Neurofilament light chain binding aptamers

Abstract

To provide an aptamer capable of simply, rapidly and highly accurately detecting a neurofilament light chain (NfL) which is a biomarker correlated with neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease, and having high binding affinity to the NfL.SOLUTION: An aptamer capable of binding to a neurofilament light chain (NfL), comprising an oligonucleotide described in any of the following (a) to (c): (a) an oligonucleotide consisting of a specific base sequence, (b) an oligonucleotide consisting of a base sequence in which one to several bases are deleted, substituted, inserted, and / or added in a specific base sequence, and (c) an oligonucleotide consisting of a base sequence having a sequence identity of 90% or more with a specific base sequence SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an aptamer having binding ability to neurofilament light chain. [Background technology]

[0002] Neurofilament light chain (hereinafter referred to as "NfL") is a protein with 543 amino acids and a theoretical molecular weight of 61.5 kDa that is expressed exclusively in neurons of the central and peripheral nervous systems. NfL is present throughout neurons, including the cell body, dendrites, and synapses, but is known to be particularly abundant in myelinated axons.

[0003] NfL plays an important role in the maintenance and formation of neuronal axons, and it has been shown that the amount of NfL increases in cerebrospinal fluid (CSF) and blood when neurodegeneration occurs in the brain. For example, the NfL concentration in CSF is more than 10 times higher than that of healthy individuals in patients with human immunodeficiency virus (HIV)-associated dementia, amyotrophic lateral sclerosis, and Creutzfeldt-Jakob disease, and more than double that in patients with multiple sclerosis (MS), multiple system atrophy, mild head trauma, frontotemporal dementia, and dementia with Lewy bodies. It has also been reported that NfL is elevated in Alzheimer's disease and Parkinson's disease (Non-Patent Document 1). Therefore, NfL can be used as a biomarker for various neurodegenerative diseases.

[0004] To date, a large, ultrasensitive ELISA device called the Single Molecule Array (Simoa) has been developed to measure blood NfL levels using anti-NfL antibodies (Non-Patent Document 2). Measurements of blood NfL concentrations using this device have revealed sub-pM changes in NfL levels, from 0.56 pM in healthy subjects to 0.83 pM in patients with dementia (Non-Patent Document 3). This measurement uses the anti-NfL antibodies UD1 and UD2, first reported by Norgren et al. (Non-Patent Document 4). Shaw et al. also performed epitope mapping of UD1 and UD2 using multiple recombinant and synthetic NfL fragments. They reported that amino acids 331-357 in coil 2b of NfL are important for UD1 binding, and amino acids 318-330 in coil 2b of NfL are important for UD2 binding (Non-Patent Document 5).

[0005] In Alzheimer's disease (AD), abnormal aggregation and accumulation of Aβ and tau proteins leads to neurodegeneration and progressive decline in cognitive function. With the start of use of the AD disease-modifying drug lecanemab in 2023, there is an increasing need to diagnose AD before neurodegeneration in the brain progresses and cognitive function begins to decline. Accordingly, there is a demand for methods to detect neurodegeneration in the brain, rather than cognitive decline. Therefore, measuring NfL levels in the blood would aid in the diagnosis of AD and enable assessment of its progression, which would be of social significance.

[0006] However, the ultrasensitive ELISA device using the anti-NfL antibody is large and not widely used. Therefore, a compact sensor device that can be widely used to measure plasma NfL levels is desired, but this has not yet been developed. Furthermore, the development of new ligands is desirable for the development of such a compact central device, but no ligands other than antibodies have been reported to date.

[0007] On the other hand, DNA aptamers, polynucleotide molecules that specifically bind to target molecules, are known. DNA aptamers are ligands that specifically bind to target molecules by forming a unique three-dimensional structure. DNA aptamers can be chemically synthesized in vitro using commercially available nucleic acid synthesizers in a short time, which has the advantage of reducing batch-to-batch variations and lowering manufacturing costs. Furthermore, because DNA aptamers are easily chemically synthesized, chemical modifications such as thiol groups, amino groups, and biotinylation can be easily performed. Therefore, when immobilizing ligands on electrode surfaces, there is greater flexibility in the immobilization method and immobilization site compared to antibodies, and they are expected to be used as sensor elements. Aptamers that specifically bind to desired target molecules can be created using a method called SELEX (Systematic Evolution of Ligands by Exponential Enrichment). [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Tanimoto School Toxicity Question Box No. 24 2022, 2. Utilization of biomarkers in research and development of drugs acting on the nervous system, Kiyoshi Katsumata [Non-patent document 2] DM Rissin et al., "Single-molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations," Nat. Biotechnol., vol. 28, no. 6, Art. no. 6, Jun. 2010, doi: 10.1038 / nbt.1641. [Non-patent document 3] N. Mattsson, U. Andreasson, H. Zetterberg, K. Blennow, and for the Alzheimer's Disease Neuroimaging Initiative, "Association of Plasma Neurofilament Light With Neurodegeneration in Patients With Alzheimer Disease," JAMA Neurol., vol. 74, no. 5, pp. 557-566, May 2017, doi: 10.1001 / jamaneurol.2016.6117. [Non-patent document 4] N. Norgren, J.-E. Karlsson, L. Rosengren, and T. Stigbrand, "Monoclonal Antibodies Selective for Low Molecular Weight Neurofilaments," Hybrid. Hybridomics, vol. 21, no. 1, pp. 53-59, Feb. 2002, doi: 10.1089 / 15368590252917647. [Non-Patent Document 5] G. Shaw et al., "Uman-type neurofilament light antibodies are effective reagents for the imaging of neurodegeneration," Brain Commun., vol. 5, no. 2, p. fcad067, Apr. 2023, doi: 10.1093 / braincomms / fcad067. Summary of the Invention [Problem to be solved by the invention]

[0009] The objective of the present invention is to provide an aptamer with high binding affinity to neurofilament light chain (NfL), which is a biomarker correlated with neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease, that can be detected simply, quickly, and with high accuracy. [Means for solving the problem]

[0010] As a result of extensive research aimed at solving the above problems, the present inventors have succeeded in obtaining multiple aptamers with high binding affinity (low binding dissociation constant) for NfL.

[0011] That is, the present invention includes the following inventions. [1] An aptamer having binding ability to neurofilament light chain (NfL), comprising an oligonucleotide according to any one of (a) to (c) below: (a) an oligonucleotide consisting of a nucleotide sequence shown in any one of SEQ ID NOs: 1 to 30 (b) an oligonucleotide consisting of a base sequence in which one to several bases are deleted, substituted, inserted, and / or added in any of the base sequences shown in SEQ ID NOs: 1 to 30; (c) an oligonucleotide consisting of a nucleotide sequence having 80% or more sequence identity with any of the nucleotide sequences set forth in SEQ ID NOs: 1 to 30; [2] The aptamer according to [1], which has the ability to bind to neurofilament light chain (NfL), and which comprises an oligonucleotide according to any one of (d) to (f) below: (d) an oligonucleotide consisting of a nucleotide sequence shown in any one of SEQ ID NOs: 4, 5, 10, 12, 13, 20, 22, 24, and 25 (e) an oligonucleotide consisting of a base sequence in which one to several bases are deleted, substituted, inserted, and / or added in any of the base sequences shown in SEQ ID NOs: 4, 5, 10, 12, 13, 20, 22, 24, and 25; (f) an oligonucleotide consisting of a nucleotide sequence having 80% or more sequence identity with any of the nucleotide sequences set forth in SEQ ID NOs: 4, 5, 10, 12, 13, 20, 22, 24, and 25; [3] A kit for detecting neurofilament light chain (NfL), comprising the aptamer according to [1] or [2]. [4] A kit for testing for neurodegenerative diseases, comprising the aptamer described in [1] or [2]. [5] The kit according to [4], wherein the neurodegenerative disease is Alzheimer's disease. [6] The following steps: (1) contacting a biological sample collected from a subject with the aptamer described in [1] or [2] to form a complex between the neurofilament light chain (NfL) in the sample and the aptamer; and (2) detecting NfL in the complex formed in step (1); A method for testing for a neurodegenerative disease, comprising: [7] The method according to [6], wherein the aptamer used in step (1) is immobilized on a solid phase. [8] The method according to [6], wherein the aptamer used in step (1) is labeled with a labeling substance. [9] The method according to [6], wherein the neurodegenerative disease is Alzheimer's disease. [Effects of the Invention]

[0012] The present invention provides a DNA aptamer that can easily, rapidly, and accurately detect neurofilament light chain (NfL) in a sample, which is useful as a biomarker for neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease. The DNA aptamer of the present invention is easier to synthesize and chemically modify than anti-NfL antibodies, is suitable for application to small biosensor devices, and is advantageous in terms of production cost. [Brief explanation of the drawings]

