]-chain monoclonal antibody, preparation method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0037] Embodiment 1: Antibody preparation:

[0038] Select 2 male BALB / c mice, mix the interleukin-6 receptor β chain antigen with Freund's complete adjuvant, and inject it intraperitoneally after the emulsification is complete. The immunization dose for each mouse is 20ug. After 1 month, the immunization was repeated twice, and one week after the third immunization, the mouse serum was collected, and the binding ability of the serum to the interleukin-6 receptor β chain antigen was identified by indirect enzyme-linked immunosorbent assay, and the normal BABL / c mouse serum was used as As a negative control, mice with higher serum titers were selected for booster immunization before fusion (peritoneal injection only). One week before fusion, a large number of myeloma cells SP2 / 0 were cultured with MD6 medium, so that the cells grew well and were in the logarithmic growth phase. On the day of fusion, the spleens of immune BALB / c mice were aseptically removed and ground to make t...

Embodiment 2

[0039] Example 2: Titer determination of anti-human interleukin-6 receptor β chain monoclonal antibody

[0040] The titer of the anti-human interleukin-6 receptor β chain monoclonal antibody (produced by the hybridoma cell line of experiment number 10.51) was determined by indirect enzyme-linked immunosorbent assay: 0.1ug / ml interleukin-6 receptor β chain antigen Coat a 96-well plate, add 100ul per well, place in a wet box for overnight coating, wash twice with phosphate buffered saline (PBST) containing 0.05% Tween 20, and wash with 1.5% bovine serum albumin (BSA) Block with PBST at room temperature for 1 hour, dilute the purified mouse serum after immunization with interleukin-6 receptor β chain antigen, add to the plate, add 100ul to each well, incubate at room temperature for 1 hour, and wash 4 times with PBST. Add horseradish peroxidase-labeled goat anti-mouse IgG-Fc secondary antibody, add 100ul per well, incubate at room temperature for 1 hour, and wash 4 times with PBS...

Embodiment 3

[0042] Example 3: Subclass Identification of Monoclonal Antibodies

[0043] Purify the anti-human interleukin-6 receptor β chain (10.51) monoclonal antibody using the affinity chromatography column standard kit of Pall Company (USA), and the monoclonal antibody rich liquid is obtained by ultrafiltration membrane, MICON50ml dialysis ultrafiltration device and PBS dialysis. Concentrate, and finally use NANODROP2000 to measure the IgG concentration of the solution containing the monoclonal antibody after dialysis, filter through a 0.22 μm microporous filter, and store it. The purified monoclonal antibody was identified by the mouse monoclonal antibody subtype rapid identification kit of Roche Company, and the anti-human interleukin-6 receptor β chain (10.51) monoclonal antibody heavy chain was identified as IgG2b, and the light chain was κ chain. .

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Abstract

The invention provides an anti-human interleukin-6 (IL-6) receptor [beta]-chain monoclonal antibody. The cDNA and the amino sequence of a light-chain variable region at a Fab-fragment antigen-binding site of the antibody molecule are represented as the Seq ID No.1 and the Seq ID No.2, and the cDNA and the amino sequence of a heavy-chain variable region at the Fab-fragment antigen-binding site of the antibody molecule are represented as the Seq ID No.3 and the Seq ID No.4. The invention also provides a preparation method of the monoclonal antibody, and the applications of the monoclonal antibody in a drug for detecting and intervening in diagnosis and therapy of rheumatoid arthritis. In-vivo and in-vitro tests prove that the monoclonal antibody has a high affinity on positive cells of the IL-6 receptor [beta]-chain and can regulate and control the molecular mechanism of fibroblast-like synoviocytes IL-6 receptor [beta]-chain-RANKL-WNT5A signaling pathway of rheumatoid arthritis patients through antagonism, thereby achieving a result of alleviating immune function disorder and joint injury due to mediated bone metabolic imbalance of the rheumatoid arthritis patients.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, and in particular relates to a monoclonal antibody, in particular to an anti-human interleukin-6 receptor beta chain monoclonal antibody and its preparation method and application. Background technique [0002] The molecular weight is 130kD, and its cell exterior is composed of 6 continuous β-sheet domains, which are immunoglobulin-like domain (functional domain 1), CHR molecule (functional domain 2, functional domain 3) and 3 fibronectin III-like domains (functional domains 4-6), different domains play an important role in ligand connection and signal transduction. Interleukin-6 receptor beta chain as interleukin-6 family cytokines - interleukin-6, cytostatic factor (LIF), oncostatin M (OSM), ciliary neurotrophic factor (CNTF), interleukin-11, cardiotrophic Cardiotrophin-1 and interleukin-27 co-receptors are involved in various diseases by mediating the signal transduction of different inflamm...

Claims

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Application Information

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IPC IPC(8): C07K16/28A61K39/395A61P19/02A61P29/00G01N33/68G01N33/577
Inventor 张冬青陆梅生许荣苗平周晓薇陈呢喃赵蓉钱柳余奇文张继英陈雪华肖涟波何东仪
Owner SHANGHAI INST OF IMMUNOLOGY
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