Method of detecting abnormal proteins and testing method for neurodegenerative diseases

Abstract

To provide a technique for detecting abnormal proteins in a sample with higher sensitivity.SOLUTION: A method of detecting abnormal proteins in a sample is provided, the method comprising (A) stirring a solution containing a sample, a self-aggregating protein as a matrix, and a surfactant with a concentration that is equal to or greater than the critical micelle concentration, and (B) detecting aggregates of abnormal proteins after the step (A).SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for detecting an abnormal protein, a method for testing a neurodegenerative disease, and the like. [Background technology]

[0002] The RT-QUIC method was developed in 2011 as a diagnostic method for prion disease, a neurodegenerative disease, and is a technology that can detect abnormal prions that accumulate in the brains and cerebrospinal fluid of prion disease patients with high sensitivity (several fg) (Non-Patent Document 1). There have been successful examples of highly sensitive detection of abnormal α-synuquine and abnormal tau using this technology (Non-Patent Documents 2 and 3). On the other hand, although there have been reports of detection of abnormal amyloid β and abnormal TDP-43, which accumulate in Alzheimer's disease and ALS, respectively, by the RT-QUIC method, the sensitivity is low (Non-Patent Document 4). [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Atarashi, Ryuichiro, et al. "Ultrasensitive human prion detection in cerebrospinal fluid by real-time quaking-induced conversion." Nature medicine 17.2 (2011): 175-178 [Non-Patent Document 2] Groveman, Bradley R., et al. "Rapid and ultra-sensitive quantitation of disease-associated α-synuclein seeds in brain and cerebrospinal fluid by αSyn RT-QuIC." Acta neuropathologica communications 6.1 (2018): 1-10. [Non-Patent Document 3] Saijo, E., et al. (2020). "4-Repeat tau seeds and templating subtypes as brain and CSF biomarkers of frontotemporal lobar degeneration." Acta Neuropathol 139(1): 63-77.Dong TT, Satoh K. The Latest Research on RT-QuIC Assays-A Literature Review. Pathogens. 2021 Mar 5;10(3):305 [Non-Patent Document 4] Scialo, C., et al. (2020). "TDP-43 real-time quaking induced conversion reaction optimization and detection of seeding activity in CSF of amyotrophic lateral sclerosis and frontotemporal dementia patients." Brain Commun 2(2): fcaa142. Summary of the Invention [Problem to be solved by the invention]

[0004] The principle of RT-QUIC is to reproduce the phenomenon in which abnormal proteins form aggregates using normal proteins as substrates (prion phenomenon) in a test tube, amplifying abnormal proteins millions of times and detecting them with high sensitivity. However, if the normal protein substrate, such as amyloid beta or abnormal TDP-43, has a high tendency to self-aggregate, aggregation will proceed even in the absence of abnormal proteins. In other words, negative control samples that do not contain abnormal proteins will also be positive, resulting in low sensitivity to detect abnormal proteins and a high false positive rate.

[0005] An objective of the present invention is to provide a technique for detecting an abnormal protein in a sample with higher sensitivity. [Means for solving the problem]

[0006] The present inventors have conducted intensive research in view of the above problems, and have found that the above problems can be solved by a method for detecting an abnormal protein in a sample, comprising: (A) stirring a solution containing a sample, a self-aggregating protein as a substrate, and a surfactant, the concentration of the surfactant being equal to or higher than its critical micelle concentration; and (B) detecting aggregates of the abnormal protein after step (A). The present inventors have conducted further research based on this finding, and have completed the present invention. That is, the present invention encompasses the following aspects.

[0007] Item 1. (A) Stirring a solution containing a sample, a substrate self-aggregating protein, and a surfactant, the concentration of the surfactant being equal to or higher than its critical micelle concentration; and (B) detecting aggregates of the abnormal protein after step (A); A method for detecting an abnormal protein in a sample, comprising:

[0008] Item 2. The detection method according to Item 1, wherein the surfactant is a nonionic surfactant.

[0009] Item 3. The detection method according to Item 2, wherein the nonionic surfactant is a nonionic surfactant containing a polyethylene glycol chain and a hydrocarbon chain.

[0010] Item 4. The detection method according to any one of Items 1 to 3, wherein the concentration of the surfactant in the solution is three times or more the critical micelle concentration.

[0011] Item 5. The detection method according to any one of Items 1 to 4, wherein the concentration of the surfactant in the solution is 0.01% by mass or more.

[0012] Item 6. The detection method according to any one of Items 1 to 5, wherein the solution contains urea.

[0013] Item 7. The detection method according to any one of Items 1 to 6, wherein the self-aggregating protein is TDP-43 and / or amyloid β.

[0014] Item 8. The detection method according to any one of Items 1 to 7, wherein the solution is stirred for 2 hours or longer.

[0015] Item 9. The detection method according to any one of Items 1 to 8, wherein the solution contains a dye that binds to aggregates of abnormal protein.

[0016] Item 10. A method for testing a neurodegenerative disease, comprising detecting an abnormal protein in a sample by the detection method according to any one of items 1 to 9, wherein the sample is derived from a subject.

[0017] Item 11. The testing method according to Item 10, wherein, when the sample contains the abnormal protein, the subject is determined to have a high probability of developing the neurodegenerative disease or to be suffering from the neurodegenerative disease.

[0018] Item 12. A sensitizer for the QUIC method which contains a surfactant and uses a self-aggregating protein as a substrate.

