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Heavy and light chain variable region gene of monoclonal antibody resisting human amyloid protein, and its uses

A technology of monoclonal antibody and amyloid protein, applied in the direction of anti-animal/human immunoglobulin, application, antibody, etc., to achieve the effect of reducing cerebral hemorrhage, good immune effect, and high titer of immune serum

Active Publication Date: 2011-04-20
BEIJING JIAOTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Targeting 16.5-25KDAβ for diagnostic and therapeutic use 1-42 Oligomeric monoclonal antibody, not yet reported

Method used

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  • Heavy and light chain variable region gene of monoclonal antibody resisting human amyloid protein, and its uses
  • Heavy and light chain variable region gene of monoclonal antibody resisting human amyloid protein, and its uses
  • Heavy and light chain variable region gene of monoclonal antibody resisting human amyloid protein, and its uses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1 Preparation of oligomer high affinity anti-human Aβ monoclonal antibody A8

[0060] 1. Preparation of Aβ Oligomerization Mixture

[0061] 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-propannol, 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 the oligomeri...

Embodiment 2

[0079] Embodiment 2 Indirect ELISA experiment

[0080] 1. Method

[0081] (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.

[0082] (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.

[0083] (3) Blocking: add PBS-T containing 0.2% BSA, 100 μl per well. 37°C, 2h.

[0084] (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).

[0085] (5) Plate washing with PBS-T: 3 times, 5 minutes each time.

[0086] (6) Add HRP-labeled goat anti-mouse IgG (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.) diluted 1:10000, 100 μl p...

Embodiment 3

[0095] Example 3 Western blot (Western blot) experiment

[0096] 1. Method

[0097] 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.

[0098] After the ...

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Abstract

The invention relates to a heavy and light chain variable region gene of monoclonal antibody resisting human amyloid and coded polypeptide thereof. The invention also relates to the application of thegene and the polypeptide in preparing reagents and drugs for diagnosing Alzheimer's disease, and clones the light and heavy chain variable region genes of the antibody from cultured oligomer high affinity antibody A8 hybridoma. The obtained gene can correctly code a mouse antibody variable region. Based on the cloned A8 antibody light and heavy chain variable region genes, a gene recombination method can be adopted to construct and express the chimeric antibody, single chain antibody, Fab and other genetic engineering anbodies of a plurality of micromolecules in the hope of being used for diagnosing and treating Alzheimer's disease.

Description

Technical field: [0001] The present invention relates to the heavy chain and light chain variable region genes of anti-human amyloid monoclonal antibody, in particular to the polypeptide encoded by the heavy chain and light chain variable region genes of oligomer high affinity anti-human Aβ monoclonal antibody A8 . The invention also relates to the application of the gene and polypeptide in the preparation of diagnostic reagents and medicines. Background technique: [0002] The deposition of β-amyloid plaques in the brains of patients with Alzheimer's disease is a typical lesion of the disease. [0003] Immunotherapy targeting amyloid (Aβ) molecules to treat or prevent Alzheimer's disease is an important approach to treatment. At present, some people have carried out some work and achieved different degrees of curative effect. However, it is not yet possible to specifically recognize Aβ oligomers, so the immune effect is not ideal, and monoclonal antibodies that specifica...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/13C07K16/18G01N33/577A61K39/395A61P25/28C12P21/08
Inventor 何金生王鑫张莹洪涛
Owner BEIJING JIAOTONG UNIV
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