Alpha-synuclein detection using bead

Abstract

To provide a method for determining the presence of soluble misfolded α-synuclein protein in a biological sample.SOLUTION: A method comprises bringing a biological sample into contact with a pre-incubation mixture to form an incubation mixture, where the pre-incubation mixture includes monomeric α-synuclein protein, a buffer composition, a salt, and an indicator. An incubation cycle is performed on the incubation mixture in the presence of either silicon nitride beads or borosilicate glass beads having a diameter of about 1 mm to about 5 mm. The method further comprises determining whether a detectable amount of misfolded α-synuclein aggregates is present in the biological sample.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 62 / 895,535, filed on September 4, 2019; U.S. Provisional Patent Application No. 63 / 040,144, filed on June 17, 2020; U.S. Provisional Patent Application No. 63 / 042,679, filed on June 23, 2020; U.S. Provisional Patent Application No. 63 / 045,593, filed on June 29, 2020; U.S. Provisional Patent Application No. 63 / 073,420, filed on September 1, 2020; and U.S. Provisional Patent Application No. 63 / 073,424, filed on September 1, 2020, each of which is hereby incorporated by reference in its entirety.

[0002] Sequence Listing The sequence listing is submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy was created on September 2, 2020, named Amprion - AS - FA - US - 1_ST25.txt, and is 26,468 bytes in size.

Background Art

[0003] The accumulation and deposition of α - synuclein (α - syn or α - S) aggregates in brain tissue are major events in the etiology of various neurodegenerative disorders called synucleinopathies. Synucleinopathies include Parkinson's disease (PD), Lewy body dementia (LBD), multiple system atrophy (MSA), and pure autonomic failure (PAF). Currently, the diagnosis of these disorders mainly relies on the recognition of clinical symptoms and, unfortunately, typically provides a diagnosis only when neurodegeneration is already in an advanced stage.

[0004] Various protein misfolding cyclic amplification (PMCA) assays provide ultrasensitive methods for detecting misfolded aggregates through the artificial acceleration and amplification of misfolding and aggregation processes in vitro. The basic concept of PMCA has been previously disclosed (Soto et al., WO 2002 / 04954; Estrada et al., US 2008 / 0118938). PMCA assays have been successfully used to detect misfolded α-syn protein with very high sensitivity and specificity. See US 2016 / 0077111, which is hereby incorporated by reference in its entirety.

[0005] The PMCA assay is an important advance over previous diagnostic methods. However, assay conditions that promote amplification while avoiding undesirable self-aggregation of protein monomers, as well as devices that facilitate the use of such assays, are still needed. SUMMARY OF THE INVENTION MEANS FOR SOLVING THE PROBLEM

[0006] In one aspect, a method for determining the presence of soluble misfolded α-syn in a biological sample is provided. The method includes contacting the biological sample with monomeric α-syn protein and one or more beads having an average diameter of from about 1.0 mm to about 5.0 mm to form an incubation mixture; incubating the incubation mixture to form misfolded α-syn aggregates from the monomeric α-syn protein and soluble misfolded α-syn of the biological sample; dissociating at least a portion of the misfolded α-syn aggregates; repeating the incubating and dissociating steps a sufficient number of times to amplify the soluble misfolded α-syn of the sample to obtain a detectable amount of misfolded α-syn aggregates; and determining whether a detectable amount of misfolded α-syn aggregates is present in the biological sample, wherein detection of the misfolded α-syn aggregates indicates the presence of soluble misfolded α-syn in the biological sample.

[0007] In some embodiments, the step of determining whether a detectable amount of misfolded α-synuclein aggregates are present in a biological sample includes contacting the incubation mixture with a protein aggregation indicator. An example of a suitable protein aggregation indicator is Thioflavin T (ThT). In further embodiments, the method includes the step of obtaining a biological sample from a subject. In still further embodiments, the biological sample is a cerebrospinal fluid (CSF) sample. In additional embodiments, the method includes determining the amount of soluble misfolded α-synuclein protein in the biological sample.

[0008] The incubation mixture includes one or more beads for accelerating the formation of misfolded α-synuclein aggregates. In some embodiments, the beads include silicon nitride (Si3N4). In some embodiments, the beads include borosilicate glass. In some embodiments, the beads have an average diameter in the range of about 1 mm to about 3.5 mm. In some embodiments, the beads have an average diameter in the range of greater than 2.3 mm to about 3.5 mm. In some embodiments, such as when the beads include Si3N4, the beads have an average diameter of about 2.38 mm (3 / 32 inch). In some embodiments, such as when the beads include borosilicate glass, the beads have an average diameter of about 2.45 mm. In some embodiments, beads having an average diameter of 2.3 mm or less are specifically excluded. In some embodiments, glass beads having an average diameter of 2.3 mm or less are specifically excluded. In some embodiments, the incubation mixture is housed in a multi-well plate including a plurality of wells. In further embodiments, each well of the multi-well plate includes a single bead. In further embodiments, such as when the beads include Si3N4, a portion or all of the surface of the beads is blocked with protein.