[0019] Item 13. A reagent for use in the detection method according to any one of Items 1 to 9 or the testing method according to Item 10, comprising a surfactant. Effect of the Invention

[0020] According to the present invention, it is possible to provide a technique for detecting an abnormal protein in a sample with higher sensitivity. Specifically, it is possible to provide a method for detecting an abnormal protein in a sample, a method for testing a neurodegenerative disease, a sensitizer for the QUIC method using a self-aggregating protein as a substrate, and the like. [Brief description of the drawings]

[0021] [Figure 1] This is a schematic diagram showing the lag time difference (Δtlag). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] 1.Definition In this specification, the expressions "contain" and "comprise" include the concepts of "contain", "include", "consist essentially of" and "consist only of".

[0023] In this specification, the upper limit value and the lower limit value can be combined in any manner.

[0024] As used herein, the "identity" of an amino acid sequence refers to the degree of amino acid sequence agreement between two or more comparable amino acid sequences. Thus, the higher the agreement between two amino acid sequences, the higher the identity or similarity between those sequences. The level of identity of an amino acid sequence can be determined, for example, using the sequence analysis tool FASTA with default parameters. Alternatively, it can be determined using the BLAST algorithm by Karlin and Altschul (Karlin S, Altschul SF. "Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes" Proc Natl Acad Sci USA. 87: 2264-2268 (1990); Karlin S, Altschul SF. "Applications and statistics for multiple high-scoring segments in molecular sequences" Proc Natl Acad Sci USA. 90: 5873-7 (1993)). A program called BLASTX based on such a BLAST algorithm has been developed. Specific techniques for these analysis methods are known and can be found on the National Center of Biotechnology Information (NCBI) website (http: / / www.ncbi.nlm.nih.gov / ). The "identity" of a base sequence is also defined in the same manner as above.

[0025] In this specification, an amino acid mutation specifically refers to an amino acid deletion, substitution, insertion, or addition.

[0026] In this specification, "conservative substitution" means that an amino acid residue is substituted with an amino acid residue having a similar side chain. For example, substitution between amino acid residues having basic side chains such as lysine, arginine, and histidine is considered to be conservative substitution. In addition, substitution between amino acid residues having acidic side chains such as aspartic acid and glutamic acid; amino acid residues having non-charged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; amino acid residues having non-polar side chains such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; amino acid residues having β-branched side chains such as threonine, valine, and isoleucine; and amino acid residues having aromatic side chains such as tyrosine, phenylalanine, tryptophan, and histidine are also considered to be conservative substitutions.

[0027] 2. Detection Method The present invention relates to a method for detecting an aberrant protein in a sample, comprising: (A) stirring a solution containing a sample, a self-aggregating protein as a substrate, and a surfactant, the concentration of the surfactant being equal to or higher than its critical micelle concentration; and (B) detecting aggregates of the aberrant protein after step (A), as described below.

[0028] An abnormal protein is a protein, oligomer, or aggregate whose structure has changed due to misfolding or the like, and is a protein that forms / amplifies aggregates (e.g., amyloid aggregates) using normal and / or abnormal proteins as substrates.

[0029] Abnormal proteins are known to be causative proteins of neurodegenerative diseases, such as Parkinson's disease, Alzheimer's disease, frontotemporal dementia, corticobasal degeneration, progressive supranuclear palsy, dementia with Lewy bodies, amyotrophic lateral sclerosis (ALS), Huntington's disease, multiple system atrophy, and Creutzfeldt-Jakob disease.

[0030] The sample is not particularly limited as long as it is a sample that may contain an aberrant protein. For example, the sample may be a sample suspected of containing an aberrant protein, a sample that is known to contain (or not contain) an aberrant protein, or a sample that has been determined by another method to contain (or not contain) an aberrant protein.

[0031] The sample can be, for example, a sample derived from a subject (biological sample), an artificially prepared sample, or the like.

[0032] The subject may be any mammal, including, for example, rodents such as mice, rats, hamsters, and guinea pigs, laboratory animals such as rabbits, pets such as dogs and cats, livestock such as cows, pigs, goats, horses, and sheep, primates such as monkeys, orangutans, and chimpanzees, and humans, with humans being particularly preferred.

[0033] The biological sample is not particularly limited. Examples of biological samples include body fluids such as cerebrospinal fluid, blood, follicular fluid, menstrual blood, saliva, synovial fluid, urine, interstitial fluid, sweat, and tears, internal tissues such as brain tissue, and samples derived from these body fluids. Samples derived from body fluids / tissues are not particularly limited as long as they are samples prepared from body fluids / tissues, and examples include samples obtained by concentrating and purifying proteins contained in the body fluids / tissues from the body fluids / tissues, and diluted samples.

[0034] An artificially prepared sample is, for example, a sample to which an artificially prepared abnormal protein (eg, a recombinant protein) has been added, or a sample to which the abnormal protein has not been added.

[0035] A substrate is a protein that forms aggregates together with an abnormal protein of the same type (abnormal TDP-43 if the substrate is TDP-43, or abnormal amyloid beta if the substrate is amyloid beta) when the same type of abnormal protein is present in a sample in a QUIC method such as the RT-QUIC method.

[0036] In the present invention, a self-aggregating protein is used as a substrate. A self-aggregating protein is a protein that forms aggregates within 2 days under shaking at 37°C under conditions of a typical protein concentration (any of 0.1 μM to 50 μM) used in an aggregate formation test, near physiological pH (any of 6.8 to 8.0), and without additives such as denaturants or surfactants. The presence or absence of aggregate formation can be determined by adding thioflavin T to the reaction solution and measuring the fluorescence derived from the substance, as in Example 1 described below.

[0037] Examples of self-aggregating proteins include TDP-43, amyloid β, amylin, calcitonin, etc. Among these, TDP-43 is preferred from the viewpoint of being detectable with higher sensitivity. Also, amyloid β is preferred from the viewpoint of being detectable with less variation.