[0013] [Figure 1] FIG. 1 is a diagram showing the procedure of the competitive SELEX method used to select the aptamer of the present invention. [Figure 2] FIG. 2 shows the amount and recovery rate of extracted ssDNA in each round of the competitive SELEX method used to select the aptamer of the present invention, and the results of electrophoresis after PCR. [Figure 3] Figure 3A shows a schematic diagram of aptamer blotting (using nitrocellulose membrane), and Figure 3B shows a schematic diagram of ELONA (Enzyme Linked Oligonucleotide Assay). [Figure 4] FIG. 4 shows the results of aptamer blotting analysis of the binding ability of aptamers MN711 and MN734 to NfL and Tag ((a) exposure time 0.25 seconds, (b) exposure time 15 seconds, (c) exposure time 60 seconds). [Figure 5] Figure 5A shows the binding ability of aptamer MN711 to NfL analyzed by ELONA, and plots of NfL concentration versus chemiluminescence. Figure 5B shows the binding ability of aptamer MN734 to NfL analyzed by ELONA, and plots of NfL concentration versus chemiluminescence. [Figure 6] Figure 6A shows a Scatchard plot of the NfL-binding ability of aptamer MN711 analyzed by ELONA, plotting the NfL concentration and chemiluminescence. Figure 6B shows a Scatchard plot of the NfL-binding ability of aptamer MN734 analyzed by ELONA, plotting the NfL concentration and chemiluminescence. [Figure 7] FIG. 7 shows the results of SDS-PAGE of various recombinant NfLs and BSA (boxed band: recombinant NfL). [Figure 8] FIG. 8 shows the results of aptamer blotting analysis of the binding ability of aptamers MN711 and MN734 to various recombinant NfLs (NfL2-338, NfL281-396, NfL381-543, and two types of NfL2-543). [Figure 9] FIG. 9 shows the predicted binding site of the aptamer of the present invention for NfL. [Figure 10] FIG. 10 shows the results of Native-PAGE analysis of multimer formation of aptamer candidates (86 sequences). [Figure 11] FIG. 11 is a graph comparing the binding ability of the aptamers of the present invention to NfL with MN711 as a reference. [Figure 12]FIG. 12 shows an alignment of the top aptamer sequences for their ability to bind to NfL. [Figure 13] FIG. 13 shows the results of aptamer blotting analysis of the binding ability of the aptamer of the present invention to various recombinant NfLs (NfL2-338, NfL281-396, NfL381-543, and two types of NfL2-543). [Figure 14] FIG. 14 shows a comparison of the binding ability of aptamer MN711 and MN711 mutants (MN711_1m, MN711_2m, MN711_3m) to NfL. [Figure 15] Figure 15 shows the results of aptamer blotting analysis of the binding ability of aptamers MN711 and MN734 to Aβ40 monomer, Aβ42 monomer, pTau, and NfL (exposure time: 40 s). Poly-A sequences were used as a negative control to demonstrate nonspecific adsorption of nucleic acids. Anti-Aβ antibodies, anti-pTau181 antibodies, and anti-His tag antibodies were used as positive controls to confirm the immobilization of Aβ40 monomer, Aβ42 monomer, pTau, and NfL to the nitrocellulose membrane. [Figure 16] FIG. 16 is a graph plotting the NfL concentration and chemiluminescence obtained by analyzing the binding ability of MN711 to NfL spiked in plasma using ELONA. DETAILED DESCRIPTION OF THE INVENTION

[0014] The present invention will be described in detail below. 1. Aptamer capable of binding to neurofilament light chain The present invention relates to an aptamer capable of binding to neurofilament light chain (NfL). Neurofilament light chain (NfL) is a protein with a total of 543 amino acids and a theoretical molecular weight of 61.5 kDa that is expressed exclusively in neurons of the central and peripheral nervous systems. It is known that the concentration of NfL increases in cerebrospinal fluid (CSF) and blood in association with axonal damage or degeneration of neurons. The aptamer of the present invention can detect NfL in biological samples simply, quickly, and with high accuracy, and can therefore be used for the early diagnosis and monitoring of neurodegenerative diseases such as Alzheimer's disease at a stage that cannot be detected by imaging tests.

[0015] In the present invention, "aptamer" refers to a single-stranded nucleic acid that specifically binds to a target substance. The single-stranded nucleic acid may be single-stranded DNA or single-stranded RNA. The nucleic acid that constitutes an aptamer has advantages not found in antibodies, which are proteins, such as being able to be produced relatively inexpensively by chemical synthesis, being able to be stored or transported at room temperature in a dry state, and being easily chemically modified.

[0016] In the present invention, "having the ability to bind to NfL" means having the ability to bind to NfL selectively and with a sufficiently high affinity to maintain stable binding. Specifically, the binding dissociation constant (K D value) is 100 nM or less, preferably 20 nM or less, more preferably 15 nM or less, and even more preferably 10 nM or less.

[0017] Furthermore, the region of NfL to which the aptamer of the present invention binds refers to the region from positions 281 to 338 of the base sequence of the NfL gene.

[0018] The aptamer of the present invention capable of binding to NfL (hereinafter referred to as "aptamer of the present invention") comprises an oligonucleotide capable of binding to NfL, preferably as a single-stranded nucleic acid component (typically as the only nucleic acid component). With respect to the aptamer of the present invention, "oligonucleotide" refers to a nucleotide polymer (polynucleotide) of 80 bases or less. The length of the oligonucleotide constituting the aptamer of the present invention is 80 bases or less, 70 bases or less, 60 bases or less, 50 bases or less, or 40 bases or less, and 20 bases or more, 25 bases or more, or 30 bases or more; specifically, 25 to 35 bases are preferred, and 27 to 32 bases are more preferred.

[0019] One embodiment of the aptamer of the present invention comprises an oligonucleotide described in any one of (a) to (c) below: (a) an oligonucleotide consisting of a nucleotide sequence shown in any one of SEQ ID NOs: 1 to 30 (b) an oligonucleotide consisting of a base sequence in which one to several bases are deleted, substituted, inserted, and / or added in any of the base sequences shown in SEQ ID NOs: 1 to 30; (c) an oligonucleotide consisting of a nucleotide sequence having 80% or more sequence identity with any of the nucleotide sequences set forth in SEQ ID NOs: 1 to 30;

[0020] Regarding (a) above, the base sequences 1 to 30 of the 30 types of aptamers are shown in Table 1 below.

[0021] [Table 1]

[0022] With regard to (b) above, the range of "1 to several" in "a base sequence in which one to several bases are deleted, substituted, inserted, and / or added" is not particularly limited as long as it has the same binding ability to NfL as the oligonucleotides consisting of the base sequences shown in SEQ ID NOs: 1 to 30, respectively, but refers to, for example, 1 to 5, preferably 1 to 4, and more preferably 1 to 3.

[0023] With respect to (c) above, "a base sequence having 80% or more sequence identity" refers to a base sequence having at least 80% sequence identity. While there are no particular limitations on the identity, as long as the base sequence has the same binding ability to NfL as the oligonucleotides consisting of the base sequences shown in SEQ ID NOS: 1 to 30, the identity is preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, and most preferably 97% or more. The sequence identity of a base sequence can be determined using methods well known to those skilled in the art, such as sequence analysis software. Examples include the blastn program of the BLAST algorithm and the fasta program of the FASTA algorithm. Here, the sequence identity of a base sequence is determined by comparing the base sequence to be evaluated with the base sequences shown in SEQ ID NOS: 1 to 30, and expressing the frequency, in percentage, of identical bases appearing at the same site.

[0024] A more preferred embodiment of the aptamer of the present invention comprises an oligonucleotide described in any one of (d) to (f) below. (d) an oligonucleotide consisting of a nucleotide sequence shown in any one of SEQ ID NOs: 4, 5, 10, 12, 13, 20, 22, 24, and 25 (e) an oligonucleotide consisting of a base sequence in which one to several bases are deleted, substituted, inserted, and / or added in any of the base sequences shown in SEQ ID NOs: 4, 5, 10, 12, 13, 20, 22, 24, and 25; (f) an oligonucleotide consisting of a nucleotide sequence having 80% or more sequence identity with any of the nucleotide sequences set forth in SEQ ID NOs: 4, 5, 10, 12, 13, 20, 22, 24, and 25;

[0025] The aptamers designated MN711, MN712, MN718, MN732, MN734, MN758, MN763, MN774, and MN780, which are represented by the above SEQ ID NOs: 4, 5, 10, 12, 13, 20, 22, 24, and 25, respectively, have particularly high binding affinity (low binding dissociation constant) for NfL and are therefore preferred.

[0026] The aptamers of the present invention can be prepared by the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) method and its improved methods. The SELEX method, which uses single-stranded DNA as the library source, is outlined below. First, a template DNA containing a random sequence of appropriate length sandwiched between two arbitrary primer sequences is synthesized. The length of the random sequence is preferably 25 to 100 bases. This template DNA is amplified by PCR (Polymerase Chain Reaction) to obtain a random DNA aptamer pool. Next, this random DNA aptamer pool is associated with a target substance, and after removing unbound DNA, the bound DNA aptamer is extracted. The obtained DNA aptamer is amplified by PCR using the above primer sequences. At this time, 5 to 8 mM Mg 2+ By performing PCR in the presence of ATP, replication fidelity is reduced, facilitating the introduction of mutations, resulting in a DNA aptamer pool containing new DNA aptamers not present in the DNA aptamer pool prior to binding to the target substance. The new DNA aptamers may have higher binding affinity, i.e., the DNA aptamers may evolve. By repeating the above series of operations for 5 to 15 rounds on this evolved DNA aptamer pool, DNA aptamers that bind to the target substance are obtained. After the final round, the resulting DNA aptamer pool is cloned and then sequenced using procedures commonly performed by those skilled in the art. In this SELEX method, template DNA synthesis, PCR and other operations, as well as cloning and sequencing, are performed using methods commonly used by those skilled in the art.

[0027] The aptamer of the present invention was obtained by competitive SELEX, a modified method of SELEX, which is described in the Examples below, and the base sequence including the primer sequence is as shown in Table 2 below (the underlined part indicates the sequence selected by the SELEX method).

[0028] [Table 2] TIFF2026009074000003.tif97141

[0029] The above-mentioned oligonucleotides constituting the aptamer of the present invention can be synthesized by those skilled in the art based on the base sequence by a conventional method, for example, using an automatic nucleic acid synthesizer.

