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Method for detecting hemoglobin-combined phosphorylated alpha-synuclein

A technology for synuclein and hemoglobin, applied in the field of detecting hemoglobin-binding phosphorylated alpha-synuclein, can solve the problem of inconclusive diagnosis of Parkinson's disease

Active Publication Date: 2014-12-17
贵安新区康顺生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, limited by the sensitivity and stability of detection techniques and the influence of other interference factors, the detection results of α-synuclein in serum and plasma and its diagnostic significance in Parkinson's disease are still inconclusive

Method used

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  • Method for detecting hemoglobin-combined phosphorylated alpha-synuclein
  • Method for detecting hemoglobin-combined phosphorylated alpha-synuclein
  • Method for detecting hemoglobin-combined phosphorylated alpha-synuclein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0097] Preparation of erythrocyte cytoplasmic protein ( figure 1 )

[0098] step 1,

[0099]Take 5-10ml anticoagulant blood (EDTA, heparin or citrate anticoagulant). Gently mix the anticoagulated blood upside down, add it into a 50ml centrifuge tube, record the volume of whole blood, add PBS at a ratio of 1:1, and mix well.

[0100] Step 2,

[0101] Slowly add the diluted whole blood to the lymphocyte separation medium, centrifuge at 400×g for 20min. At this time, the order of the liquid in the tube from top to bottom is the plasma layer, the buffy coat layer (peripheral blood mononuclear cells), the separation liquid layer and the red blood cell layer.

[0102] Step 3,

[0103] Aspirate off the plasma layer, buffy coat layer and separation liquid layer, transfer the red blood cells at the bottom layer to a new 50ml centrifuge tube, add PBS to 40ml, centrifuge at 2000rpm for 10min, and repeat this 3 times. Aliquot and store at -80°C for later use.

[0104] Step 4,

[0...

Embodiment 2

[0107] ELISA detection process of hemoglobin-binding phosphorylated α-synuclein

[0108] step 1,

[0109] Coating: with NaHCO 3 The buffer solution dilutes the anti-human hemoglobin antibody to a final concentration of about 0.1-2 μg / mL. Add 100 μL of the antibody dilution to each well of the microtiter plate and incubate overnight at 4°C. Rinse each well of the microtiter plate with PBST (which is PBS containing Tween-20), and determine the number and time of washing as required.

[0110] Step 2,

[0111] Blocking: Add 100 μL of blocking solution (PBS containing gelatin and Tween-20) to each well of the microtiter plate, and incubate at 37° C. for 1 to 2 hours. Rinse each well of the microtiter plate with PBST, and determine the number and time of washing as needed.

[0112] Step 3,

[0113] Adding samples: Add 100 μL of the prepared red blood cell cytoplasm sample to each well at a concentration of 8-10 μg / mL, and incubate at 37°C for 1-2 hours. Rinse each well of the...

Embodiment 3

[0122] The detection of real samples by ELISA method ( figure 2 )

[0123] The relative quantitative ELISA method was used to detect the amount of phosphorylated α-synuclein combined with hemoglobin in the cytoplasm of erythrocytes in 15 clinically diagnosed Parkinson's patients and 15 healthy controls. The results showed that the amount of phosphorylated α-synuclein combined with hemoglobin in the erythrocytes of Parkinson's patients was significantly higher than that of healthy controls (p<0.01).

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Abstract

The invention relates to a method for detecting the content of phosphorylated alpha-synuclein combined with hemoglobin in erythrocytes of blood. The method comprises the following steps: taking an anti-human hemoglobin monoclonal antibody as a capturing antibody; taking an anti-phosphorylation human alpha-synuclein monoclonal antibody as a detection antibody; and detecting the content of the phosphorylated alpha-synuclein combined with the hemoglobin of Parkinson patients and normal healthy persons by utilizing an antigen-antibody reaction principle, and comparing.

Description

technical field [0001] The invention relates to a method for detecting the content of phosphorylated alpha-synuclein combined with hemoglobin in red blood cells in blood, and the method can be used for the diagnosis of Parkinson's disease. Background technique [0002] Parkinson's disease is a common degenerative disease of the nervous system, clinically manifested mainly by motor symptoms such as resting tremor, slowness of movement, muscle stiffness, and postural instability. The cause of these symptoms is the massive death of dopamine neurons in the substantia nigra of the midbrain and the resulting substantial reduction of dopamine neurotransmitters in the striatum. The main pathological feature of Parkinson's disease is the presence of eosinophilic inclusion bodies in nerve cells - Lewy bodies and Lewyneurites. The main components of Lewy bodies and Lewy nerve cords are fibrous α-synuclein (alpha-synuclein). Numerous studies have shown that α-synuclein is a protein th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
CPCG01N33/721
Inventor 于顺杨巍巍李昕
Owner 贵安新区康顺生物科技有限公司
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