[0038] The gene encoding TDP-43 is known, for example, in the case of humans, the gene is the gene with NCBI Gene ID: 23435, and in the case of mice, the gene is the gene with NCBI Gene ID: 230908. Genes of other organisms are also known, or can be easily identified according to or in accordance with known methods based on the base sequence of the known gene or the amino acid sequence encoded by the base sequence.

[0039] Genes encoding amyloid β are known, for example, in the case of humans, the gene is the gene with NCBI Gene ID: 351, and in the case of mice, the gene is the gene with NCBI Gene ID: 11820. Genes of other species are also known, or can be easily identified according to or in accordance with a known method based on the base sequence of a known gene or the amino acid sequence encoded by the base sequence. In the present invention, for example, a partial fragment of full-length amyloid β can be used.

[0040] The self-aggregating protein is not particularly limited as long as it contains a domain involved in aggregate formation, and may be not only a wild type but also an amino acid sequence containing a mutation relative to the wild type amino acid sequence. The mutation includes a mutation at a site other than the domain and a mutation in the domain. The domain involved in aggregate formation is publicly known or can be easily identified according to publicly known information.

[0041] The self-aggregating protein is, for example, a wild-type self-aggregating protein, a mutant self-aggregating protein containing a domain involved in aggregate formation, or a mutant self-aggregating protein that has 70% or more (preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, and even more preferably 99% or more) identity thereto (or has one or more, for example 1 to 10, preferably 1 to 5, more preferably 1 to 2, and even more preferably 1 amino acid mutation thereto) and has the ability to form aggregates.

[0042] The TDP-43 used as a substrate is, for example, (A) a protein comprising an amino acid sequence shown in any one of SEQ ID NOs: 1 to 3 (preferably SEQ ID NO: 2 or 3), and (B) a protein comprising an amino acid sequence having 70% or more (preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, and even more preferably 99% or more) identity to the amino acid sequence shown in any one of SEQ ID NOs: 1 to 3 (preferably SEQ ID NO: 2 or 3) (or having one or more, for example, 1 to 10, preferably 1 to 5, more preferably 1 to 2, and even more preferably 1 amino acid mutation therein), and having the ability to form aggregates; It may be at least one selected from the group consisting of:

[0043] Examples of protein (B) include proteins containing the amino acid sequence shown in any one of SEQ ID NOs: 4 to 18, and mutants thereof (which have the following single amino acid mutation).

[0044] SEQ ID NOs: 1 to 3 are, respectively, the full length (1-414) of human wild-type TDP-43, a partial sequence (267-414) of human wild-type TDP-43, and a partial sequence (282-414) of human wild-type TDP-43. SEQ ID NOs: 4 to 18 are, respectively, single amino acid mutants (G288P, G290P, N291P, S292P, R293P, G309P, G314P, A315P, G338P, N345P, Q346P, S347P, G348P, N352P, and R361P) of the partial sequence (267-414) of human wild-type TDP-43.

[0045] The amyloid β used as the substrate is, for example, (C) a protein comprising an amino acid sequence shown in any one of SEQ ID NOs: 19 to 20, and (D) a protein comprising an amino acid sequence having 70% or more (preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, and even more preferably 99% or more) identity to an amino acid sequence shown in any one of SEQ ID NOs: 19 to 20 (or having one or more, for example, 1 to 10, preferably 1 to 5, more preferably 1 to 2, and even more preferably 1 amino acid mutation therein), and having the ability to form aggregates; It may be at least one selected from the group consisting of:

[0046] Examples of protein (D) include proteins comprising the amino acid sequence shown in any one of SEQ ID NOs: 21 to 28, and mutants thereof (which have the following single amino acid mutation).

[0047] SEQ ID NOs: 19 to 20 are, respectively, the full-length (1-42) sequence of human wild-type amyloid β and a partial sequence (1-39) of human wild-type amyloid β. SEQ ID NOs: 21 to 28 are, respectively, single amino acid mutants (F4P, H6P, Y10P, H14P, E22P, D23P, I31P, G38P) of the full-length (1-42) sequence of human wild-type amyloid β.

[0048] In one aspect, the present invention provides a substrate protein for use in the QUIC method, comprising: (BX) A protein comprising an amino acid sequence shown in any one of SEQ ID NOs: 4 to 18, or a mutant having 70% or more (preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, and even more preferably 99% or more) identity thereto (or having one or more, for example, 1 to 10, preferably 1 to 5, more preferably 1 to 2, and even more preferably 1 amino acid mutation thereto) (provided that the above-mentioned single amino acid mutation is retained), or (DX) a protein comprising an amino acid sequence shown in any one of SEQ ID NOs: 21 to 28, or a mutant having 70% or more (preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, and even more preferably 99% or more) identity thereto (or having one or more, for example, 1 to 10, preferably 1 to 5, more preferably 1 to 2, and even more preferably 1 amino acid mutation thereto) (provided that the above-mentioned single amino acid mutation is retained), The present invention relates to a substrate protein,

[0049] When the sample is a biological sample, the biological species of the specimen and the biological species of the substrate from which the self-aggregating protein is derived do not have to be the same. Previous reports have shown that abnormal proteins can be detected by the RT-QUIC method using substrates from different species. In one embodiment of the present invention, the biological species of the specimen and the biological species from which the substrate self-aggregating protein is derived are preferably of the same genus, and more preferably of the same species.

[0050] The self-aggregating protein may have another amino acid sequence, such as a secretory signal sequence, a protein tag, a fluorescent protein, a luminescent protein, a signal sequence such as a protease recognition sequence (e.g., a TEV protease recognition sequence), or a peptide added thereto, so long as the ability to form an aggregate is not significantly impaired. Examples of protein tags include biotin, His tag, FLAG tag, Halo tag, MBP tag, HA tag, Myc tag, V5 tag, and PA tag.