[0009] Using beads, the incubation conditions can be optimized to reduce the amount of self-aggregation of monomeric α-syn protein while accelerating the process. In some embodiments, the monomeric α-syn protein in the incubation mixture has a concentration range of about 10 μM to about 30 μM. In a further embodiment, the incubation mixture contains one or more of Tris-HCL, MES, PIPES, MOPS, BES, TES, and HEPES and has a pH of about 6 to about 8, such as about 6.2 to about 6.5. In a further embodiment, the incubation of the incubation mixture is carried out at a temperature of about 35°C to about 42°C. Additionally, in a further embodiment, incubating the incubation mixture and deaggregating at least a portion of the misfolded α-syn aggregates includes an incubation cycle that lasts from 0.3 to 1 hour. In a further embodiment, the deaggregation is carried out by shaking (including periodic agitation), stirring, or sonication. In some embodiments, the method also includes a step of concentrating soluble misfolded α-syn in the sample before incubating the sample using an antibody that specifically binds to soluble misfolded α-syn.

[0010] Another aspect provides a method of diagnosing a disease associated with α-synuclein aggregation in a subject. The method comprises contacting a biological sample with monomeric α-synuclein protein and one or more beads having an average diameter of about 1 mm to about 5 mm, greater than 2.3 mm to about 5 mm, greater than 3 mm to about 5 mm, about 2.38 mm, or about 2.45 mm to form an incubation mixture; incubating the incubation mixture to form misfolded α-synuclein aggregates from the monomeric α-synuclein protein and soluble misfolded α-synuclein of the biological sample; at least partially de-aggregating the misfolded α-synuclein aggregates; repeating the incubating and de-aggregating steps a sufficient number of times to amplify the soluble misfolded α-synuclein of the biological sample to obtain a detectable amount of misfolded α-synuclein aggregates; and determining whether a detectable amount of misfolded α-synuclein aggregates is present in the biological sample, wherein detection of misfolded α-synuclein aggregates indicates that the biological subject has a disease associated with α-synuclein aggregation.

[0011] In some aspects, the disease associated with α-synuclein aggregation is PD. In other aspects, the disease associated with α-synuclein aggregation is LBD, MSA, or PAF. In further aspects, the method also comprises treating a subject diagnosed as having a disease associated with α-synuclein aggregation with an α-synuclein modulating therapy.

[0012] In another aspect, a kit is provided for determining the presence of soluble misfolded α-syn in a biological sample. The kit includes a known amount of monomeric α-syn protein; a known amount of a protein aggregation indicator; a container for incubating the incubation mixture; a buffer composition; one or more beads having a diameter of about 1 mm to about 5 mm, greater than 2.3 mm to about 5 mm, greater than 3 mm to about 5 mm, about 2.38 mm, or about 2.45 mm; instructions for guiding a user in performing a method for detecting the presence of soluble misfolded α-syn in a biological sample as described herein; and a package for holding the components of the kit. In some aspects, the container included in the kit includes a multi-well plate including a plurality of wells.

[0013] The instructions include contacting a biological sample in the container with a known amount of monomeric α-syn protein and one or more beads to form an incubation mixture; incubating the incubation mixture to form misfolded α-syn aggregates from the monomeric α-syn protein and soluble misfolded α-syn of the biological sample; dissociating at least a portion of the misfolded α-syn aggregates; repeating the incubating and dissociating steps a sufficient number of times to amplify the soluble misfolded α-syn of the biological sample to obtain a detectable amount of misfolded α-syn aggregates; and contacting the incubation mixture with a known amount of a protein aggregation indicator to determine whether a detectable amount of misfolded α-syn aggregates is present in the biological sample, wherein detection of misfolded α-syn aggregates indicates the presence of soluble misfolded α-syn in the biological sample.

[0014] In another aspect, an apparatus for biochemical assays, such as enzyme-linked immunosorbent assay (ELISA) and PMCA, is provided. The apparatus is configured for use with beads and includes a tray. The horizontal upper surface of the tray has an opening sized to receive the beads. The opening extends vertically through the tray. The upper surface is unobstructed such that the beads can rotate and move across the upper surface and into the opening from the upper surface.

[0015] The device further includes a gate. The opening extends vertically through the gate. The blocking portion of the gate is located between the openings. The gate is supported to move horizontally between a first position and a second position. In the first position, the blocking portion of the gate is vertically aligned below the opening of the tray. In the second position, the opening of the gate is vertically aligned below the opening of the tray. Thus, by moving the gate from the first position to the second position, it becomes possible to drop the beads from the tray through the opening of the gate and further into a bead receptacle such as a well of an ELISA plate.

[0016] Each opening of the tray can be sized such that when the gate is in the first position, it contains only one of the beads. Further, the tray may have a vertical wall surrounding the upper surface. The vertical wall can have an exit passage sized such that the beads can roll down from the upper surface through the vertical wall. This makes it possible to remove excess beads from the tray once the desired number of beads has rolled into the openings of the tray. By removing the excess beads, the user can visually confirm by inspection that each of the openings contains one bead.