[0030] In the aptamer of the present invention, the oligonucleotide may be chemically modified. The chemical modification is typically labeling with a labeling substance that enables detection. The oligonucleotide may also be bound to other substances (for example, non-nucleic acid substances), which is also included in the chemical modification. Examples of chemical modifications include low molecular weight labeling molecules (biotin, avidin, streptavidin, etc.), functional groups (phosphate groups, phosphorothioate groups, thiol groups, amino groups, aminoallyl groups, etc.), biocompatible polymers (polyethylene glycol (PEG) and its derivatives, etc.), fluorescent substances (fluorescein (FAM) and its derivatives, various Alexa Fluor (R) dyes, rhodamine derivatives such as carboxytetramethylrhodamine (TAMRA), cyanine dyes such as Cy3 and Cy5, quenchers used in combination with fluorescent substances, redox probes such as methylene blue and chlorogenic acid, enzymes (horseradish peroxidase (HRP), peroxidase, alkaline phosphatase, etc.), radioisotopes ( 3 H, 14 C. 32 P, 35 S or 125 I), particles (gold particles, latex particles, etc.), electron transfer substances (phenazine methosulfate (PMS), etc.).

[0031] Furthermore, the oligonucleotide constituting the aptamer of the present invention may contain at least one of a modified nucleotide and an artificial base. In the present invention, a "modified nucleotide" refers to a nucleotide in which the sugar moiety, phosphate moiety, and / or base moiety of a natural nucleotide has been chemically modified. The modified nucleotide may also be a nucleotide containing a modified nucleoside. Examples of modified nucleotides include, but are not limited to, nucleotides containing a halogenated base such as 2'-fluoropyrimidine, a methylated base such as 2'-O-methylated base, and a modified nucleoside such as deoxyuridine, inosine, or 2-amino-deoxyadenosine.

[0032] 2. Neurofilament light chain detection kit and neurodegenerative disease testing kit The present invention also provides a kit for detecting neurofilament light chains and a kit for testing for neurodegenerative diseases, each containing the aptamer of the present invention. The kit of the present invention may contain other reagents in addition to the aptamer of the present invention, and may be similar to known kits except for the inclusion of the aptamer of the present invention. The reagents may be contained together with the aptamer of the present invention or in separate containers. Examples of the reagents include labeling substances (e.g., fluorescent dyes, enzymes, radioactive substances, chemiluminescent substances), substrates, antibodies, reaction stoppers, buffer solutions, positive controls, negative controls, etc. These reagents may be mixed in advance. The kit may also contain a solid phase on which the aptamer of the present invention is immobilized and instructions for use.

[0033] Examples of solid phases that can be used in the kit of the present invention include insoluble carriers such as polymers such as polystyrene, glass beads, magnetic particles, microplates, immunochromatographic filter paper, glass filters, etc. Among these, microplates used in sandwich ELISA are preferred.

[0034] Use of the kit of the present invention enables detection of NfL and testing for neurodegenerative diseases, particularly early diagnosis of neurodegenerative diseases, to be carried out simply, quickly, and with high accuracy.

[0035] Here, neurodegenerative diseases are not particularly limited as long as they are diseases that show a correlation with the amount of NfL in biological samples such as blood, but examples include Alzheimer's disease (AD), dementia with Lewy bodies, Parkinson's dementia, frontotemporal dementia, vascular dementia, Huntington's disease, multiple sclerosis, and amyotrophic lateral sclerosis.

[0036] 3. Testing methods for neurodegenerative diseases The method for testing for a neurodegenerative disease of the present invention comprises the following steps: (1) contacting a biological sample collected from a subject with the aptamer described in (1) or (2) to form a complex between a neurofilament light chain (NfL) in the sample and the aptamer; and (2) detecting NfL in the complex formed in step (1); Includes:

[0037] The testing method of the present invention may include, after the above step (2), a step (3) of comparing the level of NfL detected in step (2) with a reference value, and a step (4) of determining whether or not the subject has developed a neurodegenerative disease based on the comparison result of step (3).

[0038] In the present invention, "test" or "diagnosis" typically refers to determining whether or not a subject is suffering from a neurodegenerative disease, but is not limited to this and includes determining the degree of symptoms or progression of the disease, determining the effectiveness of treatment for the disease, and determining whether or not there is a risk of the disease recurring after treatment. Furthermore, "determination" does not include diagnosis by a physician, but refers to assistance in diagnosis, and specifically refers to obtaining data for diagnosis.

[0039] The subject in the testing method of the present invention is a human suspected of developing the above-mentioned neurodegenerative disease.

[0040] A biological sample refers to an organ, tissue, cell, body fluid, or a mixture thereof contained in a living body. The biological sample used in the test method of the present invention is not particularly limited as long as it is a biological sample containing NfL. For example, blood or cerebrospinal fluid (CSF) can be used, but blood is preferred because of its simplicity and the small burden it places on the subject. The blood may be any of whole blood, plasma, and serum. Furthermore, the biological sample may be a sample collected from a subject and processed by methods such as concentration, purification, extraction, isolation, or physical / chemical treatment before being subjected to NfL measurement.

[0041] First, in step (1), a biological sample collected from a subject is contacted with the aptamer of the present invention to form a complex between NfL in the sample and the aptamer. The manner, order, and specific method of contacting the aptamer with the biological sample from the subject are not particularly limited, as long as the method allows interaction between the aptamer and NfL in the biological sample. In one embodiment of the present invention, contact can be carried out, for example, by adding the biological sample to a plate on which the aptamer is immobilized. Alternatively, for example, the biological sample may be separated by means such as SDS-PAGE, transferred to a membrane and immobilized, and then contacted with the aptamer.

[0042] The contact time is not particularly limited as long as it is sufficient for the aptamer to bind to NfL contained in the biological sample derived from the subject and form a complex, but is typically several seconds to several tens of hours. The temperature conditions for the contact are typically 4°C to 50°C, preferably 4°C to 37°C, and more preferably room temperature of about 15°C to 30°C. The pH conditions for the reaction are preferably 5.0 to 9.0, and more preferably 6.0 to 8.0.

[0043] Although aptamers can be used in a soluble, unbound state, they are preferably bound to a solid phase. Examples of "solid phases" include plates (microwell plates), tubes, beads (plastic beads, magnetic beads, etc.), chromatography supports (filter paper, nitrocellulose membranes, etc.), membranes (nitrocellulose membranes, PVDF membranes, etc.), gels (polyacrylamide gels, etc.), and metal membranes. Among these, plates, beads, chromatography supports, and membranes are preferred, with plates being more preferred for their ease of handling. Examples of the bond include covalent bonds, ionic bonds, and physical adsorption, and are not particularly limited. However, covalent bonds and / or physical adsorption are preferred because they provide sufficient binding strength. The bond to the solid phase may be direct or indirect, utilizing a known substance.

[0044] Step (2) is a step of detecting NfL in the complex formed in step (1). If NfL is detected in step (2), it can be determined that the subject is highly likely to have developed a neurodegenerative disease. If NfL is not detected or is below the detection limit, it can be determined that the subject is low likely to have developed a neurodegenerative disease.

[0045] For the above detection, any method capable of detecting NfL can be used, including any nucleic acid or protein detection technique such as enzyme-linked oligonucleotide assay (ELONA), enzyme-linked immunosorbent assay (ELISA), immunoblot, immunoprecipitation, immunocapture, biolayer interferometry (BLI), flow cytometry, protein array technology, spectroscopy, mass spectrometry, chromatography, surface plasmon resonance (SPR), fluorescence extinction, and fluorescence energy transfer (FRET), and these analytical methods are well known to those skilled in the art.

[0046] In a preferred embodiment of steps (1) and (2) above, an aptamer of the present invention containing a labeled oligonucleotide is contacted (e.g., mixed) with a biological sample containing NfL to form an NfL-aptamer complex, and NfL is detected based on the labeling signal derived from the NfL-aptamer complex. Specifically, an aptamer of the present invention containing a labeled oligonucleotide is incubated in solution with a biological sample containing NfL (e.g., blood) to allow the aptamer to bind to NfL and form an NfL-aptamer complex. NfL can then be detected by detecting the labeling signal derived from the NfL-aptamer complex, more specifically, the labeling signal derived from the label of the aptamer in the NfL-aptamer complex. The resulting NfL-aptamer complex is preferably separated from free components in the solution by washing or the like, followed by detection of the labeling signal. The labeling signal to be detected can be easily identified by those skilled in the art based on the label of the aptamer used and the measurement system. For example, when an aptamer labeled with a fluorescent substance is used, the label signal is typically fluorescence from the fluorescent substance, and when an aptamer labeled with a radioisotope is used, the label signal is typically radioactivity from the radioisotope. For example, when an aptamer labeled with biotin is used, the label signal is typically chemiluminescence in a chemiluminescence measurement system using HRP-labeled avidin or an avidin derivative (e.g., streptavidin) and a chemiluminescent substrate. For example, when an aptamer labeled with biotin is used, the label signal is typically color intensity in a color reaction measurement system using HRP-labeled avidin or an avidin derivative (e.g., streptavidin) and a color-developing substrate such as 3,3',5,5'-tetramethylbenzidine (TMB).

[0047] The detection step (2) may be a step of quantitatively detecting NfL. Quantification can be performed by a method known per se, such as by converting the signal from the biological sample detected based on the label used into a numerical value using a calibration curve prepared using a standard sample of NfL. The standard sample of NfL may be NfL prepared from a biological sample, or may be prepared by a genetic engineering technique known per se based on the genetic information encoding NfL.

[0048] When the detection in step (2) yields a qualitative result, a positive NfL result can be determined to indicate a high possibility of the subject suffering from a neurodegenerative disease, and a negative NfL result (below the detection limit) can be determined to indicate a low possibility of the subject suffering from a neurodegenerative disease. When the detection in step (2) yields a quantitative result using enzyme-linked oligonucleotide assay (ELONA) or the like, the concentration of NfL in a biological sample (blood sample) can be used to not only determine whether or not a subject suffers from a neurodegenerative disease, but also to estimate the severity of the neurodegenerative disease in subjects with NfL concentrations above a certain level in the biological sample. Furthermore, the level can be compared with previously measured levels for the same subject.

[0049] The presence or absence of a neurodegenerative disease may be determined by setting an appropriate standard NfL level (cutoff value) according to the purpose. The cutoff value is a value that satisfies both high diagnostic sensitivity (positive case rate) and high diagnostic specificity (negative case rate) when the disease is determined based on that value. Usually, if the level is below the detection limit, it can be determined that the patient does not have a neurodegenerative disease.

