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Anti-Alzheimer disease monoclonal antibody and application thereof

A technology for Alzheimer's disease and monoclonal antibodies, applied in the field of new anti-Alzheimer's disease monoclonal antibodies, can solve the problems of inability to specifically recognize Aβ oligomers, immunization methods and effects are not ideal, and achieve immune High serum titer, good immune effect, and the effect of reducing cerebral hemorrhage

Active Publication Date: 2009-09-16
BEIJING JIAOTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the immunization methods and effects of the prior art are still not ideal, especially the inability to specifically recognize specific Aβ oligomers

Method used

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  • Anti-Alzheimer disease monoclonal antibody and application thereof
  • Anti-Alzheimer disease monoclonal antibody and application thereof
  • Anti-Alzheimer disease monoclonal antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1 Preparation of oligomer high affinity anti-human Aβ monoclonal antibody A8

[0051] 1. Preparation of Aβ Oligomerization Mixture

[0052] Aβ 1-42 The peptide was synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd. Refer to the literature (Lambert M, 1998) method, and make necessary adjustments, assemble it in vitro to obtain Aβ under similar natural conditions 1-42 Oligomeric mixture. Aβ 1-42 Peptide 1mg was dissolved in ice-cold hexafluoroisopropanol (1,1,1,3,3,3-hexafluoro-2-βroβannol, HFIP) (Sigma) to make Aβ 1-42 Peptide monomerization, room temperature, after 1h, the HFIP was completely evaporated. Then, 20 μl of anhydrous dimethyl sulfoxide (DMSO) (Sigma) was used to dissolve the Aβ1-42 monomer, and finally it was placed in F12 medium (Sigma) or phosphate buffer system (the volume was supplemented by 1 ml), and placed at 4°C. 24h, let it naturally aggregate, and use Western blot to detect Aβ 1-42 Preparation of oligomeric mi...

Embodiment 2

[0070] Example 2 Indirect ELISA experiment

[0071] 1. Method

[0072] (1) Coating: 10μg·ml -1 The Aβ oligomerization mixture is the coating antigen, which is added to a microtiter plate (SUNRISE Company), 100 μl per well, and left overnight at 4°C.

[0073] (2) PBS-T (NaCl 8g, KCl 0.2g, NaCl 2 HPO 4· 1.44g, KH 2 PO 4· 0.44g, Tween-20 0.05ml, add ddH 2 0 to 1L, pH7.2~7.4) Wash the plate: 3 times, 5min each time.

[0074] (3) Blocking: add PBS-T containing 0.2% BSA, 100 μl per well. 37°C, 2h.

[0075] (4) Add the double-diluted monoclonal antibody A8 into the 96-well plate, 100 μl per well. 37°C, 2h. At the same time, a blank control, a negative control and a positive control were set up (mouse anti-human Aβ serum and Calbiochem anti-human Aβ1-17 were used respectively).

[0076] (5) Plate washing with PBS-T: 3 times, 5 minutes each time.

[0077] (6) Add 1:10000 dilution of HRP-labeled goat anti-mouse IgG (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.), 100 μ...

Embodiment 3

[0086] Example 3 Western blot (Western blot) experiment

[0087] 1. Method

[0088] Take 50-100μg sample, 5×sample buffer, mix well and load the sample, first make the protein pass through the stacking gel with a voltage of 100V. When the sample enters the separating gel, adjust the voltage to keep it constant at 120V. When the bromophenol blue swims to the bottom of the gel, end the electrophoresis, remove the gel, and stain it with Coomassie Brilliant Blue R-250 routinely; put the gel and nitrocellulose membrane into containers containing blotting buffer Equilibrate in the chamber for 10 minutes, put filter paper, gel, NC membrane, and filter paper in order to form a "sandwich" shape, pour the transfer buffer, with the gel side facing the negative electrode and the NC membrane facing the positive electrode, carefully avoiding and driving away air bubbles. Turn on the power, make the constant current 80mA transfer continuously for 2h, cut off the power.

[0089] After the ...

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Abstract

The invention provides an anti-Alzheimer disease monoclonal antibody which can specifically identify and combine Abeta oligomers and can be used in the preparation of diagnostic reagents and treatment drugs for Alzheimer disease.

Description

Technical field: [0001] The present invention relates to a novel anti-Alzheimer's disease monoclonal antibody. The present invention also relates to the application of the monoclonal antibody in the preparation of diagnostic reagents and therapeutic drugs for Alzheimer's disease. Background technique: [0002] Alzheimer's disease (AD for short) is the leading cause of dementia in the elderly over 60 years old in the world. Early diagnosis, prevention and treatment of AD are of great significance. Since Kohler and Milstein created the B lymphocyte hybridoma technology in 1975, various monoclonal antibodies have appeared one after another, and have played an important role in the basic and clinical research of disease diagnosis and treatment. [0003] It is known that Aβ oligomers are the main cause of early AD pathology and changes in learning and memory behavior. In 1999, Schenk et al. used Aβ42 molecule as a polypeptide vaccine to immunize PDAPP transgenic mice. After tr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18G01N33/577A61K39/395A61P25/28
Inventor 何金生张莹王鑫洪涛
Owner BEIJING JIAOTONG UNIV
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