[0017] The tray may be formed of a transparent material such that the gate is visible through the tray. The color contrast between the beads and the gate can help the user visually confirm that a single bead is provided in each well. For example, a white gate visible through a transparent panel helps to more clearly display beads of a darker hue (such as Si3N4 beads).

[0018] The claimed invention can be more readily understood by reference to the following figures.

Brief Description of the Drawings

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DETAILED DESCRIPTION OF THE INVENTION

[0041] Methods and kits are provided for determining the presence of soluble misfolded α-syn in a biological sample. The methods and kits involve contacting the biological sample with monomeric α-syn protein and one or more beads having an average diameter of from about 1 mm to about 5 mm, greater than 2.3 mm to about 5 mm, greater than 3 mm to about 5 mm, about 2.38 mm, or about 2.45 mm to form an incubation mixture; incubating the incubation mixture to form misfolded α-syn aggregates from the monomeric α-syn protein and soluble misfolded α-syn of the biological sample; at least partially deaggregating the misfolded α-syn aggregates; and repeating the incubating and deaggregating steps a sufficient number of times to amplify the soluble misfolded α-syn of the biological sample to obtain a detectable amount of misfolded α-syn aggregates. It can then be determined whether a detectable amount of misfolded α-syn aggregates, indicative of the presence of soluble misfolded α-syn in the biological sample, is present in the biological sample.

[0042] In an exemplary example shown in the schematic description of FIG. 1, a method for determining the presence of soluble misfolded α-syn protein in a biological sample is provided. Hereinafter, the order of the steps described in FIG. 1 and in the claims is provided for clarity only and should not be considered limiting. With that caveat, the method comprises: (A) contacting a biological sample with a pre-incubation mixture comprising: (1) monomeric α-syn protein; (2) a buffer composition; (3) a salt; and (4) an indicator to form an incubation mixture; (B) an incubation cycle, each incubation cycle comprising: (i) incubating the incubation mixture for a time effective to cause at least partial misfolding and / or aggregation of the monomeric α-syn protein in the presence of soluble misfolded α-syn protein; and (ii) physically disrupting the incubation mixture, the incubation cycle being carried out: (1) more than twice on an incubation mixture effective to form an amplified portion of misfolded α-syn protein from monomeric α-syn protein; (2) in the presence of either Si3N4 beads or borosilicate glass beads having a diameter of about 1 mm to about 5 mm, greater than 2.3 mm to about 5 mm, greater than 3 mm to about 5 mm, about 2.38 mm, or about 2.45 mm; (C) determining whether a detectable amount of misfolded α-syn aggregates is present in the biological sample, the detection of misfolded α-syn aggregates indicating the presence of soluble misfolded α-syn protein in the biological sample.

[0043] In some embodiments of the method, the beads consist essentially of Si3N4 and have a diameter of about 2.38 mm. In some embodiments, the method further includes blocking the surface of the Si3N4 beads with BSA prior to implementation. In some embodiments, blocking includes immersing the Si3N4 beads in a solution of BSA in at least one of water and / or PIPES buffer. In some embodiments, the beads consist essentially of borosilicate glass and have a diameter of about 2.45. In some embodiments, the borosilicate glass is not blocked. In some embodiments of the method, the biological sample includes human CSF. In some embodiments, the monomeric α-syn protein includes at least one of SEQ ID NO: 1, SEQ ID NO: 2, or a conservative variant thereof. In some embodiments, the monomeric α-syn protein is present at a concentration of about 10 μM to about 30 μM. In some embodiments, the monomeric α-syn protein is present at a concentration of about 19.6 μM. In some embodiments, the buffer composition has a pH of about 6.2 to about 6.5, such as about 6.3. In some embodiments, the buffer composition includes PIPES. In some embodiments, the buffer composition includes about 100 mM PIPES, about 500 mM PIPES, about 600 mM PIPES, or about 700 mM PIPES. In some embodiments, the salt includes NaCl. In some embodiments, the salt includes NaCl at a concentration of about 500 mM to about 700 mM, such as about 600 mM. In some embodiments, the indicator includes ThT. In some embodiments, the detection includes measuring the ThT fluorescence at about 490 nm after excitation at about 440 nm. In some embodiments, the indicator includes ThT at a concentration of about 5 μM to about 10 μM. In some embodiments, the physical grinding includes shaking including orbital shaking.

[0044] Definitions The term "diagnosis" can encompass determining the likelihood that a subject has developed a disease or the presence or nature of a disease in a subject. The term "diagnosis" also encompasses determining the severity and expected outcome of a disease or episode of disease, or the likelihood of recovery, generally referred to as prognosis. "Diagnosis" can also encompass diagnosis in the context of rational therapy, where the diagnosis guides therapy, including the initial choice of therapy, modification of therapy (e.g., adjustment of dosage or dosing regimen), and the like.