[0051] The self-aggregating protein may be chemically modified so long as its ability to form aggregates is not significantly impaired.

[0052] Self-aggregating proteins have a C-terminus that is a carboxyl group (-COOH) or a carboxylate (-COO - ), amide (-CONH2) or ester (-COOR).

[0053] Here, R in the ester is, for example, C such as methyl, ethyl, n-propyl, isopropyl, and n-butyl. 1-6 Alkyl groups; for example, cyclopentyl, cyclohexyl, etc. 3-8 Cycloalkyl groups; for example, C phenyl, α-naphthyl, etc. 6-12 Aryl groups; for example, phenyl-C such as benzyl and phenethyl 1-2 Alkyl groups: α-naphthyl-C such as α-naphthylmethyl 1-2 C such as alkyl group 7-14 Aralkyl groups, pivaloyloxymethyl groups, etc. are used.

[0054] The self-aggregating protein may be amidated or esterified at a carboxyl group (or carboxylate) other than the C-terminus. In this case, the ester may be, for example, the C-terminus ester described above.

[0055] The self-aggregating protein may be in the form of a salt with an acid or a base. The salt is not particularly limited, and both acid salts and basic salts can be used. Examples of acid salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, nitrate, and phosphate; organic acid salts such as acetate, propionate, tartrate, fumarate, maleate, malate, citrate, methanesulfonate, and paratoluenesulfonate; amino acid salts such as aspartate and glutamate. Examples of basic salts include alkali metal salts such as sodium salt and potassium salt; and alkaline earth metal salts such as calcium salt and magnesium salt.

[0056] The self-aggregating protein may be in the form of a solvate. The solvent is not particularly limited, and examples thereof include water, ethanol, glycerol, and acetic acid.

[0057] The self-aggregating protein can be easily prepared according to the amino acid sequence by known genetic engineering techniques, for example, PCR, restriction enzyme digestion, DNA ligation, in vitro transcription / translation, recombinant protein preparation, etc. In order to prevent aggregation of the self-aggregating protein during purification, etc., it is preferable to add a denaturant such as urea.

[0058] The self-aggregating protein may be one type alone or two or more types in combination.

[0059] The concentration of the self-aggregating protein in the solution in step (A) is not particularly limited and is, for example, 0.01 μM or more. From the viewpoint of the detection sensitivity of the abnormal protein, the concentration is preferably 0.1 μM or more, more preferably 0.1 to 1 mM, even more preferably 0.2 to 200 μM, still more preferably 0.4 to 100 μM, particularly preferably 1 to 50 μM, particularly more preferably 2 to 20 μM, and particularly more preferably 2 to 10 μM.

[0060] The surfactant is not particularly limited, and a nonionic surfactant, an ionic surfactant, or the like can be used.

[0061] Examples of the surfactant include anionic surfactants, nonionic surfactants, cationic surfactants, and amphoteric surfactants.

[0062] Examples of nonionic surfactants include polyoxyethylene alkyl ethers, partial fatty acid esters of polyhydric alcohols, ethylene oxide adducts of fatty alcohols, ethylene oxide adducts of fatty acids, ethylene oxide adducts of fatty aminos or fatty acid amides, ethylene oxide adducts of alkylphenols, ethylene oxide adducts of partial fatty acid esters of polyhydric alcohols, polyethylene glycols, alkyl glucosides, fatty acid alkanolamides, and the like.

[0063] Examples of anionic surfactants include alkyl phosphate surfactants, sulfated oils, soaps, sulfated ester oils, sulfated amide oils, sulfated ester salts of olefins, fatty alcohol sulfate salts, alkyl sulfate salts, fatty acid ethyl sulfonates, alkyl naphthalene sulfonates, alkyl benzene sulfonates, oleate sulfonates, and phosphate salts.

[0064] Examples of the cationic surfactant include quaternary ammonium salts, primary amine salts, secondary amine salts, tertiary amine salts, quaternary amine salts, and pyridine derivatives.

[0065] Examples of amphoteric surfactants include betaine type surfactants, carboxylic acid derivatives, and imidazoline derivatives.

[0066] From the viewpoints of detection sensitivity of abnormal proteins, detection with less variation, etc., the surfactant is preferably a nonionic surfactant, more preferably a nonionic surfactant containing a polyethylene glycol chain and a hydrocarbon chain, even more preferably a nonionic surfactant consisting of a polyethylene glycol chain and a hydrocarbon chain, and particularly preferably a nonionic surfactant in which the polyethylene glycol chain and the hydrocarbon chain are linked by an ether bond.

[0067] The degree of polymerization of the polyethylene glycol chain in the nonionic surfactant containing a polyethylene glycol chain and a hydrocarbon chain is not particularly limited, and is, for example, 5 to 300. From the viewpoints of detection sensitivity of abnormal proteins, detection with less variation, etc., the degree of polymerization is preferably 5 to 200, more preferably 5 to 150, even more preferably 10 to 150, still more preferably 10 to 100, particularly preferably 10 to 50, particularly more preferably 15 to 50, particularly still more preferably 15 to 30, and particularly preferably 15 to 25.

[0068] The hydrocarbon chain in the nonionic surfactant containing a polyethylene glycol chain and a hydrocarbon chain may be either linear or branched, but is preferably linear from the viewpoints of detection sensitivity of abnormal proteins, detection with less variation, etc. The hydrocarbon chain may be, for example, a saturated hydrocarbon chain or an unsaturated hydrocarbon chain, but is preferably a saturated hydrocarbon chain (alkyl chain) from the viewpoints of detection sensitivity of abnormal proteins, detection with less variation, etc. The number of carbon atoms in the hydrocarbon chain is not particularly limited and is, for example, 10 to 30, but is preferably 12 to 30, more preferably 12 to 26, even more preferably 12 to 22, still more preferably 14 to 22, particularly preferably 14 to 20, particularly more preferably 16 to 20, and particularly preferably 16 to 18, from the viewpoints of detection sensitivity of abnormal proteins, detection with less variation, etc.

