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Aggregate Detection Method for Aggregate-Forming Polypeptides

A technology of aggregates and dimers, which is applied in the direction of measuring devices, biological tests, material inspection products, etc., can solve the problems of too small antigens and difficult diagnosis, and achieve the effect of automation

Active Publication Date: 2022-03-22
PEOPLEBIO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In the determination of the presence or progress of such an illness or disease, it is difficult to perform the determination because the amount of the antigen in the sample is very small or the size of the antigen is very small, or the amount of the antigen in the body and the amount of the antigen in the sample When the amount is disproportionate, for example, it is known that β-amyloid (β-amyloid, amyloid-β), which is involved in Alzheimer's disease, is also present in non-normal individuals compared with normal individuals β-amyloid High levels of protein oligomers, but may be difficult to perform when the amount of β-amyloid oligomers in the blood sample is difficult to detect or when β-amyloid oligomers are present in an amorphous manner in the blood sample diagnosis
[0006] In addition, there are cases where it is difficult to diagnose diseases by sandwich ELISA because the antigen to be measured is too small or the amount is small

Method used

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  • Aggregate Detection Method for Aggregate-Forming Polypeptides
  • Aggregate Detection Method for Aggregate-Forming Polypeptides
  • Aggregate Detection Method for Aggregate-Forming Polypeptides

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Embodiment 1: the preparation of experimental material

[0076] Coating buffer (Carbonate-Bicarbonate Buffer), phosphate Tween buffer (PBST), TBST and phosphate buffer saline (PBS) were purchased from Sigma. Purchased BlockAce from Bio-rad. As a buffer, Block Ace was prepared by diluting to 0.4% in phosphate Tween buffer. Prepared as a blocking buffer by diluting 1% Block Ace in D.W. HBR1 was purchased from ScantibodiesLaboratory. 6E10 antibody was purchased from Antibody (Biolegend). WO2-horseradish peroxidase (WO2-HRP) antibody was purchased from Absolute Antibody. FF51-horseradish peroxidase (FF51-HRP) was purchased from The H lab Co., Ltd. WO2-horseradish peroxidase antibody was purchased from Absolute Antibody. Recombinant β-amyloid (Aβ) 1-42 was purchased from Antibody Corporation. Recombinant S26C-β-amyloid (1-40) dimer was purchased from JPT Corporation. Plasma samples were received from Seoul National University Bundang Hospital and Chung-Ang University...

Embodiment 2

[0077] Embodiment 2: Preparation of 6E10 plate

[0078] Dilute 30 μg of 6E10 antibody (anti-β-amyloid protein, Biolegend) in 10 ml of coating buffer (Sigma Co.), inoculate 100 μl in each well of a plate (Nunc Co.), and place in a refrigerator at 4° C. The reaction was carried out for 1 day. The plate was washed 3 times in phosphate buffer, and 240 µl of blocking buffer in which 1% Block Ace was dissolved was inoculated in D.W. Then, the reaction was carried out at room temperature for 2 hours. The plate was washed three times with a phosphate buffer solution, dried at room temperature for 30 minutes, and used.

Embodiment 3

[0079] Example 3: Preparation of Control Groups

[0080]As a positive control (positive control), 990 µl of phosphate Tween buffer was added to 10 µl of recombinant β-amyloid 1-42 (rec.Aβ) (1 µg / ml) and 100 µl was used. 100 µl of phosphate buffer was used as a negative control.

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Abstract

The present invention relates to a method for detecting aggregates of polypeptides used to form aggregates in a biological sample (biosample). The method for detecting aggregates of polypeptides used to form aggregates in a biological sample includes: step (a), to an analysis object The biological sample is spiked (spiking) the dimer of the above-mentioned polypeptide for forming aggregates; step (b), by incubating the result of the above-mentioned step (a), to also form the above-mentioned dimer of the polypeptide for forming aggregates Aggregates; step (c) of contacting a binding agent-marker that is aggregated from a signal-producing marker and the above-mentioned polypeptide for forming aggregates with the result of step (b) above and a step (d) of detecting a signal generated from the binding agent-marker bound to the aggregate of the polypeptide used to form the aggregate.

Description

technical field [0001] This application claims priority from Korean Patent Application No. 10-2016-0031534 filed on March 16, 2016, the entire contents of which are incorporated herein by reference. [0002] The present invention relates to a method or a kit for detecting aggregates of aggregate-forming polypeptides in a biosample. Background technique [0003] First of all, there are cases where the polypeptides constituting the protein form multimers to form functional proteins, but under normal conditions, they exist as monomers. mismatched type)), it often induces diseases by forming multimers (Massimo Stefani, et al., J. Mol. Med. 81:678-699 (2003); and Radford SE, et al. , Cell. 97:291-298 (1999)). [0004] For example, disorders or diseases associated with abnormal aggregation or mismatching of proteins include: Alzheimer disease, Creutzfeldt-Jakob disease, spongiform encephalopathy (Spongiform encephalopathies), Parkinson`s disease, Huntington`s Disease, Amyotroph...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/58G01N33/68G01N33/50
CPCG01N33/6896G01N2333/4709G01N2800/2821G01N33/542G01N33/54306
Inventor 李炳燮李官修金信元林君泽金光济柳志宣
Owner PEOPLEBIO
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