[0045] The term "prognosis" refers to the prediction of the likely course and outcome of a disease or the likelihood of recovery from a disease. Prognosis is distinguished from diagnosis in that it is generally already known that the subject has the disease, although prognosis and diagnosis can be performed simultaneously. In the case of the prognosis of PD, the prognosis can classify the relative severity of PD and use this to guide the selection of appropriate therapy.

[0046] The terms "treatment", "treating", etc. refer to obtaining a desired pharmacological or physiological effect. The effect can be curative, wholly or partly, of a disease or prophylactic against adverse effects caused by a disease. "Treatment" encompasses any treatment of a disease in a mammal, particularly a human, and can include inhibiting a disease or condition, i.e., preventing its onset, and alleviating a disease, i.e., causing regression of the disease.

[0047] Prevention or prophylaxis refers to preventing the occurrence of a disease or symptoms of a disease in a subject who may have a predisposition to the disease but has not yet been diagnosed as having it (e.g., including diseases that can be associated with or caused by a primary disease). Prevention can include completely or partially preventing the disease or symptoms.

[0048] The terms "therapeutically effective" and "pharmacologically effective" are intended to delimit the amount of an agent that achieves the goal of improving disease severity and incidence beyond that of treatment with each agent alone, while avoiding the adverse side effects typical of alternative therapies. The effectiveness of the treatment can be measured by assessing a decrease in the level of soluble misfolded α-syn or a reduction of other symptoms associated with a particular synucleinopathy.

[0049] Where a range of values is provided, each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limits of that range, and any other stated value or intervening value within the stated range is understood to be included, unless the context clearly indicates otherwise. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, subject to any specifically excluded limits within the stated range. Where the stated range includes one or both of the limits, ranges excluding one or both of those included limits are also included.

[0050] Unless otherwise defined, all technical and scientific terms have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0051] The term "about" in relation to a number is intended to include ±10% of that number. This applies whether "about" modifies an independent number or numbers at either or both ends of a range of numbers. In other words, "about 10" means 9 to 11. Similarly, "about 10 to about 20" means 9 to 22. Where the term "about" is not present, an exact number is intended. In other words, "10" means 10.

[0052] The singular forms "a", "and", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a sample" includes one sample and a plurality of such samples. Similarly, reference to "the monomeric α-syn protein" includes reference to one or more protein molecules and the like.

[0053] To determine the presence of soluble misfolded α-syn protein in a biological sample A method for determining the presence of soluble misfolded α-syn protein in a biological sample is provided. In some embodiments, determining the presence includes detecting whether soluble misfolded α-syn protein is present in the sample, and in other embodiments, determining the presence includes determining the amount of soluble misfolded α-syn protein in the sample. The method includes contacting a biological sample with monomeric α-syn protein and one or more beads having an average diameter of about 1 mm to about 5 mm, greater than 2.3 mm to about 5 mm, greater than 3 mm to about 5 mm, about 2.38 mm, or about 2.45 mm to form an incubation mixture; incubating the incubation mixture to form misfolded α-syn aggregates from the monomeric α-syn protein and the soluble misfolded α-syn of the biological sample; and at least partially deaggregating the misfolded α-syn aggregates. The steps of incubating and deaggregating the incubation mixture are repeated a sufficient number of times to amplify the soluble misfolded α-syn protein of the sample to obtain a detectable amount of misfolded α-syn aggregates. The method then includes determining whether a detectable amount of misfolded α-syn aggregates is present in the biological sample. Detection of the misfolded α-syn aggregates indicates the presence of soluble misfolded α-syn protein in the biological sample.

[0054] As used herein, "α-S", "α-syn", or "α-synuclein", such as soluble misfolded α-synuclein protein, can refer to the full-length 140 amino acid wild-type α-synuclein protein, i.e., "αS-140". Other isoforms or fragments can include, for example, alpha-syn-126, "αS-126", which lacks residues 41-54 due to deletion of exon 3, and alpha-syn-112, "αS-112", which lacks residues 103-130 due to deletion of exon 5. Various αS isoforms can include, but are not limited to, αS-140, αS-126, and αS-112. Various α-syn peptides can be associated with neuronal damage associated with synucleinopathies such as PD.

[0055] "Soluble misfolded α-synuclein protein" refers to misfolded monomeric or aggregated α-synuclein protein that remains in solution. Examples of soluble misfolded α-synuclein protein can include any number of aggregated misfolded α-synuclein protein monomers as long as the misfolded α-synuclein protein remains soluble. For example, soluble misfolded α-synuclein protein can include aggregates of 2 to about 50 units of misfolded α-synuclein protein monomers. In some examples, the aggregates can be referred to as oligomers or polymers. In some examples, aggregation can be referred to as oligomerization or polymerization.

