Method of examining alzheimer s disease and diagnostic reagent

A technology for Alzheimer's disease and testing methods, which can be used in disease diagnosis, material inspection products, biological testing, etc., and can solve problems such as a certain understanding of the amount of Aβ1-42

Inactive Publication Date: 2007-10-03
三光纯药株式会社
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0011] In this way, compared with healthy people, the amount of Aβ1-42 in the cerebrospinal fluid of AD patients tends to decrease, but so far there is no certain understanding of the amount of Aβ1-42 in the blood. become a big subject

Method used

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  • Method of examining alzheimer s disease and diagnostic reagent
  • Method of examining alzheimer s disease and diagnostic reagent
  • Method of examining alzheimer s disease and diagnostic reagent

Examples

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experiment example 1 and 2

[0119] (Experimental example 1 and 2) Electrochemiluminescence double antibody sandwich assay

[0120] Experimental examples 1 and 2 are analyzes by the electrochemiluminescence double-antibody sandwich assay using two kinds of Aβ-specific antibodies. As the primary antibody, the 21F12 mouse monoclonal antibody (manufactured by Innogenetics), which was prepared by immunizing mice with the 33-42 amino acid site of Aβ1-42, was used as an Aβ1-42 specific reactive antibody. As the secondary antibody, ruthenium-coordinated A 3D6 mouse monoclonal antibody (manufactured by Innogenetics) prepared by immunizing mice with the 1-5 amino acid site of Aβ1-42 was labeled with the compound and used.

[0121] The preparation method of each constituent component of the reagent is described below.

[0122] (1) Preparation method of 21F12 antibody-bound magnetic beads

[0123] Dilute 21F12 mouse monoclonal antibody to 1 mg / mL antibody concentration with 10 mmol / L potassium phosphate buffer (pH...

experiment example 1

[0127] (Experimental example 1) Determination of Aβ1-42 synthetic peptide

[0128] Add 150 μL of reaction solution [50mmol / L Tris HCl, 1% BSA, 0.15mol / L NaCl, 0.01% (W / V) Tween20, 10mmol / L to a 500 μL polystyrene cup (hereinafter referred to as reaction cup) EDTA2Na, 0.1% normal mouse serum, pH 7.5] (hereinafter referred to as the sandwich measurement reaction solution), prepare 7 parts, and mix and dilute the Aβ1-42 synthetic peptide with the sandwich measurement reaction solution in each cuvette 50 μL of each sample at 0, 0.5, 1, 5, 10, 25, 50 or 100 pg / mL. To this was added 25 μL each of 21F12 antibody-bound magnetic beads diluted to a concentration of 2 mg / mL with a sandwich measurement reaction solution, and reacted at 30° C. for 9 minutes (first reaction).

[0129] Then, after trapping the magnetic beads with a magnet, remove the liquid in the cuvette, and wash with 350 μL of cleaning solution [50 mmol / L Tris HCL, 0.01% (W / V) tween20, 0.15 mol / L NaCl, pH7.5] Magnetic b...

experiment example 2

[0137] (Experimental Example 2) Determination of AD patient and healthy human serum by electrochemiluminescence double antibody sandwich assay

[0138] Add 150 μL of the solution for the sandwich measurement reaction to the cuvette, prepare the necessary amount, mix the solution for the sandwich measurement reaction in each cuvette, and dilute the Aβ1-42 synthetic peptide to 0, 0.5, 1, 5, 10, 25, 50 μL each of samples obtained at 50 or 100 pg / mL (standard for calibration curve preparation), 25 AD patient serum samples, and 25 healthy human serum samples. To this was added 25 μL each of 21F12 antibody-bound magnetic beads diluted to a concentration of 2 mg / mL with a sandwich measurement reaction solution, and reacted at 30° C. for 9 minutes (first reaction).

[0139] Then, after capturing the magnetic beads with a magnet, remove the liquid in the cuvette, and wash the magnetic beads with 350 μL of washing solution [50 mmol / L Tris HCL, 0.01% (W / V) tween20, 0.15 mol / L NaCl, pH7.5...

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Abstract

It is intended to devise a technique of measuring a ss-amyloid in a biological specimen such as blood and apply the same to the diagnosis of Alzheimer's disease. Alzheimer's disease can be examined by measuring the total content of ss-amyloid 1-42 and ss-amyloid 1-42 fragments having the C-terminal part of ss-amyloid 1-42 in a biological specimen by using an immunoassay method wherein an antibody capable of recognizing the C-terminal part of ss-amyloid 1-42 is employed. It is preferable that the above immunoassay method is a competitive immunoassay method.

Description

technical field [0001] The present invention relates to a method for measuring the total amount of β-amyloid 1-42 in biological samples, especially blood, and β-amyloid 1-42 fragments retaining the C-terminus of β-amyloid 1-42 Test methods and diagnostic reagents for testing diseases related to β-amyloid protein such as Alzheimer's disease. Background technique [0002] β-Amyloid protein (hereinafter referred to as Aβ) is the main constituent of amyloid plaques that are characteristically recognized in the brains of Alzheimer's disease (AD) patients, and Aβ is known to utilize cleavage of its precursor Produced by the action of β-secretase at the β site of the N-terminal of protein (APP) and the action of γ-secretase that cleaves APP-C-terminal presenilin present in the cell membrane. [0003] It is known that the molecular species of Aβ have various molecular weights. Among them, the most famous ones related to neurocytotoxicity are Aβ composed of 40 amino acids (hereinaft...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543G01N33/53G01N33/541
CPCG01N2800/2821G01N2333/4709G01N33/6896C07K16/18G01N33/53G01N33/541
Inventor 高山茂雄山田雄二
Owner 三光纯药株式会社
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