[0069] The hydrocarbon chain in the nonionic surfactant containing a polyethylene glycol chain and a hydrocarbon chain is preferably represented by the general formula (1): HO[-(CH2)2-O] n -R (wherein n is an integer of 5 to 300, and R is a monovalent chain hydrocarbon group). The surfactant may be a single type, or a combination of two or more types.

[0070] The concentration of the surfactant in the solution in step (A) is equal to or higher than the critical micelle concentration. In this specification, the critical micelle concentration of each surfactant is the upper CMC value measured and estimated according to the method described in Reference Example 4 described later (see "upper limit" in Table 1). Although not wishing to limit the interpretation, by setting the surfactant concentration to equal to or higher than the critical micelle concentration, the process of forming nuclei for aggregate formation by the substrate self-aggregating protein is suppressed, while there is no or only a weak degree of suppression of aggregate formation based on the abnormal protein, so that the difference in aggregate formation rate in the presence / absence of the abnormal protein can be made larger, which is considered to realize an improvement in detection sensitivity.

[0071] The concentration of the surfactant in the solution in step (A) is, from the viewpoint of the detection sensitivity of the abnormal protein, preferably 1.5 times or more, more preferably 2 times or more, even more preferably 3 times or more, still more preferably 5 times or more, particularly preferably 10 times or more, particularly more preferably 20 times or more, 30 times or more, 40 times or more, 50 times or more, or 100 times or more of the critical micelle concentration. From the viewpoint of the detection sensitivity of the abnormal protein, the concentration is preferably 1000 times or less, more preferably 800 times or less, even more preferably 600 times or less, still more preferably 400 times or less, and particularly preferably 250 times or less of the critical micelle concentration.

[0072] From the viewpoint of the detection sensitivity of abnormal proteins, etc., the concentration of the surfactant in the solution in step (A) is preferably 0.001% by mass or more, more preferably 0.002% by mass or more, even more preferably 0.005% by mass or more, still more preferably 0.01% by mass or more, particularly preferably 0.02% by mass or more, particularly more preferably 0.05% by mass or more, and particularly more preferably 0.07% by mass or more. From the viewpoint of the detection sensitivity of abnormal proteins, etc., the concentration is preferably 2.0% by mass or less, more preferably 1.0% by mass or less, even more preferably 0.8% by mass or less, still more preferably 0.6% by mass or less, particularly preferably 0.5% by mass or less, particularly more preferably 0.4% by mass or less, particularly more preferably 0.3% by mass or less, and particularly more preferably 0.25% by mass or less.

[0073] The solution in step (A) preferably further contains urea. By using urea in combination with a surfactant having a critical micelle concentration equal to or higher, the detection sensitivity can be further improved. From the viewpoint of the detection sensitivity of abnormal proteins, the concentration of urea is preferably 0.05 to 10 M, more preferably 0.1 to 6 M, even more preferably 0.2 to 4 M, even more preferably 0.3 to 3 M, particularly preferably 0.3 to 2 M, and particularly preferably 0.4 to 1.2 M.

[0074] The solvent of the solution in step (A) is not particularly limited as long as it does not denature the self-aggregating protein and allows the formation of aggregates based on the abnormal protein. For example, water can be used as the solvent. A mixed solvent of water and an organic solvent (e.g., alcohol, etc.) can also be used.

[0075] The solvent has, for example, a water content of, relative to 100% by volume of the solvent, for example, 50% by volume or more, preferably 70% by volume or more, more preferably 80% by volume or more, even more preferably 90% by volume or more, still more preferably 95% by volume or more, and particularly preferably 99% by volume or more.

[0076] The pH of the solution in step (A) is not particularly limited as long as the self-aggregating protein is not denatured and aggregate formation based on the abnormal protein is possible. The pH is preferably 5.0 to 9.0, more preferably 7.0 to 8.0.

[0077] The solution in step (A) preferably contains a buffer for adjusting or controlling the pH, and examples of the buffer include, but are not limited to, HEPES buffer, Tris buffer, phosphate buffer, MOPS buffer, etc.

[0078] The solution in step (A) preferably contains a dye that binds to the abnormal protein aggregates in order to facilitate detection of the aggregates. Examples of such dyes include, but are not limited to, thioflavin T (ThT), 8-(phenylamino)-1-naphthalenesulfonic acid (ANS), etc. The fluorescence derived from these dyes serves as an indicator of aggregates.

[0079] The solution in step (A) may further contain additives, such as preservatives such as EDTA and NaN3.

[0080] The method of stirring the solution in step (A) is not particularly limited, and either a method using a stirrer or a method not using a stirrer can be adopted. As an example, the stirring is performed by shaking the solution. The intensity of the stirring is not particularly limited as long as aggregates based on the abnormal protein are formed, but in the case of shaking, the stirring speed can be, for example, 100 to 250 rpm, preferably 150 to 200 rpm. The stirring may be performed continuously or intermittently. In general, the stronger the stirring intensity, the faster the aggregate formation, and the weaker the stirring intensity, the slower the aggregate formation.

[0081] The temperature during stirring is not particularly limited, but is, for example, 20 to 45° C., and preferably 30 to 42° C. In general, a higher temperature speeds up the formation of aggregates, and a lower temperature slows down the formation of aggregates.