[0056] Soluble misfolded α-synuclein can aggregate or oligomerize to form insoluble aggregates and / or higher oligomers, resulting in misfolded α-synuclein aggregates in the form of protofibrils, fibrils, and ultimately plaques or inclusions. Nucleation-dependent polymerization can typically be characterized by a slow lag phase during which aggregated nuclei can form, followed by the rapid formation of additional aggregates and / or larger aggregates. The lag phase can be minimized or eliminated by the addition of pre-formed "nuclei" or "seeds". "Seeds" or "nuclei" refer to misfolded α-synuclein or fragmented short fibrils, particularly soluble misfolded α-synuclein, that have the ability to induce further misfolding, oligomerization, and / or aggregation. In some examples, "seeds" or "nuclei" may exclude non-aggregated monomers of α-synuclein. Without wishing to be bound by theory, at least under some conditions, monomeric α-synuclein may be unstable, and the minimum stable size of pathogenic misfolded α-synuclein may be considered to be an aggregate of two monomer units of misfolded α-synuclein.

[0057] As used herein, "soluble" species include soluble misfolded α-syn and can form a solution in a biological fluid under physiological conditions, while "insoluble" species can exist as precipitates, fibrils, deposits, condensates, or other non-dissolved forms in such biological fluids. Examples of insoluble species include fibrils of Aβ, α-S, tau, etc. Species that dissolve in non-biological fluids but not in biological fluids under physiological conditions can be considered insoluble. For example, fibrils such as α-syn can be dissolved in an aqueous solution of a surfactant such as sodium dodecyl sulfate (SDS), but are still insoluble in one or more of the described biological fluids under physiological conditions and are therefore considered insoluble herein.

[0058] In some embodiments, the biological sample can exclude precipitates, fibrils, deposits, concentrates, plaques, or insoluble species of misfolded proteins in other forms that can be insoluble in one or more of the biological fluids described under physiological conditions. The sample can exclude insoluble forms of misfolded α-syn protein. For example, the biological sample can exclude precipitates, fibrils, deposits, concentrates, plaques, or misfolded α-syn protein in other insoluble forms, such as fibril form.

[0059] As used herein, a "misfolded protein" is a protein that no longer contains all or part of the structural conformation of the protein that exists in its typical non-pathogenic normal function within a biological system. A misfolded protein can be an aggregate. A misfolded protein can be localized in a protein aggregate. A misfolded protein can be a non-functional protein. A misfolded protein can be a pathogenic conformational isomer of a protein. A monomeric α-syn protein composition can be provided with confirmed native, non-pathogenicity, having no catalytic activity for misfolding, oligomerization, and aggregation related to seeds. A monomeric α-syn protein composition can be provided in a seedless form.

[0060] The phrases "monomeric α-syn protein" and "monomeric α-syn substrate" are used interchangeably and refer to one or more α-syn protein molecules in their native non-pathogenic structure. In some embodiments, the monomeric α-syn protein has 140 amino acids, has a molecular mass of 14,460 Da, and comprises, consists essentially of, or consists of a wild-type or recombinant human α-syn protein represented by the following sequence.

[0061] SEQ ID NO: 1 MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH GVATVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA

[0062] In some embodiments, the monomeric α-syn protein comprises, consists essentially of, or consists of a conservative variant of SEQ ID NO:1. A conservative variant is a peptide or amino acid sequence that deviates from SEQ ID NO:1 only in amino acid substitutions by one or more amino acids having similar biochemical properties and having a minimal or beneficial effect on the activity of the protein obtained in the PMCA assay. The conservative variant must function essentially in the same way as the basic component, i.e., SEQ ID NO:1. For example, a conservative variant of SEQ ID NO:1 aggregates with misfolded α-syn and forms aggregates with substantially the same kinetics under similar reaction conditions. The conservative variant may have, for example, from 1, 2, 3, 4, 5, 6, or 7 (5%), up to 14 (10%) substitutions in the amino acid sequence. In some embodiments, the monomeric α-syn protein comprises six additional histidine amino acids (i.e., a polyHis purification tag) at the C-terminus of SEQ ID NO:1, has a molecular mass of about 15,283 Da, and comprises a recombinant α-syn protein represented by the following sequence.

[0063] SEQ ID NO:2 MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH GVATVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA HHHHHH

[0064] Accordingly, SEQ ID NO:2 is distinguishable from SEQ ID NO:1 by six additional histidine amino acids at the C-terminus. SEQ ID NO:2 is further distinguishable from, for example, variants of SEQ ID NO:1 in which one or more amino acids are added at the N-terminus. In some embodiments, variants of SEQ ID NO:1 in which one or more amino acids are added at the N-terminus are excluded. However, some embodiments include N-terminal adducts. Accordingly,

[0065] SEQ ID NO:3 HHHHHH MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH GVATVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA

[0066] For example, additional purification tags including FLAG, HA, Myc and V5 are contemplated, and thus generate the following SEQ ID NOs.