[0050] Specifically, if the NfL level in the subject's biological sample is above the cutoff value, the subject is determined to have a high probability of suffering from a neurodegenerative disease, and if it is below the cutoff value, the subject is determined to have a low probability of suffering from a neurodegenerative disease.

[0051] Furthermore, when a simple test such as immunochromatography is performed, the presence or absence of NfL in a biological sample can be determined based on the presence or absence of a visually observed signal (e.g., color development). For example, if a signal (e.g., color development) is confirmed by visual observation, the subject is determined to have a high probability of suffering from a neurodegenerative disease, and the presence or absence of a neurodegenerative disease can be further determined by combining this with other diagnostic methods.

[0052] The method of the present invention can also be used to evaluate the effectiveness of treatment for patients undergoing treatment for neurodegenerative diseases. Biological samples are collected from such patients before, during, and / or after treatment, and the effectiveness of the treatment can be determined by examining changes in NfL concentration. For example, if the NfL concentration in the later biological sample is lower than that in the earlier biological sample, the treatment can be evaluated as effective. [Example]

[0053] The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples. The compositions of the buffers used in the examples are shown below.

[0054] <Composition of each buffer> TE buffer (10mM Tris, 1mM EDTA, pH8.0) 50x TE buffer (500mM Tris-HCl, 50mM EDTA, pH 8.0) PBS buffer (137mM NaCl, 8.1mM Na2HPO4, 2.68mM KCl, 1.47mM KH2PO4, pH 7.4) TBE buffer (44.5mM Tris-HCl, 44.5mM H3BO3, 1mM EDTA, pH 8.28) PBS-T buffer (0.05% (v / v) Tween20 in PBS buffer) TAE buffer (40mM Tris, 20mM Acetic acid, 1mM EDTA) Column buffer (30mM HEPES, 500mM NaCl, 5mM EDTA, pH7.0)

[0055] Example 1: Screening of NfL-binding DNA aptamers using nitrocellulose membranes 1. Reagents and Equipment <Reagents> Recombinant human neurofilament light chain (abcam) N-terminal 10xHis-SUMO-tag and C-terminal Myc-tag Negative Control (Tag) (MyBioSource) Oligo DNA (Eurofins) Disodium dihydrogen ethylenediaminetetraacetate dihydrate (EDTA), trishydroxymethylaminomethane (Tris), sodium chloride (NaCl), Tween 20, disodium hydrogen phosphate (NaHPO), potassium dihydrogen phosphate (KHPO), potassium chloride (KCl) (all from Kanto Chemical Co., Ltd.) TaKaRa Ex Taq (R) Hot Start Version (Takara Bio) sterile water Phenol:chloroform:isoamyl alcohol (125:24:1) (Sigma-Aldrich) Chloroform:isoamyl alcohol (24:1) (Sigma-Aldrich) Ethachinmate (Nippon Gene) Ethanol (EtOH) (99.5%) (Kanto Chemical Co., Ltd.) Pierce (R) Avidin Agarose (Thermo Scientific) SYBR Green I (Bio-Rad) SYBR TM Gold Nucleic Acid Gel Stain (Thermo Fisher Scientific) Skim Milk (Snow Brand Milk Products Co., Ltd.) Bovine serum albumin (BSA) (Fujifilm) Amersham Protran Premium 0.45 NC nitrocellulose Western blotting membranes (GE Healthcare Life Sciences) Agarose 21 (Wako Pure Chemical Industries)

[0056] <Equipment> ROTARY MIXER MRC-20D (Nissin Rika Co., Ltd.) Thermal Cycler PC-801-05 (Astec Co., Ltd.) Electrophoresis equipment Thermal cycler Veriti (Applied Biosystems) LightCycler (R) 96 System (Roche) Thermo Scientific TM NanoDrop TM 2000 / 2000c Spectrophotometers (Thermo Scientific) Gel Doc EZ Gel Photographer (BIO RAD)

[0057] <Oligonucleotides used in screening> The sequences of the oligonucleotides used in the screening are shown in Table 3. All of the oligonucleotides were synthesized by Eurofins Genomics, Inc.

[0058] [Table 3]

[0059] 2. Method Screening was performed for a total of seven rounds by repeating the following steps (1) to (4) (Figure 1). (1) Selection of ssDNA that binds to NfL The ssDNA library was heated in PBS buffer with equal amounts of 5'-block and 3'-block at 95°C for 10 minutes, then slowly cooled to 25°C over 30 minutes for folding. In the first to fourth rounds, only full-length human NfL was spotted onto the nitrocellulose membrane. In the fifth to seventh rounds, full-length human NfL and a peptide consisting of full-length human NfL linked to a tag protein (hereafter referred to as Tag) were spotted onto the nitrocellulose membrane and air-dried at 25°C. Blocking was performed by shaking in PBS-T containing 2% w / v BSA at 25°C for 1 hour. The membrane was then washed three times for 5 minutes with PBS-T and then shaken for 1 hour at 25°C in 1 mL of the folded ssDNA library solution diluted to 1 μM in PBS-T. The membrane was then washed three times for 5 minutes with PBS-T. The amount of ssDNA library used in each round, the amount of NfL immobilized and the number of spottings, the amount of Tag immobilized, and the air-drying time are shown in Table 4 below.

[0060] [Table 4]

[0061] (2)-1 Extraction and purification of ssDNA that binds to NfL The NfL-bound DNA was extracted from the membrane containing NfL and DNA (1). Using tweezers and scissors thoroughly washed with detergent, sterile water, and alcohol, the membrane corresponding to the NfL-immobilized region was excised and collected in 400 μL of sterile water. DNA was then isolated from the NfL by phenol-chloroform extraction. 400 μL of phenol:chloroform:isoamyl alcohol was added to the membrane in 400 μL of sterile water, vortexed for 3 minutes, then shaken at 1,400 rpm at 25°C for 5 minutes, and allowed to stand for 10 minutes. After centrifugation at 12,000 rpm at 25°C for 5 minutes, the supernatant was collected. An equal volume of chloroform:isoamyl alcohol was added to the collected supernatant, vortexed again for 3 minutes, and centrifuged at 12,000 rpm at 25°C for 3 minutes. Finally, the ssDNA extracted in the supernatant was purified by ethanol precipitation. The collected supernatant was mixed with 19.8 μL of 3 M sodium acetate and 3 μL of Ethachinmate and vortexed. Then, 1 mL of 100% ethanol was added and vortexed again. After centrifugation at 12,000 rpm at 4°C for 3 minutes, the supernatant was removed and the pellet was collected. 1 mL of 70% ethanol was added to the collected pellet and centrifuged again at 12,000 rpm at 4°C for 3 minutes. The supernatant was removed and the pellet was completely dried using an aspirator. The pellet was then dissolved in 25 μL of TE buffer.

[0062] (2)-2 Confirmation of the amount of extracted ssDNA The recovery rate of ssDNA was calculated by qPCR. 10 μL of 100-fold diluted extracted ssDNA was used as a template, and 20 pmol each of forward primer and reverse primer were used. (R) Hot Start Version, 5000x diluted SYBR TM20 μL of qPCR reaction mixture was prepared in a 96-well plate. 10 μL of the initial ssDNA library, serially diluted 10-fold from 1 nM to 1 fM, was used as a template. 20 μL of qPCR reaction mixture was prepared in a 96-well plate. Each prepared qPCR reaction mixture was run on a LightCycler. (R) After heating at 98°C for 1 minute using the 96 System, 40 cycles of 98°C for 15 seconds, 50°C for 30 seconds, and 72°C for 20 seconds were repeated. TM The fluorescence derived from Green I was detected. The C of each reaction solution was calculated from the obtained cycle number and fluorescence intensity. t The value was calculated. Then, the concentration of the initial ssDNA library and C t A standard curve was created by plotting the values. t The concentration of the extracted ssDNA was calculated from this value. Furthermore, the amount of extracted ssDNA and the recovery rate of the extracted ssDNA relative to the ssDNA library used in SELEX were calculated from this concentration.

[0063] (3)-1 Amplification and confirmation of extracted ssDNA PCR was performed using the extracted ssDNA as a template. In the first to fifth rounds, 1 μL of ssDNA was used as a template, and in the sixth and seventh rounds, 1 μL of 10,000-fold diluted ssDNA was used as a template for PCR. (R) Sixteen 100 μL PCR reaction mixtures were prepared, each containing 100 pmol each of Hot Start Version, forward primer, and biotin-modified reverse primer. After heating at 98°C for 1 minute using a thermal cycler, the extracted ssDNA was amplified by 26 cycles of 98°C for 15 seconds, 50°C for 30 seconds, and 72°C for 20 seconds. The PCR product was mixed with 1 / 10 the volume of 50x TE buffer and 1 / 5 the volume of 5 M NaCl, and electrophoresed at 125 V for 20 minutes on a 3% agarose 21 gel in TAE buffer. After electrophoresis, the gel was washed with SYBR 10000-fold diluted in TAE buffer.TM The DNA was stained with Fluorescent Gold for 20 minutes to confirm amplification of the DNA.

