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CFH antibody related with alternative pathway

A bypass pathway and antibody technology, applied in the direction of antibodies, antibody medical components, anti-inflammatory agents, etc., can solve the problem that the comprehensive effect of autoantibodies cannot be completely replaced

Active Publication Date: 2017-03-22
GUANGZHOU TAINUODI BIOTECH +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Some studies have completed the expression of human anti-CFH antibody fragments Fab and scFv, but these small molecule antibodies cannot completely replace the comprehensive effects of autoantibodies in vivo, including antibody-dependent cytotoxicity and antibody-mediated cell lysis, etc. Therefore, the production of a complete fully human anti-CFH antibody has become the goal of further research

Method used

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  • CFH antibody related with alternative pathway
  • CFH antibody related with alternative pathway
  • CFH antibody related with alternative pathway

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Example 1 PBMC isolation and single memory B cell sorting

[0078] Sample acquisition: peripheral blood mononuclear cells (PBMC) were pooled from 11 patients expressing CFH autoantibodies.

[0079] Sorting of memory B cells:

[0080] Avidin-labeled CFH 15-mer polypeptide (sequence shown in SEQ ID NO: 10) was used as bait, and single memory B cells were sorted from pooled PBMCs by flow cytometry.

[0081] Select P1 (lymphocyte gate)→P2(IgG+)→P3(CD3- / CD14- / CD16- / CD20+ / CD27ALL) in turn. Select P1&P2&P3 gate (IgG+ / CD3- / CD14- / CD16- / CD20+ / CD27ALL), count cells (5000 cells / well), sort multiwell. CD3- / CD14- / CD16- / CD20+ / CD27ALL cell population was sorted by flow cytometry. To minimize false positives, Streptavidin Fluor647 and Brilliant Violet 421 were labeled with Alexa. Each fluorophore was labeled with streptavidin on a separate aliquot which was then mixed together and interacted with the biotinylated antigenic peptide. Cells of both fluorophores showing elevated fluore...

Embodiment 2

[0082] Example 2 Single memory B cell RT-PCR isolation antibody variable region gene

[0083] Synthesize cDNA first strand:

[0084] A 96-well plate containing a single B cell was added with 0.5 μM constant region primers of heavy and light chains of each subtype and Superscript III reverse transcriptase, and incubated at 37°C for 1 hour; PCR amplification was carried out under the following conditions: 95°C for 15 minutes; 95°C for 1min, 55°C for 1min, 72°C for 1min, 30cycles; 72°C for 10min; 4°C for 5min. The product cDNA was stored at -20°C.

[0085] Antibody gene isolation by Nest-PCR

[0086] Each single cell amplifies the heavy chain and light chain sequences separately. The 50 μL system contains 5 μL of RT reaction products, HotStarTaq enzyme, dNTPs, and 0.5 μM specific primers for heavy chain and light chain antibodies of each subtype. Reaction conditions: pre-denaturation at 95°C for 5 minutes, and then 35 PCR cycles: 95 30s at ℃, 60s at 55℃, 90s at 72℃, and final...

Embodiment 3

[0087] Example 3 PCR product clone identification and antibody expression

[0088] PCR product purification clone identification:

[0089] Take 2 μL of the amplified product and detect it by 1.2% agarose gel electrophoresis. The gel electrophoresis is identified as positive, and the PCR product of the antibody variable region gene whose heavy chain and light chain can be paired is connected to pcDNA3 by the method of TA cloning. 3 On the carrier (already modified and containing antibody leader and constant region), transform the ligation product into DH5α competent bacteria, culture on a plate containing ampicillin at 37°C overnight, then pick 10 single colonies and perform PCR with specific primers . Reaction conditions: Pre-denaturation at 94°C for 3 minutes, denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 1 minute and 40 seconds, 28 cycles, and finally extension at 72°C for 5 minutes; take 5 μL of PCR products for electrophoresi...

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Abstract

The invention discloses a CFH antibody related with an alternative pathway. The antibody is fully human CFH monoclonal antibody trn1005, and the antibody can be specially combined with the antigen CFH. The invention provides a drug containing the antibody and application of the drug in hyperplasia, or inflammation or diseases or symptoms related to immunity.

Description

technical field [0001] The invention belongs to the field of cellular immunity, and relates to a CFH antibody related to an alternative pathway, in particular to a CFH fully human monoclonal antibody TRN1005. Background technique [0002] The complement system is an important component of innate immunity against microbial infection and contains a variety of plasma and membrane-bound proteins. Complement is widely involved in the body's antimicrobial defense response and immune regulation, and can also mediate the damage response of immune pathology. It is an effector system and effect amplification system with important biological significance in the body. [0003] Complement factor H is an important complement regulatory substance, which determines the fate of complement C3b, whether in blood vessels or on the cell surface, and controls the production and stability of C3 convertase. In addition, it also has functions other than complement regulation. It can act as an adhes...

Claims

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Application Information

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IPC IPC(8): C07K16/18C12N15/13A61K39/395A61P29/00A61P37/02
CPCC07K16/18A61K39/00C07K2317/92C07K2317/565C07K2317/21
Inventor 廖化新王月明袁晓辉昝利鹏刘彤吴昌文张远旭
Owner GUANGZHOU TAINUODI BIOTECH
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