[0067] SEQ ID NO:4: MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH GVATVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA DYKDDDD

[0068] SEQ ID NO:5: DYKDDDD MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH GVATVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA

[0069] SEQ ID NO:6: MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH GVATVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA DYKDDDDK

[0070] Sequence number 7: DYKDDDDK MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH GVATVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA

[0071] Sequence number 8: MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH GVATVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA DYKDDDK

[0072] Sequence number 9: DYKDDDK MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH GVATVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA

[0073] Sequence number 10: MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH GVATVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA YPYDVPDYA

[0074] Sequence number 11: YPYDVPDYA MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH GVATVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA

[0075] Sequence number 12: MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH GVATVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA YAYDVPDYA

[0076] Sequence number 13: YAYDVPDYA MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH GVATVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA

[0077] Sequence number 14: MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH GVATVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA YDVPDYASL

[0078] Sequence number 15: YDVPDYASL MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH GVATVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA

[0079] Sequence number 16: MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH GVATVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA EQKLISEEDL

[0080] Sequence number 17: EQKLISEEDL MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH GVATVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA

[0081] Sequence number 18: MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH GVATVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA GKPIPNPLLGLDST

[0082] Sequence number 19: GKPIPNPLLGLDST It should be noted that the "70" and "71" in the translation should be the same format as the original tags, but the font here may not display properly. You can adjust it according to the actual situation to ensure the correct display of the tags. MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH GVATVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA

[0083] In some embodiments, the method may include providing the monomeric α-synuclein protein in a labeled form. The labeled monomeric α-synuclein protein may be considered a conservative variant. The labeled form of the monomeric α-synuclein protein may include one or more of a covalently incorporated radioactive amino acid, a covalently incorporated isotopically labeled amino acid, a covalently incorporated fluorophore, etc. Thus, detection of the soluble misfolded α-synuclein protein may include detecting the labeled form of the monomeric α-synuclein protein incorporated into the amplified portion of the misfolded α-synuclein protein.

[0084] Incubation conditions The method includes contacting a biological sample with monomeric α-syn protein and one or more beads having an average diameter of 1 mm to 5 mm, greater than 2.3 mm to 5 mm, greater than 3 mm to 5 mm, 2.38 mm, or 2.45 mm to form an incubation mixture, and incubating the incubation mixture to form misfolded α-syn aggregates from monomeric α-syn protein and soluble misfolded α-syn of the biological sample. As used herein, "contacting" refers to bringing various agents into proximity and placing them under conditions where they can interact to produce a desired effect. For example, by contacting a biological sample with monomeric α-syn protein, the monomeric α-syn protein can interact with any soluble misfolded α-syn protein present in the biological sample, thereby enabling stimulation of the aggregation of the monomeric α-syn protein.

[0085] Incubation conditions for forming misfolded α-syn aggregates include various variable factors such as bead type and number, monomeric α-syn protein identity and concentration, container type used for carrying out the incubation, temperature, pH, buffer composition, salt concentration or ionic strength, and other properties of the liquid medium used for the incubation. Inclusion of beads in the incubation mixture enables incubation to occur under conditions that are faster and also avoid self-aggregation by the monomeric α-syn protein.

[0086] The incubation mixture can contain monomeric α-synuclein at various different concentrations. By including beads, the concentration of the monomeric α-synuclein substrate required to perform the PMCA assay can be reduced. In some embodiments, the incubation mixture can contain monomeric α-synuclein at a concentration or concentration range of about 500 nM to about 500 μM; about 1 μM to about 200 μM; about 5 μM to about 100 μM; about 10 μM to about 50 μM; about 65 μM; about 10 μM to about 30 μM; greater than 10 μM and less than 30 μM; about 20 μM; about 19.6 μM; or 19.6 μM.

[0087] The incubation mixture can contain various buffer compositions. The buffer composition is effective to maintain the pH of the incubation mixture in the range of about pH 5 to about pH 9, about pH 6 to about pH 8, about pH 6 to about pH 7, about pH 7 to about pH 8, about pH 7, about pH 7.4, about pH 6.2 to about pH 6.5 (including pH 6.3, 6.4, and 6.5). In some embodiments, the incubation mixture contains one or more of the buffers Tris-HCL, MES, PIPES, MOPS, BES, TES, and HEPES. In some embodiments, the incubation buffer contains PIPES at a concentration of about 100 mM, about 500 mM, about 600 mM, and about 700 mM.

[0088] In some embodiments, the incubation mixture contains a salt at a given concentration. The salt can, for example, enhance the signal-to-noise ratio in fluorescence detection. In one embodiment, the salt contains NaCl. Other suitable salts can include KCl. In one embodiment, the salt, such as NaCl, may be present at a concentration of about 50 mM to about 1,000 mM, about 50 mM to about 500 mM, about 50 to about 150 mM, about 150 mM to about 500 mM, about 50 mM, about 150 mM, about 300 mM, about 500 mM, about 600 mM, or about 700 mM. In one embodiment, the salt, such as NaCl, is present at a concentration of about 500 mM.

[0089] In one embodiment, the incubation mixture excludes a surfactant or detergent, such as SDS.

[0090] A variety of temperatures are suitable for carrying out the incubation cycle. The temperature of the incubation mixture in °C can be, independently for each incubation cycle, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or a range between any two of the foregoing values, for example, about 15°C to about 50°C, or about 25°C to about 45°C, or about 30°C to about 42°C. In some embodiments, the incubation is carried out at a temperature that is approximately the normal physiological temperature of a warm-blooded animal. In further embodiments, the incubation of the incubation mixture is carried out at a temperature of about 35°C to about 40°C or about 37°C to about 42°C.