[0064] (3)-2 Column purification of amplified dsDNA and re-amplification of purified dsDNA (5th round only) 100 μL of the 5th round PCR product was applied to each well of a 3% agarose 21 gel in 10 μL portions and electrophoresed at 125 V for 30 minutes in TAE buffer cooled to 4°C. TM The DNA band of interest, observed around 90 bp, was excised using a cutter thoroughly washed with ethanol and then purified using FastGene. TM The target DNA was purified from the agarose gel using the Gel / PCR Extraction kit. Then, 1 μL of the purified DNA solution was diluted 10,000 times with sterile water and diluted with TaKaRa Ex Taq (R) Sixteen 100 μL PCR reaction mixtures containing Hot Start Version, forward primer, and biotin-modified reverse primer were prepared. After heating at 98°C for 1 minute using a thermal cycler, the column-purified DNA was re-amplified by PCR using 26 cycles of 98°C for 15 seconds, 50°C for 30 seconds, and 72°C for 20 seconds. The PCR product was mixed with 1 / 10 the volume of 50x TE buffer and 1 / 5 the volume of 5 M NaCl, and electrophoresed at 125V for 20 minutes on a 3% agarose 21 gel in TAE buffer. The gel after electrophoresis was then washed with SYBR 10000-fold diluted in TAE buffer. TM The amplified DNA was stained with HCl for 20 minutes to confirm whether it was observed as a single band.

[0065] (4) Single-stranded amplified dsDNA A 150 μL avidin-immobilized agarose beads were washed twice with 5 volumes of column buffer. The PCR product was added to the washed avidin-immobilized agarose and incubated at 25°C for 30 minutes with gentle agitation. After incubation, the supernatant was removed, and the agarose beads were washed twice with 5 volumes of column buffer. A 4 / 3 volume of 0.15 M NaOH solution was added and agitated for 15 minutes. The supernatant was then collected. The same volume of 0.15 M NaOH was added to the agarose beads and agitated for 15 minutes to elute the ssDNA. The supernatant was then collected. The ssDNA-containing supernatant was neutralized with 2 M HCl and subjected to ethanol precipitation as in (2). The ssDNA was recovered. The recovered ssDNA was resuspended in 25 μL of TE buffer. This ssDNA solution was used as the ssDNA library for the next round. The ssDNA concentration was determined by measuring absorbance at 260 nm using a Nanodrop™.

[0066] 3.Results Figure 2 shows the amount and recovery of ssDNA extracted in each round, calculated by qPCR, as well as the results of electrophoresis after PCR. The recovery rate increased in the first to third rounds, suggesting that ssDNA that bind to NfL was amplified and enriched. Furthermore, the recovery rate significantly decreased in the fourth round, likely due to the reduced amount of NfL immobilized. Normally, in SELEX, the recovery rate increases with each round as the target-binding DNA is amplified and enriched. However, the recovery rate in the fifth round was almost the same as that in the fourth round. This is likely due to the removal of DNA that binds to the tag in the library by immobilizing the tag on the nitrocellulose membrane and performing competitive SELEX. The recovery rate increased in the sixth round, suggesting that DNA that specifically binds only to NfL but not to the tag was amplified and enriched. The decrease in recovery rate in the seventh round, similar to the fourth round, is likely due to the reduced amount of NfL immobilized, suggesting that DNA with higher NfL-binding ability was successfully screened.

[0067] Furthermore, in the first through fourth, sixth, and seventh rounds of electrophoresis, a band believed to be the target DNA was observed only around 90 bp. On the other hand, in the fifth round, in addition to the band believed to be the target DNA around 90 bp, a band believed to be DNA nonspecifically amplified during PCR was observed around 140 bp. However, when PCR was performed again after gel purification of the fifth round PCR product, a band was observed only around 90 bp, albeit as a smear. These results suggest that gel purification was able to remove most of the nonspecifically amplified DNA other than the target DNA from the PCR product.

[0068] (Example 2) Sequence analysis of the obtained ssDNA (7th round) 1. Reagents and Equipment <Reagents> pGEM (R) -T Easy vector system (Promega) Big Dye Terminator V3.1 cycle sequencing kit (Applied Biosystems) jet competent cell DH5α(BioDynamics laboratory inc) MaXtract High Density Tubes (Qiagen) TempliPhi Amplification Kits (illustra) Ampicillin Gel / PCR Extraction Kit (Fast Gene) Agarose 21 (Wako Pure Chemical Industries) SYBR TM Gold Nucleic Acid Gel Stain (Thermo Fisher Scientific)

[0069] <Equipment> Thermal Cycler PC-801-05 (Astec Co., Ltd.) Electrophoresis equipment Thermal cycler Veriti (Applied Biosystems) Gel Doc EZ Gel Photographer (BIO RAD)

[0070] 2. Method The ssDNA from the 7th round of SELEX after phenol-chloroform extraction and ethanol precipitation was purified using TaKaRa Ex Taq (R) PCR amplification was performed using the Hot Start Version and forward and reverse primers. The resulting PCR product was analyzed using FastGene TM The purified PCR product was then purified using a column with the Gel / PCR Extraction Kit. (R) -T Easy vector at a molar ratio of 4:1, and pGEM (R) Ligation was performed by TA cloning using the -T Easy Vector Systems and incubation at 16°C overnight. 10 μL of the ligation product was transferred to DynaCompetent (R) The resulting mixture was added to 100 μL of Cells Jet DH5α and allowed to stand on ice for 5 minutes for transformation. Then, 900 μL of SOC medium was added and allowed to recover for 30 minutes. In parallel, 200 μL of X-gal (final concentration 5 ng / μL) diluted 4-fold with SOC medium was spread on LB agar medium containing ampicillin and allowed to stand at room temperature for 30 minutes. After the recovery culture, 150 μL of E. coli was spread on agar medium and incubated at 37°C for 16 hours. Blue / white screening was then performed to select a total of 94 white colonies containing the plasmid with the target DNA inserted, which were then added to a 96-well plate. Illustrated. TM Templiphi TMThe DNA Amplification Kit's sample buffer was added and the mixture was heated at 95°C for 3 minutes. The reaction buffer and enzyme mix were then added, and the mixture was incubated at 30°C for 12 hours and then at 65°C for 10 minutes. The template DNA for sequencing analysis was amplified by rolling cycle amplification (RCA). The M13 forward primer was then added, and the base sequence was analyzed.

[0071] 3.Results Sequence analysis of the DNA obtained in the 7th round yielded 88 DNA sequences ranging from 27 to 32 mers. Alignment of these 88 DNA sequences revealed no significant homology, but all were guanine-rich with consecutive guanine sequences. This suggests that the aptamer binding to NfL has a sequence capable of forming a G4 structure. Furthermore, many thymines were present, and these sequences can be broadly divided into two types: those with a central thymine cluster and those with a cluster at the 3' end.

[0072] Furthermore, two identical sequences were identified among the 88 sequences, designated MN711 and MN734. These MN711 and MN734 were selected as NfL-binding aptamer candidates with excellent binding ability and were used for further evaluation.

[0073] The sequences of the selected MN711 and MN734 (including primer sequences) are shown below. MN711: 5'-TACATTCATTCAGAGGTTtttGGTGGTGTGGTGGGTGGGTTGTCTGTGTGGtttTGTGACTGAATAACTGAA-3' (SEQ ID NO: 34) MN734: 5'-TACATTCATTCAGAGGTTtttCGTGGGGTGGGAGGGTGGTTTTCTTGGTTCtttTGTGACTGAATAACTGAA-3' (SEQ ID NO: 43)

[0074] (Example 3) Binding analysis of aptamers MN711 and MN734 to NfL 1. Reagents and Equipment <Reagents> Recombinant human neurofilament light chain (abcam) N-terminal 10xHis-SUMO-tag and C-terminal Myc-tag Negative Control (Tag) (MyBioSource) Oligo DNA (Eurofins) Disodium dihydrogen ethylenediaminetetraacetate dihydrate (EDTA) Trishydroxymethylaminomethane (Tris), sodium chloride (NaCl), Tween 20, disodium hydrogen phosphate (Na2HPO4), potassium dihydrogen phosphate (KH2PO4), potassium chloride (KCl) (all from Kanto Chemical Co., Ltd.) NeutrAvidin TM Protein HRP Conjugates (Thermo Fisher Scientific) Penta·His HRP Conjugate Kit (QIAGEN) Bovine serum albumin (Sigma-Aldrich) Immobilon TM Western Chemiluminescent HRP Substrate (MILLIPORE)

[0075] <Equipment> Shaker Amersham Protran Premium NC 0.45 (200×200 mm) (GE Healthcare) ImageQuant LAS 4000 mini (GE Healthcare)

[0076] 2. Method Two DNA fragments, MN711 and MN734, were selected after the seventh round of sequencing, revealing identical sequences. Their binding to NfL was assessed by aptamer blotting. Aptamer candidates containing primer binding sites for SELEX at both ends and 10 pmol of Poly-T (final concentration 100 nM) were combined with equal amounts of 10 pmol of 5'-block and 10 pmol of biotin-modified 3'-block in PBS buffer at 95°C for 10 minutes, then gradually cooled to 25°C over 30 minutes for folding. Nitrocellulose membranes were spotted with 0.25, 0.50, or 1.0 pmol of full-length human NfL and 1.0 pmol of Tag and air-dried at 25°C for 15 minutes. The membranes were then blocked by shaking in PBS-T containing 5% (w / v) skim milk or 2% (w / v) BSA at 25°C for 1 hour. The membrane was washed three times for 5 minutes with PBS-T and then incubated for 1 hour in 1 mL of a 10 nM solution of the folded aptamer candidate in PBS-T. After washing three times for 5 minutes with PBS-T, a 5000-fold diluted NeutrAvidin-HRP solution was added and the membrane was then incubated for 1 hour. Two membranes were also incubated in 1 mL of PBS-T without the aptamer candidate solution for 1 hour. One membrane was incubated with a 5000-fold diluted NeutrAvidin-HRP solution as a negative control to confirm nonspecific adsorption of the HRP-labeled molecule, and the other was incubated with a 15000-fold diluted HRP-labeled anti-His antibody as a positive control to confirm protein immobilization. The membrane was then washed three times for 5 minutes with PBS-T, and Immobilon Western Chemiluminescent HRP Substrate was added. The membrane was then incubated for 5 minutes, and the HRP-derived chemiluminescence was detected using a LAS4000 mini. A schematic diagram of the aptamer blotting analysis is shown in Figure 3A.

