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Abeta-oligomer measurement method
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An assay method, oligomer technology, applied in biological testing, chemical instruments and methods, anti-animal/human immunoglobulin, etc., which can solve the problem of difficulty in analyzing multiple samples by Western blotting
Inactive Publication Date: 2011-06-08
TAKEDA PHARMA CO LTD
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Problems solved by technology
[0006] Aβ oligomers can be measured by Western blotting, but since it is difficult to analyze multiple samples by Western blotting, it is desirable to develop a method for measuring Aβ oligomers by ELISA
Method used
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Embodiment 1
[0119] The following procedure was carried out by ELISA method (quantification of oligomeric Aβ) using BAN50a and HRP-labeled BAN50a.
[0120] (1) Dissolve the BAN50a antibody in coating buffer A to a concentration of 10 μg / ml (75 μl / well), add it to an EIA 96-well plate (high maximum binding clear plate, Greiner), and store it in a refrigerator (about 4°C) Keep warm all day and night.
[0121] (2) Wash 3 times with PBS (excluding calcium and magnesium; hereinafter referred to as PBS(-)), then add 100 μL / well of blocking buffer B, and store.
[0122] (3) On the day of the experiment, the blocking buffer B was discarded, washed twice with PBS(-), and 25 μl of buffer C was added.
[0123] (4) Dissolve standard monomeric Aβ1-42 [Aβ1-42 TFA salt (PEPTIDE INSTITUTE INC.) in DMSO to a concentration of 1 mM, further dilute to 10 μM with F12 medium (GIBCO), and distribute in 1 ml, Let it stand in a refrigerator (about -4°C) for a day and night, and use buffer C to dilute the above-m...
Embodiment 2
[0132] The effect of the test compound on oligomer formation was studied as follows.
[0133] (1) Aβ1-42 was dissolved in DMSO to a concentration of 300 μM.
[0134] (2) Prepare a reaction solution with an appropriate composition and add it to a test tube.
[0135] [Composition example of the reaction solution (in the final reaction solution of Aβ1-42, the concentration of Aβ1-42 is 3 μM, and the compound is 3 μM)]
[0136] Aβ1-42 (300μM) 1μL
[0137] Curcumin solution (300μM) 1μL
[0138] F12 medium 98μL
[0139] 100 μL total
[0140] (3) Incubate the test tube at 4°C for 1 day and night.
[0141] (4) The reaction solution was diluted 100-fold with buffer C (above), and measured according to the above-mentioned ELISA method.
[0142] (5) Taking the vehicle group as 100%, calculate the inhibition rate.
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Abstract
Disclosed is a method for measuring an Abeta-oligomer by ELISA method, particularly a method for selectively measuring an Abeta-oligomer having a relatively high molecular weight. The method for measuring an Abeta-oligomer comprises the following steps (a) and (b): (a) contacting a monoclonal antibody (a first antibody) which can recognize a partial peptide comprising the amino acid sequence depicted in SEQ ID NO:1 and is bound to an insoluble carrier with a sample which is suspected to contain the Abeta-oligomer, thereby binding the Abeta-oligomer to the first antibody; and (b) reacting the Abeta-oligomer bound to the first antibody in step (a) with a labeled monoclonal antibody (a second antibody) which can recognize a partial peptide comprising the amino acid sequence depicted in SEQ ID NO:1.
Description
technical field [0001] The present invention relates to the assay method of Aβ oligomer. Background technique [0002] Alzheimer's disease (AD) is a neurodegenerative disease characterized by neuropathological features such as progressive loss of cognitive ability, amyloid deposits in the brain, neurofibrillary degeneration, and loss of nerve cells. [0003] In recent years, the role of amyloid β (Aβ) peptide oligomers in neurological diseases (eg, Alzheimer's disease, mild cognitive impairment MCI (mild cognitive impairment), Down syndrome) has received much attention. Interestingly, it has been reported that Aβ(1-42) oligomer is the structural basis of pathogenicity, and its formation is the initial pathological event of Alzheimer's disease (Journal of Neurochemistry, 2005, 95, 834-847). [0004] In addition, it has been reported that relatively high molecular weight oligomers, especially 12-mers, have a greater effect on memory impairment (Vol440, Number 7082, 2006, p.35...
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