[0082] The stirring time is not particularly limited as long as it is long enough to determine the presence or absence of the abnormal protein in the sample. The time can be determined each time a negative control (a sample not containing the abnormal protein) and a positive control (a sample containing a predetermined amount of the abnormal protein) are stirred in the same manner, based on the time and degree of aggregate formation in each case, or can be determined in advance. The stirring time can be, for example, 2 hours or more, 5 hours or more, 10 hours or more, 20 hours or more, 30 hours or more, 40 hours or more, or 50 hours or more, and can be, for example, 200 hours or less, 150 hours or less, 100 hours or less, or 80 hours or less.

[0083] In step (A), aggregates of abnormal proteins corresponding to the substrate self-aggregating protein (abnormal TDP-43 when the substrate is TDP-43, and abnormal amyloid β when the substrate is amyloid β) are formed / amplified. In step (B), the aggregates are detected.

[0084] The detection method is not particularly limited, and a method of detecting fluorescence derived from the above-mentioned dye is simple and preferable, but it is also possible to adopt detection methods based on immune reactions using aggregate-specific antibodies (e.g., ELISA, Western blotting), etc.

[0085] In step (B), it is possible to determine whether or not a sample contains an aberrant protein, and to measure the amount of the aberrant protein, based on an index value relating to the rate of formation of aberrant protein aggregates, such as the time required for the aberrant protein aggregates to reach a certain amount. When quantifying the aberrant protein, the index value is measured using a sample containing a predetermined amount of the aberrant protein, and the amount of the aberrant protein in the test sample can be quantified by comparing the index values.

[0086] 3. Testing Method In one aspect, the present invention relates to a method for testing for a neurodegenerative disease, which comprises detecting an abnormal protein in a sample by the detection method of the present invention, and the sample is derived from a subject.

[0087] The terms are as explained in "2. Detection method" above.

[0088] The above test method can provide values ​​for testing for neurodegenerative diseases, and therefore can assist in the testing and diagnosis of neurodegenerative diseases.

[0089] The above-mentioned testing method can further include determining that the subject has a high probability of developing the neurodegenerative disease or is suffering from the neurodegenerative disease when the sample contains the abnormal protein.

[0090] For example, when a sample contains abnormal TDP-43, it can be determined that the patient is highly likely to develop or suffers from frontotemporal dementia, amyotrophic lateral sclerosis (ALS), inclusion body myositis, etc. As another example, when a sample contains abnormal amyloid β, it can be determined that the patient is highly likely to develop or suffers from Alzheimer's dementia, amyloid β-associated vasculitis, etc.

[0091] The above judgment can also be made based on a cut-off value. The cut-off value is set in advance based on a statistical analysis or ROC analysis of the data of the above values ​​by preparing a database that tracks the index value for the generation rate of abnormal protein aggregates, the amount or concentration value of abnormal protein, and the possibility of developing a neurodegenerative disease and / or the presence or absence of a neurodegenerative disease for the evaluation population. Alternatively, the cut-off value can be set each time. When the cut-off value is determined by statistical analysis, for example, the median, arithmetic mean, or other average value of the data of the above values ​​of the evaluation population can be used. When the cut-off value is determined by ROC analysis, for example, the cut-off value based on ROC analysis can be the above value of the point on the ROC curve where the distance between the point on the vertical axis (sensitivity or true positivity) of the ROC curve graph that is 1.0 and the point on the horizontal axis (1-specificity) that is 0.0 is the smallest, or the cut-off value can be derived from the Youden index of the ROC curve (Cancer 1950;3:32-35.).

[0092] 4. Reagents In one aspect, the present invention relates to a sensitizer for the QUIC method using a self-aggregating protein as a substrate, the sensitizer containing a surfactant. Also, in one aspect, the present invention relates to a reagent for use in the detection method of the present invention or the testing method of the present invention, the reagent containing a surfactant.

[0093] The terms are as explained in "2. Detection method" above.

[0094] The above sensitizer / reagent is used so that the surfactant concentration in the solution containing the sample and substrate is equal to or higher than the critical micelle concentration.

[0095] The sensitizer / reagent may be in the form of a kit, which may include, in addition to the surfactant, various reagents (e.g., buffer solutions, substrates, positive controls, negative controls, etc.) and instruments (e.g., reaction vessels, and instruments for collecting, purifying, and isolating biological samples) used in the QUIC method.

[0096] The above sensitizers / reagents can be used as companion diagnostics for preventive or therapeutic drugs for neurodegenerative diseases (eg, inhibitors of the production of abnormal proteins, agents promoting the degradation of abnormal proteins, antibody therapy against abnormal proteins, etc.). EXAMPLES

[0097] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0098] Reference Example 1. Preparation of recombinant TDP-43 protein A DNA fragment encoding human TDP-43 (aa. 267-414: SEQ ID NO: 2) was inserted into pRSET A Bacterial Expression Vector (Thermo Fisher Scientific #V35120) at the NheI and BamHI sites. Point mutations in TDP-43 (SEQ ID NO: 4-18) were introduced using inverse PCR. Plasmids encoding TDP-43 and its mutants were transformed into ECOS Competent E. coli BL21 using the manufacturer's protocol (NIPPON GENE #314-06533). The next day, a single colony was inoculated into 5 mL of LB medium containing 100 μg / mL ampicillin and precultured at 37 °C with shaking at 180-240 rpm until mid-log phase. The precultured E. coli was inoculated into 1.25 L of LB medium containing 50 μg / mL ampicillin and shaken under the same conditions until mid-log phase. Protein expression was then induced with 1 mM IPTG and shaking at 37°C for 4 h. E. coli were harvested by centrifugation at 7,000g for 12 min, resuspended in 20 mM Tris-HCl (pH 8) buffer, and sonicated on ice using a VP-300N ultrasonic processor (TAITEC, #0075955-001). Disrupted E. coli were centrifuged at 30,000g for 25 min, and the supernatant fraction was discarded. The pellet fraction was solubilized in 6M GdHCl / 50mM Tri-HCl buffer (pH 8) for 3–24 h with shaking at 100 rpm at room temperature, after which the solution was centrifuged at 30,000g for 60 min, and the supernatant fraction was collected for subsequent purification procedures.