[0091] The incubation mixture includes one or more beads. The beads are typically small spherical objects, for example, high-density beads having a low-friction surface commonly used as bearing beads. Inclusion of beads in the incubation mixture increases the rate of formation of misfolded α-syn aggregates from monomeric α-syn protein and soluble misfolded α-syn of the biological sample. These beads should be distinguished from antibody-coated magnetic or paramagnetic beads or particles (e.g., Dynabeads) used in known enrichment and / or immunodepletion procedures. See, for example, U.S. Patent Publication No. 2016 / 0077111.

[0092] Beads can be formed from a variety of biologically inert materials. In some embodiments, the beads are formed from synthetic or natural polymers, and in other embodiments, the beads are formed from minerals, ceramics, glass, or metals. The beads may be homogeneous or may include a core made of different materials. Examples of synthetic polymers include acrylic polymers, polyamides, polyimides, polyesters, polyethers, polymeric vinyl compounds, polyalkenes, and substituted derivatives thereof, as well as copolymers containing two or more such polymers. Examples of specific synthetic polymers include polystyrene, polytetrafluoroethylene, polymethyl methacrylate, and the like. Examples of natural polymers include carbohydrate-based polymers such as agarose.

[0093] In some embodiments, the polymer includes a non-polymeric material such as a mineral, ceramic, glass, or metal. More specific examples of non-polymeric materials for the beads include zirconium oxide, silica / zirconium, silica, glass, borosilicate glass, quartz, steel, titanium, tungsten carbide, silicon carbide, and Si3N4. In some embodiments, it may be appropriate to use materials having a darker hue, such as Si3N4, to reduce the background fluorescence present in the assay.

[0094] In some embodiments, the beads contain Si3N4, consist essentially of Si3N4, or consist of Si3N4. In some embodiments, the beads contain borosilicate glass, consist essentially of borosilicate glass, or consist of borosilicate glass. In one embodiment, zirconium / silica beads are excluded. In one embodiment, glass beads other than borosilicate glass beads are excluded. In one embodiment, steel beads are excluded. In one embodiment, polystyrene beads are excluded.

[0095] The size of the beads can significantly affect its ability to increase the protein aggregation rate. In some embodiments, the beads included in the incubation mixture can have an average diameter exceeding 0.5 mm. In some aspects, the beads have an average diameter of more than 0.5 mm to about 10 mm. In some aspects, the beads have an average diameter of more than 0.5 mm to about 5 mm. In further aspects, the beads have an average diameter in the range of more than 0.5 mm to about 3.5 mm. In some aspects, the beads have an average diameter of about 1.0 to about 10 mm, while in additional aspects, the beads have an average diameter of about 1.0 mm to about 5 mm. In further aspects, the beads have an average diameter in the range of more than 1.0 mm to about 3.5 mm. In some aspects, the beads have an average diameter of 2.38 to about 10 mm, while in additional aspects, the beads have an average diameter of 2.38 mm to about 5 mm. In further aspects, the beads have an average diameter in the range of about 2.3 mm or more to about 3.5 mm, about 2.38 to about 3.5 mm, or about 2.45 mm to about 3.5 mm. In further aspects, the beads can have an average diameter of about 1 mm to about 5 mm, more than 2.3 mm to about 5 mm, more than 3 mm to about 5 mm, about 2.38 mm, or about 2.45 mm. In some aspects, the beads contain Si3N4, consist essentially of Si3N4, or consist of Si3N4, have an average diameter of 2.38 mm, and are blocked with BSA. In some aspects, the beads contain borosilicate glass, consist essentially of borosilicate glass, or consist of borosilicate glass, have an average diameter of 2.45 mm, and are not blocked. In some aspects, beads having an average diameter of 2.3 mm or less are excluded from the present invention. In some aspects, glass beads having an average diameter of 2.3 mm or less are excluded from the present invention. In some aspects, glass beads having an average diameter of 3 mm or less are excluded from the present invention. The size distribution of the beads is defined such that more than 90% of the beads are between 80 - 120% of the average bead diameter, or between 90 - 110% of the average bead diameter.

[0096] The number of beads included in the incubation mixture may be changed according to the size of the incubation mixture. In some embodiments, the incubation mixture includes a plurality of beads. In some embodiments, the incubation includes one bead per 200 μL of the incubation mixture. In further embodiments, the incubation mixture includes 1 to 10, 1 to 20, 1 to 100, 5 to 50, 20 to 100, or 50 to 500 beads. In some embodiments, the incubation mixture includes a single bead.

