Method for detecting subject humoral disease related protein polymerizing power

A technology of related proteins and aggregation ability, which is applied in the field of in vitro detection of aggregation ability of disease-related proteins in body fluids of subjects, can solve problems such as unclear, decreased total content of α-synuclein, increased total content, etc., to achieve easy acceptance , Simple operation, and the effect of improving sensitivity

Inactive Publication Date: 2008-11-19
XUANWU HOSPITAL OF CAPITAL UNIV OF MEDICAL SCI
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  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

Analysis of α-synuclein in cerebrospinal fluid and blood showed that the content of α-synuclein oligomers in the blood and cerebrospinal fluid of PD patients and Lewy body dementia patients was higher than that of control patients, but α-synuclein The change of the total nucleoprotein content has opposite results among different experimenters: Lee et al. used the double-antibody sandwich ELISA method to prove that the total content of α-synuclein in the blood increased in PD, while Li and Tokuda et al. The total content of α-synuclein in blood and cerebrospinal fluid was significantly reduced by Western blot and ELISA, the reason for this difference is not clear

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  • Method for detecting subject humoral disease related protein polymerizing power
  • Method for detecting subject humoral disease related protein polymerizing power
  • Method for detecting subject humoral disease related protein polymerizing power

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Experimental program
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specific Embodiment approach

[0027] According to a specific embodiment of the present invention, the process flow of the method includes:

[0028] First, a sample of bodily fluid is taken from the subject. The subject as the body fluid sample source of the present invention can be clinically diagnosed PD patient serum or plasma (H-Y stage in I-IV), aged 40-70 years old, and the control body fluid sample can be a normal healthy physical examination 40- 70-year-old middle-aged and elderly people or other age-matched non-PD patients. In the present invention, bodily fluids may be, for example but not limited to, blood and cerebrospinal fluid. Methods for extracting body fluid samples are well known to those skilled in the art. The level of α-synuclein in the blood of normal people and patients is very low, and the content of α-synuclein oligomers in the blood of patients is very low, so it is difficult to directly measure the content of α-synuclein oligomers big. Since the method of the present invention i...

Embodiment 1

[0042] Body Fluid Sample Preparation

[0043] In the present invention, the body fluid sample is illustrated by taking serum as an example, but those skilled in the art should understand that the method for preparing body fluid samples using other body fluids such as cerebrospinal fluid is similar.

[0044] In this example, serum was obtained from 34 clinically diagnosed PD patients (17 males, 17 females, aged 54-82 years, with an average age of 65.4 years), while control serum was obtained from 5 healthy individuals with normal physical examination (3 males, 2 females, mean age 60 years). In this example, the volume of extracted serum was about 20 μL.

[0045] Prepare PBS solution

[0046] The composition of the PBS solution used in the present embodiment is as follows:

[0047] KCl

0.1g

K H 2 PO 4

0.1g

Na 2 HPO 4 .12H 2 o

1.45g

NaCl

4g

DDW (double distilled water)

500mL

[0048] The com...

Embodiment 2

[0083] In this example, the detection method and steps are the same as in Example 1, except that the volume of extracted serum is 40 μL.

[0084] As a result of the detection, the aged (oscillating) sample contains synuclein oligomers, and the kit of the present invention does not recognize fresh samples (mainly containing monomers) before 200-fold dilution of the sample, but recognizes aged samples (containing oligomeric body), but when the concentration is higher than 200 times, since oligomers can also be formed in fresh samples, the content of oligomers increases with the prolongation of the shaking time, and the trend of absorbance increase occurs, which illustrates the present invention The kit mainly recognizes oligomers.

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Abstract

The invention relates to a method for external detection of polymerizing power of disease-related protein promoted by body fluids of subjects. More specifically, the invention concerns a method for detecting the oligomer formation power of disease-related protein such as alpha-synuclein promoted by body fluids of the subjects in vitro. The method comprises: incubating purified recombinant human disease-related protein and body fluids of the subjects by stirring at a certain temperature to make the two components be polymerized, and detecting the content of oligomers of disease-related protein such as alpha-synuclein in the incubated body fluids by utilizing a method that can detect protein oligomers. The method provided by the invention has advantages that: 1) the need for body fluid sample is less, so the subjects are easy to accept; 2) operations are simple; and 3) the result obtained from the detection method reflects the synthetic action of various factors of polymerization of disease-related protein to subjects, and the result is particularly more favorable for determining comprehensive pathophysiological situations of patients with Parkinson disease caused by complex factors.

Description

[0001] Cross References to Related Applications [0002] This application claims the priority of the titled "Method for Detecting the Polymerization Ability of Disease-Related Proteins in Body Fluids of Subjects" (application number 200710107817.9) submitted to the Chinese Patent Office on May 16, 2007, and its entire contents are hereby incorporated as refer to. technical field [0003] The present invention relates to a method for in vitro detection of disease-associated protein polymerization ability in body fluids of subjects, more specifically, relates to a method for in vitro detection of diseases in body fluids (including serum, plasma or cerebrospinal fluid) of subjects with Parkinson's disease (PD) A method for the oligomer forming ability of related proteins such as α-synuclein (also known as α-Syn). Background technique [0004] Parkinson's disease (PD) is a common neurodegenerative disease with an incidence rate as high as 2% in the population over 60 years old....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/543G01N21/27
Inventor 于顺陈彪李雁李尧华
Owner XUANWU HOSPITAL OF CAPITAL UNIV OF MEDICAL SCI
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