[0099] The solubilized proteins were purified using immobilized metal affinity chromatography (IMAC) using 7.5 M urea to prevent protein aggregation during purification. 5 mL of Ni Sepharose 6 Fast Flow resin (Cytiva) was loaded onto a spin column, washed with 10 mL deionized water, and charged with 10 mL 0.1 M NiSO4. The column was washed again with 10 mL deionized water and equilibrated with 10 mL binding buffer [100 mM sodium phosphate, 10 mM Tris-HCl (pH 7.8), 20 mM imidazole, 7.5 M urea]. The solubilized proteins were then applied to the column and washed extensively with 10 mL binding buffer until the absorbance at 280 nm of the flow-through was below the detection limit. Proteins were eluted by applying 10 mL of elution buffer [100 mM sodium phosphate, 10 mM Tris-HCl (pH 7.8), 500 mM imidazole, 7.5 M urea]. IMAC-purified proteins were further purified by reversed-phase chromatography using a COSMOSIL Protein-R column (Nacalai Tesque #06532-31) connected to an AKTApurifier (GE healthcare). Proteins were eluted with a linear gradient of 20–55% acetonitrile in 0.1% trifluoroacetic acid. TDP-43-enriched fractions were collected and lyophilized to remove the solvent. Lyophilized proteins were dissolved in 6 M GuHCl at a concentration of 500 μM to remove existing aggregates. Protein concentration was determined using UV absorbance at 280 nm based on the extinction coefficient (18,350 cm-1M-1 for TDP-43). The stock solution was passed through a 0.2 μm filter, dispensed, and stored at −80° C. until use.

[0100] Reference Example 2. Preparation of recombinant amyloid β A DNA fragment encoding human amyloid beta (aa.1-42: SEQ ID NO:19) was inserted into the pET-28c(+) vector (Merck #69866) at the NcoI and SalI sites. Point mutations in amyloid beta (SEQ ID NO:21-28) were introduced using inverse PCR. Plasmids encoding amyloid beta (M1-42) and its mutants with an initiation methionine were transformed into E. coli BL21 and protein expression was induced as described above. The E. coli were then disrupted and centrifuged at 30,000g for 25 min, and the supernatant fraction was discarded. The pellet fraction was washed three times with Triton X-100 buffer (20 mM Tris-HCl, pH 8, and 4% Triton X-100) and then twice with 20 mM Tri-HCl buffer (pH 8). The pellet fraction was solubilized in 8 M urea / 20 mM Tri-HCl buffer (pH 8) by shaking at 100 rpm at room temperature for 3 to 24 hours, and then centrifuged at 30,000 g for 60 minutes. The supernatant fraction was collected and used for subsequent purification procedures.

[0101] The solubilized proteins were purified using cation exchange affinity chromatography using standard procedures. Briefly, the proteins were applied to Q Sepharose Fast Flow resin (Cytiva) loaded onto a spin column, washed with 100 mM NaCl buffer (M Urea, 20 mM Tri-HCl buffer, pH 8), and eluted with 200 mM NaCl buffer. The purified proteins were concentrated by ultrafiltration (Millipore, 3,000 MWCO) and stored at -80°C. Immediately before use, the proteins were applied to a Superdex 75 10 / 300 GL column (Cytiva) equilibrated with running buffer (10 mM sodium phosphate, pH 7.4), and the monomer fraction was collected and used for further experiments.

[0102] Reference Example 3. Preparation of seed protein A reaction solution (5 μM TDP-43 protein (recombinant TDP-43 protein (SEQ ID NO: 2) of Reference Example 1), 25 μM thioflavin T (ThT), 20 mM HEPES-NaOH (pH 7.2), 1 mM EDTA, 0.01% NaN3) was prepared and shaken for 48 hours (conditions were the same as in Example 1 described below), and the resulting solution was used as the TDP-43 seed protein in the following examples.

[0103] In addition, a reaction solution (3 μM amyloid β protein (recombinant amyloid β protein (sequence number 19) of Reference Example 2), 25 μM thioflavin T (ThT), 20 mM HEPES-NaOH (pH 7.2), 1 mM EDTA, 0.01% NaN3) was prepared and shaken for 48 hours under the same conditions as in Example 3 described below (conditions were the same as in Example 1 described below), and the resulting solution was used as amyloid β seed protein in the following examples.

[0104] Reference Example 4. Measurement of critical micelle concentration (CMC) The CMC values ​​of the surfactants were determined using Nile Red (NR) dye as previously reported (Analytical Methods, 7 (2015) 6877-6882.). Briefly, NR was added to serial dilutions of the surfactants (0.0014, 0.0041, 0.012, 0.037, 0.11, 0.33, 1%) dispersed in distilled water at a final concentration of 1 μM. After 30 min of incubation at room temperature, the fluorescence intensity at 640 nm (excitation wavelength, 580 nm) was recorded with a SpectraMax iD5 multimode microplate reader (Molecular Devices). The fluorescence intensity was plotted as a function of surfactant concentration and fitted to a sigmoidal function using Igor Pro 9.02. The second derivative was used to determine the surfactant concentration at the lower inflection point, which represents the CMC. The experiment was repeated four times to estimate the lower and upper limits of the CMC (mean ± 2 SE).

[0105] The results are shown in Table 1. In Table 1, the n in "name" or the numerical value in parentheses indicates the degree of polymerization of polyethylene glycol / polyoxyethylene.

