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Method for quantitatively detecting alpha-synuclein auto-antibodies in human sera

A technique for quantitative detection of synuclein, which is applied in the field of detection of autologous α-synuclein antibodies, can solve problems affecting the quality of life of patients, increasing the burden on society and families, etc.

Inactive Publication Date: 2010-04-07
XUANWU HOSPITAL OF CAPITAL UNIV OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At this time, there is no radical treatment except the symptomatic treatment of supplementing dopamine, which will seriously affect the quality of life of the patient and add a heavy burden to the society and family.

Method used

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  • Method for quantitatively detecting alpha-synuclein auto-antibodies in human sera
  • Method for quantitatively detecting alpha-synuclein auto-antibodies in human sera
  • Method for quantitatively detecting alpha-synuclein auto-antibodies in human sera

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] 1. Prokaryotic expression, purification and identification of recombinant human α-synuclein

[0055] The results of WESTERN BLOT showed that 50-1000ng of purified recombinant human α-synuclein combined with mouse anti-human monoclonal antibody (3D5) showed positive bands of different sizes, and the molecular weight of the protein was about It is about 18kD, which is consistent with the molecular weight of human α-synuclein, which proves that the recombinantly expressed protein is based on human α-synuclein ( image 3 ).

[0056] 2.: Production of rabbit anti-human α-synuclein polyclonal antibody

[0057] After immunizing rabbits with recombinantly expressed human α-synuclein, the results of WESTERNBLOT on the harvested antiserum showed that the antiserum could clearly display recombinantly expressed human α-synuclein at a dilution of 1:30000, indicating that The rabbit antiserum has a good affinity with the antigen; while the results of rat brain homogenate showed tha...

Embodiment 2

[0059] Kit preparation:

[0060] Kit composition: 96-well ELISA plate (Corning Company), coated antigen (recombinant human α-synuclein), standard antibody (rabbit anti-human α-synuclein polyclonal antibody), antigen diluent (0.05M Carbonic acid buffer, pH 9.6), antibody and serum diluent (10% BSA / PBS), plate washing solution (0.1% (v / v) Tween-20 / PBS), blocking solution (10% BSA / PBS), marker Secondary antibody (AP-goat anti-rabbit IgG; AP-goat anti-human IgG), chromogenic substrate (p-Nitrophenyl Phosphate Liquid Substrate System, Sigma) relevant diluent, washing solution, blocking solution, labeled secondary antibody, chromogenic The composition and preparation method of the substrate belong to the known technology.

Embodiment 3

[0062] Detection of autologous human α-synuclein antibody in serum samples

[0063] Autologous α-synuclein antibodies were detected in the serum of 116 clinically diagnosed Parkinson's patients and 78 healthy controls by relative quantitative ELISA. The results showed that the autologous α-synuclein antibody content in the serum of Parkinson's patients was 30.3±11.69 antibody units, and the highest serum autologous α-synuclein antibody in Parkinson's patients was 64.72567; It was 15.7±5.99 antibody units, and the highest value was 27.665. The content of autologous α-synuclein antibody in the serum of Parkinson's patients was significantly higher than that of normal healthy controls (p Figure 5 . Relative quantitative ELISA method to detect the content of autologous α-synuclein antibody in human serum. ** p<0.01.

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Abstract

The invention relates to a method for quantitatively detecting alpha-synuclein auto-antibodies in human sera, and prepares recombinant human alpha-synuclein as antigens and rabbit anti-human alpha-synuclein polyclonal antibodies as antibodies into a kit, wherein, the quantities of alpha-synuclein auto-antibodies in the sera of patients with Parkinson's disease (PD) and in the sera of normal healthy persons are detected by utilizing the antigen-antibody reaction principle and are compared; and the presence and quantity of alpha-synuclein auto-antibodies in human sera are judged by the kit on the basis of color reaction.

Description

Technical field: [0001] The invention relates to a method for detecting autologous α-synuclein antibody in human serum, which can be used for the diagnosis of Parkinson's disease. Background technique: [0002] Parkinson's disease (Parkinson's disease, PD) is an senile neurodegenerative disease with an incidence rate second only to senile dementia. There are eosinophilic inclusion bodies called Lewy bodies. α-synuclein is the main component of Lewy bodies, mainly in the form of aggregates. During the pathogenesis of Parkinson's disease, high levels of α-synuclein can form an allosteric form that is toxic to dopaminergic neurons, and this allosteric form may be in the local microenvironment where dopaminergic neurons die The formation can lead to the production of autoantibodies, which in turn increases the level of autoantibodies to α-synuclein in the serum of Parkinson's patients. So far, only one literature has reported that the level of autologous α-synuclein antibody ...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/543G01N33/52G01N33/531C12N15/70C12P21/02C07K16/18C07K1/30C12R1/19
Inventor 陈彪徐胜利
Owner XUANWU HOSPITAL OF CAPITAL UNIV OF MEDICAL SCI
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