[0077] 3.Results The results for an exposure time of 0.25 seconds are shown in Figure 4(a). When detected using an HRP-conjugated anti-His antibody at an exposure time of 0.25 seconds, chemiluminescence was observed in the NfL 0.50 and 1.0 pmol spots. Furthermore, when the exposure time was extended to 15 seconds (Figure 4(b)), chemiluminescence was observed in the NfL 0.25 pmol spot, and when the exposure time was extended to 60 seconds (Figure 4(c)), chemiluminescence was also observed in the Tag 1.0 pmol spot. This suggests that NfL and Tag were successfully immobilized on the membrane. The weaker chemiluminescence of Tag compared to NfL may be due to differences in the solvents used to dissolve NfL and Tag, or because the His tag portion of Tag was not exposed on the surface in a three-dimensional structure, making it difficult for the HRP-conjugated anti-His antibody to bind to it. Furthermore, when only HRP-labeled NeutrAvidin was added without the aptamer, no chemiluminescence was observed in any of the NfL or Tag spots, confirming that HRP-labeled NeutrAvidin did not nonspecifically adsorb to NfL or Tag. When MN711 or MN734 was added, strong chemiluminescence was observed only in the NfL spots, but not in the Tag spots. This suggests that SELEX enabled the acquisition of an aptamer that specifically binds to NfL but not to Tag. On the other hand, when Poly-T was added, chemiluminescence was also observed in the NfL spots. This suggests that the binding between NfL and the aptamer may be related to the consecutive thymine sequences.

[0078] (Example 4) Calculation of the binding dissociation constants of aptamers MN711 and MN734 to NfL 1. Reagents and Equipment <Reagents> Recombinant human neurofilament light chain (abcam) Tween 20 (Kanto Chemical Co., Ltd.) Bovine serum albumin (Wako Pure Chemical Industries) Nunc Immobilizer Streptavidin 96 well plate (Thermo Fisher Scientific) Anti-Myc-tag mAb-HRP-DirecT (MBL Life Sciences) BM Chemiluminescence ELISA Substrate (POD) (Roche)

[0079] <Equipment> Thermal Cycler PC-707 (Astec) Applied Biosystems TM Veriti TM Thermal Cycler 96-Well (Thermo Fischer Scientific) High-speed shaker CM-1000 (Tokyo Rikakikai Co., Ltd.) Nivo TM Multi-mode microplate reader (PerkinElmer)

[0080] 2. Method The binding dissociation constants of MN711 and MN734 were calculated by ELONA (Enzyme Linked Oligonucleotide Assay). A schematic diagram of ELONA is shown in Figure 3B. 100 pmol (final concentration 1 μM) of MN711 and MN734, each ligated with primer binding sites for SELEX at both ends, were combined with 100 pmol of 5'-block and biotin-modified 3'-block in PBS buffer. The mixture was heated at 95°C for 10 minutes and gradually cooled to 25°C over 2 hours. This cycle was repeated three times to fold the aptamer candidate solution. After folding, 150 μL of the aptamer candidate solution, diluted to 0.1 μM with PBS buffer, was added to NUNC IMMOBILIZER STREPTAVIDIN F96 WHITE and shaken at 600 rpm at 25°C for 1 hour. To confirm nonspecific adsorption of NfL and the HRP-conjugated anti-Myc tag antibody used for detection, wells containing 150 μL of PBS buffer were prepared and shaken at 600 rpm at 25°C for 1 hour. Each well was washed three times with 200 μL of PBS-T, after which 200 μL of 2% BSA (w / v) in PBS-T was added and shaken at 600 rpm at 25°C for 2 hours. After washing three times with 200 μL of PBS-T, 150 μL of full-length human NfL solutions diluted with PBS buffer to various concentrations (final concentrations: 0, 0.25, 0.50, 1.0, 2.5, 5.0, 10, and 20 nM) were added and shaken at 600 rpm at 25°C for 1 hour. After washing three times with 200 μL of PBS-T, 200 μL of HRP-labeled anti-Myc tag antibody diluted 10,000 times with PBS-T was added and the plate was shaken at 600 rpm at 25°C for 1 hour. After washing five times with 200 μL of PBS-T at 600 rpm for 1 minute, 150 μL of BM Chemiluminescence ELISA Substrate (POD) was added and the plate was left to stand for 5 minutes, shaken for 5 seconds, and then incubated for 1 hour. TM Chemiluminescence was detected using a multimode microplate reader.

[0081] 3.Results Figure 5 shows a plot of the chemiluminescence when the aptamer was immobilized minus the chemiluminescence when the aptamer was not immobilized, against the added NfL concentration. An NfL concentration-dependent increase in chemiluminescence was observed for both MN711 and MN734. Furthermore, Figure 6 shows a Scatchard plot created from the chemiluminescence observed at NfL concentrations of 1.0, 2.5, 5.0, 10, and 20 nM. From this Scatchard plot, the binding dissociation constant K D The calculated value was K D The value is 11 nM, and the K D The calculated value was 8.1 nM, demonstrating that both assays have very high binding affinity for NfL. Furthermore, when the added NfL concentration was 0.25 nM, a difference in chemiluminescence was observed between the case with the aptamer immobilized and the case without the aptamer immobilized. Furthermore, when the aptamer was immobilized, a difference in chemiluminescence was observed between the case with an added NfL concentration of 0 nM and the case without the aptamer immobilized. This suggests that even a simple assay without signal enhancement can detect 0.25 nM NfL. Furthermore, using an assay with signal enhancement may enable detection of sub-pM NfL concentrations, which are the actual concentrations found in blood.

[0082] (Example 5) Evaluation of the binding sites of aptamers MN711 and MN734 to NfL 1. Reagents and Equipment <Reagents> Recombinant neurofilament light polypeptide (Cloud-clone.corp) Recombinant Human NF-L Protein, CF Summary (R&D systems) Recombinant human neurofilament light chain (abcam) Oligo DNA (Eurofins) Disodium dihydrogen ethylenediaminetetraacetate dihydrate (EDTA), trishydroxymethylaminomethane (Tris), sodium chloride (NaCl), Tween 20, disodium hydrogen phosphate (Na2HPO4), potassium dihydrogen phosphate (KH2PO4), potassium chloride (KCl) (all from Kanto Chemical Co., Ltd.) NeutrAvidin TM Protein HRP Conjugates (Thermo Fisher Scientific) Penta·His HRP Conjugate Kit (QIAGEN) Bovine serum albumin (Sigma-Aldrich) Immobilon TM Western Chemiluminescent HRP Substrate (MILLIPORE) Any kD Mini-PROTEAN TGX Precast Protein Gels, 15-well, 15 μL (BIO RAD)

[0083] <Equipment> Shaker Amersham Protran Premium NC 0.45 (200×200 mm) (GE Healthcare) ImageQuant 500 (Cytiva) Gel Doc EZ Gel Photographer (BIO RAD)

[0084] 2. Method (Reconstitution and concentration measurement of purchased recombinant NfL) Lyophilized powders of three types of recombinant NfL purchased from Cloud-Clone Corp. were reconstituted. The lyophilized powder of the 281-396 amino acid fragment of NfL (hereafter NfL281-396) was dissolved in 200 μL of 20 mM Tris, 150 mM NaCl (pH 8.0). The lyophilized powder of the 381-543 amino acid fragment of NfL (hereafter NfL381-543) was dissolved in 200 μL of sterile water. The lyophilized powder of full-length NfL (2-543 amino acids) (hereafter full-length NfL2-543) was dissolved in 200 μL of PBS buffer. The lyophilized powder of the 2-338 amino acid fragment of NfL (hereafter NfL2-338) purchased from R&D Systems was dissolved in 100 μL of 4 mM HCl.

[0085] The concentrations of the four recombinant NfLs (NfL281-396, NfL381-543, full-length NfL2-543, and NfL2-338) and BSA were measured by SDS-PAGE. 5 μL of each of the following solutions was prepared: NfL281-396 diluted 4-fold and 8-fold with PBS buffer, undiluted NfL381-543, full-length NfL2-543 diluted 2-fold with PBS buffer, and undiluted NfL2-338. 5 μL of 2x SDS sample buffer was added to prepare 10 μL of SDS sample solution. 5 μL of BSA diluted to 100, 200, 400, or 600 μg / mL with PBS buffer was mixed with 5 μL of 2x SDS sample buffer to prepare 10 μL of SDS sample solution. These SDS sample solutions were heat-treated at 95°C for 10 minutes, and 10 μL of each was applied to an SDS-PAGE gel. Electrophoresis was performed at 200 V for 25 minutes. The gel was then stained with CBB staining solution for 10 minutes, destained with distilled water, and photographed using a GelDoc EZ scanner. The band intensities of the photographed gel were analyzed using Image J. A calibration curve was created by plotting the BSA concentration versus band intensity, and the concentration of each recombinant NfL was calculated from its band intensity.

[0086] 3.Results The results of SDS-PAGE of the recombinant NfL and BSA are shown in Figure 7. The bands enclosed in boxes represent the recombinant NfL. The theoretical molecular weights of NfL281-396 are 14.5 × 10 3 , NfL381-543 is 21.7 × 10 3 The full-length NfL2-543, which was different from the recombinant NfL2 used in SELEX, was 65.1 × 10 3 , NfL2-338 is 39 × 10 3 The full-length NfL2-543 used in SELEX was 77.4 × 10 3 Although this is thought to be the case, the target band was observed at a higher position than the theoretical molecular weight in several NfL samples. This is thought to be due to the fact that the C-terminus of NfL is rich in acidic amino acid residues, resulting in a negative charge, which inhibits SDS binding and slows migration. Using a calibration curve of band intensity versus protein concentration generated by SDS-PAGE with known concentrations of BSA, the concentrations of each recombinant NfL were calculated from the band intensity. The NfL concentrations after reconstitution of each lyophilized powder were 152 μM for NfL281-396, 5.8 μM for NfL381-543, 26 μM for full-length NfL2-543 (not the recombinant used in SELEX), and 11 μM for NfL2-338. Furthermore, the concentration of full-length NfL2-543 used in SELEX was also measured and calculated to be 1.80 μM. The concentrations calculated from these SDS-PAGE results were used for subsequent evaluations.

