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Method for preparing anti-human R47H-TREM2 mutant human monoclonal antibody

A human monoclonal antibody and mutant technology, applied in the field of biomedicine, can solve the problems of difficulty and impracticality of monoclonal antibodies

Inactive Publication Date: 2016-01-06
河北翰林生物科技有限公司
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  • Abstract
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  • Application Information

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Problems solved by technology

[0004] Production of human monoclonal antibodies has been very difficult in existing research and practice due to the impracticality of immunizing humans with the antigen of interest

Method used

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  • Method for preparing anti-human R47H-TREM2 mutant human monoclonal antibody
  • Method for preparing anti-human R47H-TREM2 mutant human monoclonal antibody

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Embodiment

[0050] A method for preparing a human monoclonal antibody against a human R47H-TREM2 mutant, comprising the steps of:

[0051] 1) Synthesis of polypeptide antigen

[0052] 201 R47H-TREM2 polypeptides of 6-15 aa were artificially synthesized to make the epitope surround the mutation target of TREM2 gene and expand to both sides centered on R47 of TREM2. Each polypeptide sequence was staggered by 1-2 aa; then HPLCC-18 reversed Column purified and freeze-dried;

[0053] 2) Preparation of R47H-TREM2 mutant peptide antigen array PeptideArray

[0054] Use 96 or 184-well ELISA plates, dilute the polypeptide antigen to 1-10 μg / ml with coating buffer 0.05mol / L carbonate buffer, add 0.1ml to each well, overnight at 4°C, wash 3 times the next day and use Wet box 4C preservation;

[0055] 3) Collection of CD22+ B cells

[0056] Collect peripheral blood from 60-80-year-old healthy elderly and AD patients, 15mL / person, and perform density gradient centrifugation with FicollHypaque reage...

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Abstract

The invention provides a method for preparing an anti-human R47H-TREM2 mutant human monoclonal antibody. The method comprises the following steps that (1) a polypeptide antigen is synthesized, wherein 201 6-15aa R47H-TREM2 polypeptides are artificially synthesized; antigenic determinants are expanded to both sides with the R47 of TREM2 as the center and around TREM2 gene mutation targets, wherein each polypeptide sequence is staggered by 1-2 aa; and HPLC C-18 reverse column purification and freeze drying are carried out; (2) an R47H-TREM2 mutant polypeptide antigen array (Peptide Array) is prepared; (3) CD22+B-cells are collected; (4) cell culturing is carried out; (5) B-cell immortalization is carried out; and (6) B-cell positive monoclone is screened with the R47H-TREM2 mutant polypeptide antigen array. According to the invention, the human monoclonal antibody is prepared with the B-cell immortalization technology. The human monoclonal antibody has important early clinical diagnosis and therapeutic potentials on Alzheimer's disease (AD) patients.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a method for preparing a human monoclonal antibody against a human R47H-TREM2 mutant. Background technique [0002] Alzheimer's disease AD (Alzheimer'sdisease) is a primary degenerative brain degenerative disease of unknown etiology. , reaching 40%-50% by the age of 95, and the average survival period after diagnosis of AD is 6-8 years. According to the relevant epidemiological research data organized by the Alzheimer's Disease (AD) International Alliance and the prevalence data of Alzheimer's disease provided by the World Health Organization (WHO), there are currently about 24.3 million AD patients in the world, and it is estimated that new ones will be added every year. Alzheimer's disease cases are 4.6 million and will double every 20 years. By 2040, there will be 81.1 million AD and dementia cases worldwide. The pathogenesis of Alzheimer's disease (AD) is thought to be...

Claims

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Application Information

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IPC IPC(8): C07K16/18
CPCY02P20/55
Inventor 周希明
Owner 河北翰林生物科技有限公司
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