[0106] [Table 1]

[0107] Example 1. Analysis of the effect of surfactants on RT-QUIC reaction 1 Brij 58, Triton X-114, NP-40, or Brij Sami15 was used as a detergent to examine the effect of the detergent on the RT-QUIC reaction.

[0108] The components were mixed to prepare a reaction solution (TDP-43 seed protein (Reference Example 3), 5 μM substrate protein (recombinant TDP-43 protein (SEQ ID NO: 2) of Reference Example 1), 0.1% surfactant, 25 μM thioflavin T (ThT), 20 mM HEPES-NaOH (pH 7.2), 1 mM EDTA, 0.01% NaN3). The reaction solution was transferred to a 96-well black microplate (10 pg seed protein / 100 μL reaction solution / well), sealed with transparent film, and shaking was started (conditions: 176 rpm, continuous shaking at 42 °C). The time course of ThT fluorescence was recorded by reading from the top with a TECAN Infinite 200 F Nano+. The lag time was defined as the time from the start of shaking until the fluorescence intensity reached >150% of the initial intensity (n = 12). The lag time was also calculated in the same manner when the reaction solution did not contain seed protein (n=6). The lag time difference (Δtlag) was calculated by subtracting the average lag time when the seed protein was present from the average lag time when the seed protein was not present (see Figure 1).

[0109] The results are shown in Table 2. In Table 2, (none) indicates the case where no surfactant was added.

[0110] [Table 2]

[0111] It was found that the difference in lag time increases when the reaction solution contains a surfactant at or above the critical micelle concentration. The increase in lag time difference makes it possible to detect even smaller amounts of abnormal proteins.

[0112] Example 2. Analysis of the effect of surfactants on RT-QUIC reaction 2 The test was carried out in the same manner as in Example 1, except that Briji58 and its analogues (Brij C23, Brij S20, Brij 58, Brij S100, or Brij O20) were used as the surfactant. All of these surfactants are nonionic surfactants containing a polyethylene glycol chain and a hydrocarbon chain.

[0113] The results are shown in Table 3.

[0114] [Table 3]

[0115] It was found that the use of a nonionic surfactant containing a polyethylene glycol chain and a hydrocarbon chain resulted in a relatively large difference in lag time.

[0116] Example 3. Analysis of the effect of surfactants on RT-QUIC reaction 3 The test was performed in the same manner as in Example 1, except that Brij 58, Brij NoPh18, Tween 40, Tween 80, Tween 60, Tween 20, or cymal-7 was used as the surfactant, amyloid β seed protein (Reference Example 3) was used as the seed protein (2 ng in each well), recombinant amyloid β protein (SEQ ID NO: 19) of Reference Example 2 was used as the substrate protein (3 μM in the reaction solution), and the shaking conditions were 37°C, with repeated cycles of incubation for 5 minutes and shaking at 176 rpm for 10 seconds.

[0117] The results are shown in Table 4.

[0118] [Table 4]

[0119] It was found that for amyloid beta, which is a self-aggregating protein like TDP-43, the lag time difference becomes larger when the reaction solution contains a surfactant at or above the critical micelle concentration.

[0120] Example 4. Analysis of the effect of surfactants on RT-QUIC reaction 4 The test was performed in the same manner as in Example 1, except that the concentration of Brij 58 in the reaction solution was as shown in Table 5.

[0121] The results are shown in Table 5.

[0122] [Table 5]

[0123] It was found that the lag time extension effect was achieved by increasing the surfactant concentration above the critical micelle concentration.

[0124] Example 5. Analysis of the effect of using surfactants and urea in combination in RT-QUIC reactions The test was performed in the same manner as in Example 1, except that Brij 58 was used as the surfactant, the amount of seed protein was 10 pg or 250 fg, and urea was or was not added to the reaction solution.

[0125] The results are shown in Table 6. In the table, the detection limit is the minimum seed amount at which p<0.01 is obtained by t-test for the presence of a lag time difference.

[0126] [Table 6]

[0127] It was found that the lag time difference became even larger when urea was used in combination. Also, it was found that the detection limit was on the order of fg even without urea.

Claims

1. (A) stirring a solution containing a sample, a substrate self-aggregating protein, and a surfactant, the concentration of the surfactant being equal to or higher than its critical micelle concentration; and (B) detecting abnormal protein aggregates after step (A); A method for detecting an abnormal protein in a sample, comprising:

2. The detection method according to claim 1 , wherein the surfactant is a nonionic surfactant.

3. The detection method according to claim 2 , wherein the nonionic surfactant is a nonionic surfactant containing a polyethylene glycol chain and a hydrocarbon chain.

4. The detection method according to claim 1 , wherein the concentration of the surfactant in the solution is three times or more the critical micelle concentration.

5. The detection method according to claim 1 , wherein the concentration of the surfactant in the solution is 0.01% by mass or more.

6. The method of claim 1 , wherein the solution contains urea.

7. The detection method according to claim 1 , wherein the self-aggregating protein is TDP-43 and / or amyloid beta.

8. The detection method according to claim 1 , wherein the solution is stirred for 2 hours or more.

9. The detection method according to claim 1 , wherein the solution contains a dye that binds to aggregates of abnormal proteins.

10. A method for testing for a neurodegenerative disease, comprising detecting an abnormal protein in a sample by the detection method according to any one of claims 1 to 9, wherein the sample is derived from a subject.

11. The testing method according to claim 10, wherein the subject is determined to have a high probability of developing the neurodegenerative disease or to be suffering from the neurodegenerative disease when the sample contains the abnormal protein.

12. A surfactant-containing sensitizer for the QUIC method that uses a self-aggregating protein as a substrate.

13. A reagent for use in the detection method according to any one of claims 1 to 9 or the testing method according to claim 10, comprising a surfactant.

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