[0087] (Example 6) Aptamer blotting using multiple recombinant NfLs 1. Method Aptamer candidates with SELEX primer binding sites at both ends, 100 pmol of Poly-T and Poly-A (final concentration 1 μM), and equal amounts of 5'-block and biotin-modified 3'-block were heated in PBS buffer at 95°C for 10 minutes, then gradually cooled to 25°C over 30 minutes for folding. Recombinantly produced NfL281-396, NfL381-543, full-length NfL2-543 (recombinant not used in SELEX), NfL2-338, and full-length NfL2-543 (recombinant used in SELEX) were spotted onto a nitrocellulose membrane and air-dried at 25°C for 15 minutes. The membrane was then shaken for 1 hour in PBS-T containing 5% (w / v) skim milk or 2% (w / v) BSA. The membrane was washed three times for 5 minutes with PBS-T and then shaken for 1 hour in 1 mL of 100 nM folded aptamer candidate solution. After washing with PBS-T, 5000-fold diluted Neutravidin-HRP was added and the membrane was shaken for 1 hour. At the same time, two membranes without the aptamer candidate were prepared; one was added with 5000-fold diluted Neutravidin-HRP, and the other with 15000-fold diluted HRP-labeled anti-His antibody. The membrane was washed three times for 5 minutes with PBS-T, then added with Immobilon Western Chemiluminescent HRP Substrate and left to stand for 5 minutes. The HRP-derived chemiluminescence was detected using an Image Quant 500.

[0088] 2.Results The results of an exposure time of 30 seconds are shown in Figure 8. When detected with an HRP-labeled anti-His antibody, chemiluminescence was observed in all spots. This suggests that all recombinant NfL was successfully immobilized on the membrane. The difference in chemiluminescence intensity between NfL recombinants is thought to be due to differences in the solvent in which NfL was dissolved. Furthermore, when only HRP-labeled NeutrAvidin was added without the aptamer, no chemiluminescence was observed in any of the NfL or Tag spots, confirming that HRP-labeled NeutrAvidin does not nonspecifically adsorb to NfL or Tag.

[0089] When MN711 and MN734 were added, chemiluminescence was observed not only in the spots of NfL2-543 used in SELEX, but also in the spots of other recombinant NfL2-543, NfL281-396, and NfL2-338 that were not used in SELEX, suggesting that the region of residues 281-338 of NfL, which is a common site among these, is involved in the binding of MN711 and MN734 (Fig. 9).

[0090] (Example 7) Evaluation of DNA multimer formation for all 86 sequences analyzed in the 7th round of sequence analysis 1. Reagents and Equipment <Reagents> Acrylamide, tetramethylethylenediamine, ammonium peroxodisulfate (all from Wako Pure Chemical Industries, Ltd.) N,N'-methylenebisacrylamide HGX, disodium dihydrogen ethylenediaminetetraacetate dihydrate (EDTA), Loading buffer (6x) (Takara Bio) SYBR TM Gold Nucleic Acid Gel Stain (Thermo Fisher Scientific)

[0091] <Equipment> Electrophoresis device (BIO-RAD) Thermal Cycler PC-707 (Astec) Applied Biosystems TM Veriti TM Thermal Cycler 96-Well (Thermo Fischer Scientific) Gel Doc EZ Gel Photographer (BIO RAD)

[0092] 2. Method 100 pmol (final concentration 1 μM) of DNA with 86 sequences, each with a primer binding site for SELEX at both ends, was heated in PBS buffer at 95°C for 10 minutes with equal amounts of 5'-block and biotin-modified 3'-block, and folded by gradually cooling to 25°C over 2 hours, repeated three times. After folding, 5 μL of the aptamer solution diluted to 0.5 μM with PBS buffer was mixed with 1 μL of 6x loading dye, and 5 μL of each was applied to a 10% native acrylamide gel. Electrophoresis was then performed at 120 V for 45 minutes in TBE buffer supplemented with 4 mM KCl. After electrophoresis, the gel was purified by SYBR TM The cells were stained with Gold Nucleic Acid Gel stain for 10 minutes, and the band positions were observed using a Gel imager.

[0093] 3.Results The results of native-PAGE are shown in Figure 10. For all DNAs, the strongest signal band was observed around 100 to 110 bp. For the DNAs of MN703, MN704, MN705, MN711, MN712, MN713, MN714, MN715, MN717, MN718, MN728, MN732, MN736, MN745, MN747, MN753, MN754, MN757, MN758, MN759, MN763, MN769, MN774, MN780, MN783, MN785, MN787, MN789, and MN791 (a total of 29 types), no bands were observed above the main band around 100 to 110 bp, suggesting that they do not form oligomers. On the other hand, bands were observed above the main band at around 100 to 110 bp for the other DNAs, suggesting that they form multimers.

[0094] (Example 8) Comparison of binding ability of 29 aptamer sequences that do not form multimers In Example 7, MN711, which does not form multimers and is free from the risk of non-specific adsorption, was used as a standard to compare the binding abilities of the other 28 aptamers.

[0095] 1. Reagents and Equipment <Reagents> Recombinant human neurofilament light chain (abcam) Tween 20 (Kanto Chemical Co., Ltd.) Bovine serum albumin (Wako Pure Chemical Industries) Nunc Immobilizer Streptavidin 96 well plate (Thermo Fisher Scientific) Anti-Myc-tag mAb-HRP-DirecT (MBL Life Sciences) BM Chemiluminescence ELISA Substrate (POD) (Roche)

[0096] <Equipment> Thermal Cycler PC-707 (Astec) Applied Biosystems TM Veriti TM Thermal Cycler 96-Well (Thermo Fischer Scientific) High-speed shaker CM-1000 (Tokyo Rikakikai Co., Ltd.) Nivo TM Multi-mode microplate reader (PerkinElmer)

[0097] 2. Method 100 pmol (1 μM final concentration) of 29 NfL aptamer candidates, each with a primer binding site for SELEX at both ends, that do not form multimers, were combined with equal amounts of 5'-block and biotin-modified 3'-block in PBS buffer. Heat treatment was performed at 95°C for 10 minutes, followed by slow cooling to 25°C over 2 hours. This cycle was repeated three times for folding. After folding, 150 μL of the aptamer candidate solution, diluted to 0.1 μM with PBS buffer, was added to NUNC IMMOBILIZER STREPTAVIDIN F96 WHITE and shaken at 600 rpm at 25°C for 1 hour. To confirm nonspecific adsorption of NfL and the HRP-conjugated anti-Myc tag antibody used for detection, wells containing 150 μL of PBS buffer were prepared and shaken at 600 rpm at 25°C for 1 hour. After washing each well three times with 200 μL of PBS-T, 200 μL of 2% BSA in PBS-T was added and the plate was shaken at 600 rpm at 25°C for 2 hours. After washing three times with 200 μL of PBS-T, 150 μL of NfL solution diluted to 5 nM with PBS buffer was added and the plate was shaken at 600 rpm at 25°C for 1 hour. After washing three times with 200 μL of PBS-T, 200 μL of HRP-labeled anti-Myc tag antibody diluted 1:10,000 with PBS-T was added and the plate was shaken at 600 rpm at 25°C for 1 hour. After washing five times with 200 μL of PBS-T at 600 rpm for 1 minute, 150 μL of BM Chemiluminescence ELISA Substrate (POD) was added, the plate was left to stand for 5 minutes, and then shaken for 5 seconds. TM Chemiluminescence was detected using a multimode microplate reader.

[0098] 3.Results The chemiluminescence intensity detected when each aptamer was immobilized on a plate is shown in Figure 11, with the chemiluminescence intensity when MN711 was immobilized on a plate taken as 100%. There were seven aptamer sequences that showed chemiluminescence equivalent to or greater than that of MN711, and these seven aptamers (MN780, MN774, MN758, MN732, MN718, MN763, and MN712) had the same K as MN711.D K value lower than 11 nM D It was suggested that this may indicate a value.

[0099] Furthermore, the results of aligning MN711 with the seven aptamers are shown in Figure 12. Although extremely high homology was not observed among the eight aptamers, a similar sequence containing a thymine residue between consecutive guanine residues was identified at the 5' end. This suggests that this site may be the binding site for the aptamer.

[0100] (Example 9) Evaluation of the binding site of 30 NfL-binding aptamers by aptamer blotting 1. Method ssDNA (Table 6) (final concentration 1 μM) with primer binding sequences (Table 5) ligated to both ends was folded in PBS buffer for 45 minutes together with equal amounts of 5'-block and Bio-3'-block (Table 5).

[0101] [Table 5]

[0102] [Table 6]

[0103] Five pmol of each of the 2-338 amino acid fragment of NfL (NfL2-338), the 281-396 amino acid fragment of NfL (NfL281-396), the 381-543 amino acid fragment of NfL (NfL381-543), and a different full-length NfL (NfL2-543) from the recombinant NfL used in SELEX (5 pmol) were spotted onto a nitrocellulose membrane and air-dried for 15 minutes. The membrane was then shaken for 1 hour in PBS-T containing 2% (w / v) BSA. The membrane was then washed three times for 5 minutes with PBS-T and then shaken for 1 hour in 1 mL of 100 nM folded NfL aptamer solution. The membrane was then washed three times for 5 minutes with PBS-T, after which it was shaken for 1 hour in the presence of 1:5000 diluted Neutravidin-HRP. After washing the membrane with PBS-T three times for 5 minutes, Immobilon Western Chemiluminescent HRP Substrate was added and allowed to stand for 5 minutes, after which HRP-derived chemiluminescence was detected using Image Quant 500.

