KR-102962056-B1 - Method for Providing Information for Diagnosis of Neurodegenerative Diseases Using Exosomes Containing Neurodegenerative Disease-Associated Proteins

Abstract

The present invention enables the detection of proteins associated with neurodegenerative diseases without separately separating and extracting exosomes associated with neurodegenerative diseases from the blood, and significantly improves detection accuracy, by introducing magnetic beads to which a first antibody capable of capturing exosomes, such as an anti-CD63 antibody, is attached into the blood of a patient to capture exosomes containing proteins associated with neurodegenerative diseases, and then attaching antibodies specific to proteins associated with neurodegenerative diseases to the captured exosomes to proceed with detection.

Inventors

• 신경식
• 소오미 로이

Assignees

• 주식회사 바이오소닉스

Dates

Publication Date

20260507

Application Date

20241205

Claims (7)

1. A first step of reacting exosomes in which proteins and glycoproteins associated with neurodegenerative diseases are aggregated with magnetic beads having a first antibody adsorbed on their surface; After the first step, the exosome is reacted with the second antibody, A second step of forming a sandwich structure centered on the exosome, wherein a first antibody attached to the surface of a magnetic bead and a second antibody reacting with a protein associated with neurodegenerative diseases are surrounded; and It includes a third step of detecting the neurodegenerative disease-associated protein using the second antibody, The first step above is that the first antibody and the glycoprotein undergo an antigen-antibody reaction, and The above second step is a method for providing diagnostic information of a neurodegenerative disease, characterized by an antigen-antibody reaction between the second antibody and the neurodegenerative disease-associated protein.
2. In paragraph 1, A method for providing diagnostic information for degenerative neurological diseases, characterized in that the third step is performed through an electrical detection method.
3. In paragraph 1, A method for providing information for diagnosing a neurodegenerative disease, characterized in that the neurodegenerative disease-associated protein is one or more selected from the group consisting of amyloid beta 40 (Aβ40), amyloid beta 42 (Aβ42), tau, phosphorylated tau, apolipoprotein E, amyloid precursor protein (APP), and alpha-synuclein (a-syn), and is in an aggregated or non-aggregated form.
4. In paragraph 1, The above second antibody is characterized by having a labeling substance bound to it, A method for providing information for diagnosing degenerative neurological diseases, characterized in that the above-mentioned labeling substance is one or more selected from the group consisting of peroxidase, quantum dots, magnetic bead nanoparticles, gold nanoparticles, fluorescent dyes, fluorescent proteins, nanophosphors, and silicon nanoparticles.
5. In paragraph 1, A method for providing information for diagnosing a degenerative neurological disease, characterized in that the above-mentioned degenerative neurological disease is one or more selected from the group consisting of Alzheimer's disease, frontotemporal dementia, dementia with Lewy bodies, corticobasal degeneration, Parkinson's disease, multiple system atrophy, progressive supranuclear palsy, Huntington's disease, dentato-rubro-pallido-luysian atrophy, spinocerebellar ataxia, amyotrophic lateral sclerosis, primary lateral sclerosis, and spinal muscular atrophy.
6. In paragraph 1, A method for providing information for diagnosing degenerative neurological diseases, characterized in that the size of the magnetic beads is 1 to 10 μm.
7. In paragraph 2, A method for providing information for the diagnosis of degenerative neurological diseases, characterized in that the above electrical detection method is performed through a microwell-based electrochemical sensor.

Description

Method for Providing Information for Diagnosis of Neurodegenerative Diseases Using Exosomes Containing Neurodegenerative Disease-Associated Proteins The present invention relates to a method for providing diagnostic information on a neurodegenerative disease by introducing a magnetic bead to which a first antibody capable of capturing exosomes, such as an anti-CD63 antibody, is attached into the blood of a patient to capture exosomes containing proteins associated with neurodegenerative diseases, and then attaching a specific antibody for the neurodegenerative disease-associated protein to the captured exosomes to perform detection. Neurodegenerative diseases are a group of conditions caused by the degeneration and death of brain nerve cells, with representative examples including Alzheimer's disease, Parkinson's disease, and Huntington's disease. These neurodegenerative diseases cause various symptoms such as cognitive decline, motor disorders, and behavioral changes, making early diagnosis and treatment crucial. However, existing diagnostic methods have drawbacks, such as low accuracy or the need for invasive tests. For instance, brain imaging and cerebrospinal fluid examinations are costly and burdensome for patients. Meanwhile, it has been revealed that neurodegenerative diseases such as Alzheimer's disease can occur due to the accumulation of amyloid beta (amyloid-β, Aβ), which is produced as a result of the cleavage of amyloid precursor proteins in cell membranes. The accumulated amyloid beta activates mitochondrial division to generate reactive oxygen species (ROS) and activates the action of the BACE1 (beta-amyloid converting enzyme 1) protein, which causes Alzheimer's disease; consequently, neuronal damage occurs due to the activation of BACE1. As a result, loss of memory and language abilities occurs, leading to impaired brain function, and in severe cases, it is known to be possible to even die. Recently, a method is being studied to measure the amount of neurodegenerative disease-associated proteins, such as amyloid beta, using the ELISA method for the diagnosis of Alzheimer's disease. CD63 is one of the major membrane proteins of exosomes and is used as a representative biomarker of exosomes; exosomes containing amyloid beta and CD63 have been found in the blood of patients with Alzheimer's disease (AD). Researchers define this as "exosome-bound Aβ" (Aβ42+ CD63+). Accordingly, conventional amyloid beta detection was performed by indirectly measuring the amount of amyloid beta using a method in which an anti-CD63 antibody is attached to the surface of a sample, and then the blood of an Alzheimer's patient is brought into contact with the surface of the sample to measure the amount of exosome-bound Aβ. However, since CD63 is merely an indicator of exosomes and not a specific indicator of exosome-bound Aβ, when using the above method, even exosomes that do not contain amyloid beta are included in the detection results, resulting in low reliability of the detection results. To improve detection accuracy as described above, a method was recently devised in which an Aβ42-specific antibody is immobilized on the surface of a sample, a plasma sample is applied directly to a sensor to capture Aβ42, a CD63 antibody is attached to the captured Aβ42, and detection is performed targeting the CD63. By detecting Aβ42 bound to exosomes through this method, errors caused by exosomes that do not contain amyloid beta could be reduced; however, as Aβ aggregated on the sensor surface, oligomers and fibrils of various sizes were formed, and since these polymers may have structural characteristics different from monomeric Aβ42, problems arose in which the antibody binding site was obscured or deformed. Furthermore, in the case of large aggregates, some epitopes of Aβ42 molecules were hidden inside, making it difficult for the antibody to access them, resulting in a problem where the amount of Aβ42 measured was lower than the actual amount present. Recently, to address this, a method of detecting exosome-bound Aβ after isolating it from plasma is being studied. The present invention is intended to resolve the above-mentioned problem. The inventors have completed the present invention by confirming that high detection accuracy can be secured without separately extracting exosomes in which the neurodegenerative disease-associated protein and the labeled glycoprotein are associated in the blood, by introducing magnetic beads to which a first antibody capable of capturing exosomes, such as an anti-CD63 antibody, is attached to the blood of a patient to capture exosomes containing neurodegenerative disease-associated proteins, and then attaching an antibody specific to the neurodegenerative disease-associated protein to the captured exosomes to proceed with detection. FIG. 1(a) is used in the method for providing information for the diagnosis of neurodegenerative diseases according to the present invention, and shows a sand