[0104] 2.Results The results are shown in Figure 13. The clones, excluding MN734, were ranked in order of highest binding ability when the binding ability was compared at a single concentration by ELONA in Example 8 (Figure 11). For all clones except MN780, chemiluminescence was observed in four spots: NfL2-338, NfL281-396, and two NfL2-543. These NfLs contain the region of residues 281-338. This suggests that clones other than MN780 may bind to the region of residues 281-338 of NfL. On the other hand, for MN780, chemiluminescence was observed not only in the four spots (NfL2-338, NfL281-396, and two NfL2-543) but also in the NfL381-543 spot. This suggests that MN780, unlike the other clones, may have multiple epitopes.

[0105] (Example 10) Comparison of the binding ability of MN711 and MN711 mutants to NfL by ELONA 1. Method For the MN711 mutants (MN711_1m, MN711_2m, MN711_3m) shown in Table 7 below (* indicates the mutation site relative to MN711), aptamers (Table 8) were prepared with primer binding sequences linked to both ends.

[0106] [Table 7]

[0107] [Table 8]

[0108] 100 pmol (final concentration 1 μM) of aptamer was combined with equal amounts of 5'-block and biotin-modified 3'-block in PBS buffer and heated at 95°C for 10 minutes. The mixture was then slowly cooled to 25°C over 2 hours, and this cycle was repeated three times to allow for folding. After folding, 150 μL of the aptamer solution, diluted to 0.1 μM with PBS buffer, was added to NUNC IMMOBILIZER STREPTAVIDIN F96 WHITE and shaken at 600 rpm at 25°C for 1 hour. To confirm nonspecific adsorption of NfL and the HRP-conjugated anti-Myc tag antibody used for detection, wells were prepared by adding 150 μL of PBS buffer and shaking at 600 rpm at 25°C for 1 hour. Each well was washed three times with 200 μL of PBS-T, after which 200 μL of 2% BSA in PBS-T was added and the plate was shaken at 600 rpm at 25°C for 2 hours. After washing three times with 200 μL of PBS-T, 150 μL of NfL solution diluted to 5 nM with PBS buffer was added and the plate was shaken at 600 rpm at 25°C for 1 hour. After washing three times with 200 μL of PBS-T, 200 μL of HRP-labeled anti-Myc tag antibody diluted 10,000-fold with PBS-T was added and the plate was shaken at 600 rpm at 25°C for 1 hour. After washing five times with 200 μL of PBS-T at 600 rpm for 1 minute, 150 μL of BM Chemiluminescence ELISA Substrate (POD) was added and the plate was left to stand for 5 minutes, then shaken for 5 seconds.TM Chemiluminescence was detected using a multimode microplate reader.

[0109] 2.Results The results are shown in Figure 14. The vertical axis represents the chemiluminescence intensity when each aptamer was immobilized minus the chemiluminescence intensity when no aptamer was immobilized. Only MN711_3m showed a clear outlier due to the edge effect, so N=2 was used. When MN711_1m, MN711_2m, and MN711_3m were immobilized, chemiluminescence intensities comparable to those detected when the parent clone, MN711, was immobilized were detected. These results suggest that the binding ability does not change significantly when 90% or more of the aptamer, i.e., 27 or more bases of the sequence, are retained.

[0110] Example 11: Confirmation of binding in the presence of AD biomarkers by aptamer blotting 1. Method Primer-binding sequences of MN711, MN734, and PolyA were prepared in PBS buffer (final concentration 1 μM) with equal amounts of 5'-block and biotin-modified 3'-block, respectively. The mixture was heated at 95°C for 10 minutes and then slowly cooled to 25°C over 30 minutes for folding. One pmol each of Aβ40 monomer, Aβ42 monomer, pTau181, and NfL was spotted onto a nitrocellulose membrane and air-dried at 25°C for 15 minutes. The membrane was then shaken for 1 hour in PBS-T containing 2% (w / v) BSA. The membrane was then washed three times for 5 minutes with PBS-T and then shaken for 1 hour in 1 mL of 100 nM folded aptamer candidate solution. After washing the membrane with PBS-T, a 5000-fold diluted Neutravidin-HRP was added and the membrane was shaken for 1 hour. Simultaneously, three aptamer-free membranes were prepared and loaded with 5000-fold diluted anti-Aβ antibody (clone 82E1), 5000-fold diluted anti-pTau181 antibody (clone AT230), and 15000-fold diluted HRP-conjugated anti-His antibody. When using anti-Aβ and anti-pTau181 antibodies, 5000-fold diluted HRP-conjugated anti-mouse IgG was used as the secondary antibody. After washing the membranes three times for 5 minutes each with PBS-T, Immobilon Western Chemiluminescent HRP Substrate was added and allowed to stand for 5 minutes. HRP-derived chemiluminescence was detected using an Image Quant 500.

[0111] 2.Results The results of an exposure time of 40 seconds are shown in Figure 15. When MN711 or MN734 was added, strong chemiluminescence was detected in the NfL spots. When PolyA was added, no chemiluminescence was detected in any of the spots. This suggests that the random DNA did not bind nonspecifically to each protein. Furthermore, when antibodies binding to each protein were added, chemiluminescence was detected in each spot, suggesting that each protein was immobilized on the membrane. On the other hand, faint chemiluminescence was also detected in the pTau181 spot, but the binding affinity to pTau181 was much weaker than that to NfL, and it was thought that this would not significantly affect the detection of NfL in plasma.

[0112] Example 12: Detection of NfL spiked in plasma by sandwich ELONA using aptamer MN711 1. Method MN711 with a primer binding sequence was prepared at 1 μM in PBS buffer, heated at 95°C for 10 minutes, and then slowly cooled to 25°C over 2 hours. This folding procedure was repeated three times. After folding, 150 μL of the aptamer candidate solution, diluted to 0.1 μM with PBS buffer, was added to NUNC IMMOBILIZER STREPTAVIDIN F96 WHITE and shaken at 600 rpm at 25°C for 1 hour. Each well was washed three times with 200 μL of PBS-T, and then 200 μL of 2% BSA (w / v) in PBS-T was added and shaken at 600 rpm at 25°C for 2 hours. After blocking, the plate was washed three times with 200 μL of PBS-T, and 150 μL of NfL solution (final concentrations: 0, 2.5, 5.0, 10 nM) diluted with PBS buffer or 10% human plasma treated with heparin sodium was added and shaken at 600 rpm for 1 hour. After washing three times with 200 μL of PBS-T, 200 μL of HRP-labeled anti-Myc tag antibody diluted 1:10,000 with PBS-T was added and shaken at 600 rpm for 1 hour at 25°C. After washing five times with 200 μL of PBS-T at 600 rpm for 1 minute, 150 μL of BM Chemiluminescence ELISA Substrate (POD) was added, and the plate was left to stand for 5 minutes, then shaken for 5 seconds. TM Chemiluminescence was detected using a multimode microplate reader.

[0113] 2.Results The results are shown in Figure 16. The horizontal axis of the graph in Figure 16 represents the NfL concentration, and the vertical axis represents the value obtained by subtracting the chemiluminescence intensity due to nonspecific adsorption when MN711 was not immobilized from the value obtained when MN711 was immobilized. In the results of detecting NfL in PBS buffer, an increase in chemiluminescence dependent on the NfL concentration was observed. DThe results were similar to those obtained when calculating the R value. Although the vertical scale is different, an increase in chemiluminescence dependent on the NfL concentration was observed in the results of detecting NfL in human plasma. 2 The value was 0.994, suggesting that MN711 can quantify NfL in plasma. [Industrial Applicability]

[0114] The present invention can be used in the field of production and development of diagnostic agents for the early diagnosis of neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease.

Claims

1. An aptamer having binding ability to neurofilament light chain (NfL), comprising an oligonucleotide according to any one of (a) to (c) below: (a) an oligonucleotide consisting of a base sequence shown in any one of SEQ ID NOs: 1 to 30; (b) an oligonucleotide consisting of a base sequence in which one to several bases are deleted, substituted, inserted, and / or added in any of the base sequences shown in SEQ ID NOs: 1 to 30; (c) an oligonucleotide consisting of a base sequence having 80% or more sequence identity with any of the base sequences shown in SEQ ID NOs: 1 to 30;

2. The aptamer according to claim 1, which has the ability to bind to neurofilament light chain (NfL), and which comprises an oligonucleotide described in any one of (d) to (f) below: (d) an oligonucleotide consisting of a base sequence shown in any one of SEQ ID NOs: 4, 5, 10, 12, 13, 20, 22, 24, and 25 (e) an oligonucleotide consisting of a base sequence in which one to several bases are deleted, substituted, inserted, and / or added in any of the base sequences shown in SEQ ID NOs: 4, 5, 10, 12, 13, 20, 22, 24, and 25; (f) an oligonucleotide consisting of a nucleotide sequence having 80% or more sequence identity with any of the nucleotide sequences set forth in SEQ ID NOs: 4, 5, 10, 12, 13, 20, 22, 24, and 25;

3. A kit for detecting neurofilament light chain (NfL), comprising the aptamer of claim 1 or 2.

4. A kit for testing for neurodegenerative diseases, comprising the aptamer according to claim 1 or 2.

5. The kit of claim 4, wherein the neurodegenerative disease is Alzheimer's disease.

6. The following steps: (1) contacting a biological sample collected from a subject with the aptamer of claim 1 or 2 to form a complex between the neurofilament light chain (NfL) in the sample and the aptamer; and (2) detecting NfL in the complex formed in step (1); A method for testing for a neurodegenerative disease, comprising:

7. The method according to claim 6, wherein the aptamer used in step (1) is immobilized on a solid phase.

8. The method according to claim 6, wherein the aptamer used in step (1) is labeled with a labeling substance.

9. 7. The method of claim 6, wherein the neurodegenerative disease is Alzheimer's disease.

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