[0097] In some embodiments, the surface of one or more beads is "blocked" with a protein. Blocking the surface of the beads with a protein refers to providing a coating or layer over all or a substantial portion of the surface of the beads. Any suitable biocompatible protein can be used to coat the surface of the beads. Proteins suitable for use in blocking the surface of the beads are albumins such as BSA. Other suitable blocking proteins may include casein or nonfat dry milk. One or more beads can be blocked by immersing the one or more beads in a solution containing the protein. The solution can be an aqueous solution and / or a buffer solution such as PIPES, Tris-HCl, MES, MOPS, BES, TES, and HEPES.

[0098] The incubation mixture is held in a container of appropriate size, such as a test tube. Appropriate sterile incubation containers are known to those skilled in the art. In some embodiments, the incubating mixture is housed in a multi-well plate containing a plurality of wells. For example, the multi-well plate can contain 96 wells. The wells of the multi-well plate can have volumes of 100 - 1000 μL, 150 - 750 μL, or 200 - 350 μL. In one embodiment, for example, when the beads are Si3N4 beads, the container is a black-bottom 96-well plate (Costar 3916). In one embodiment, for example, when the beads are borosilicate glass beads, the container is a clear-bottom 96-well plate (Costar 3603).

[0099] In some embodiments, each well of the multi-well plate contains a single bead. In one such embodiment, the multi-well plate can be used with a bead dispensing device such as the bead dispensing device 10 shown in FIG. 16. The device 10 can be used in any assay performed using beads in a multi-well plate, such as ELISA, or in a PMCA assay as described and / or claimed herein. In the illustrated example, the device 10 includes a tray 12, a gate 14, and a receptor plate 18. The gate 14 is movable relative to the tray 12 and the receptor plate 18 as shown in FIGS. 17 and 18. This enables the spherical beads 20, such as Si3N4 beads having an average diameter greater than 2.3 mm, to be filled into the tray 12 and individually dispensed into the receptor plate 18 as shown in FIGS. 21 - 23.

[0100] The tray 12 in the figure example is generally rectangular, and the opposing side surfaces 30, 32 extend longitudinally between the opposing end portions 34, 36. The side surfaces 30, 32 and the end portions 34, 36 of the tray 12 are defined by corresponding portions of the peripheral wall 40. The panel 42 extends over the entire length and width of the tray 12 within the surrounding peripheral wall 40. The panel 42 has a horizontal upper surface 46 that includes an array of bead openings 49. The bead openings 49 penetrate the panel 42. In a given example, as seen in FIGS. 17 and 18, there are 96 bead openings 49 in an 8×12 array of parallel rows and columns. The bead outlet 51 penetrates the corner of the peripheral wall 40. The slot 53 penetrates the first end portion 34 of the peripheral wall 40.

[0101] The gate 14 is configured as a rectangular card. The slot 61 penetrates the gate 14 and is arranged to coincide with the columns of the bead openings 49 of the tray 12. Thus, the illustrated example has 12 parallel slots 61 that penetrate the gate 14. The slots 61 are separated by blocking portions 64 of the gate 14, each of which is located between a pair of adjacent slots 61.

[0102] The receptacle plate 18 has an array of wells 71 that corresponds to the array of bead openings of the tray 12. In this example, the receptacle plate 18 is a standard 96-well ELISA plate having a known configuration in which the wells 71 are arranged in an 8×12 array.

[0103] In use, the tray 12 is placed on top of the receptacle plate 18 as shown in FIG. 19. Next, the bead openings 49 of the tray 12 are vertically aligned with the wells 71 of the receptacle plate 18. The lower skirt portion 76 of the peripheral wall 40 is placed on the shoulder portion 78 of the receptacle plate 18. The skirt 76 supports the tray 12 at a position where the panel 42 is spaced upwardly from the receptacle plate 18. This provides a space for the gate 14 to be inserted through the slot 53 at the first end 34 of the peripheral wall 40. The inserted gate 14 is vertically interposed between the panel 42 and the receptacle plate 18. The user can slide the gate 14 inside and outside the slot 53 to move the gate 14 back and forth between the positions shown in FIGS. 17 and 18. The panel 42 can be formed of a transparent material so that, as shown in FIGS. 17 and 18, the gate 14 can be seen through the panel 42.

[0104] When the gate is in the position of FIG. 17, each blocking portion 64 of the gate 14 is located below the corresponding row of bead openings 49 of the tray 12, as shown in FIG. 20. The user can pour an indefinite number of coated beads 20 onto the tray 12, and as a result, the beads 20 freely roll across the upper surface 46 of the panel 42 between the openings 49, and a portion of the beads 20 roll from the upper surface 46 into the openings 49 as shown in FIG. 21. In the illustrated example, each bead opening 49 is sized to receive only a single bead 20. The user can then lift and tilt the tray 12 to roll and discharge excess beads 20 from the upper surface 46 through the outlet passage 51 of the peripheral wall 40. Thus, the tray 12 is filled with beads 20 as shown in FIG. 22. When the user slides the gate 14 inwardly to the position of FIG. 18, the slot 61 of the gate 14 moves below the opening 49 of the tray 12, as shown in FIG. 23. The beads 20 then fall from the opening 49 through the slot 61 into the wells 71, and each well 71 receives only a single bead 20.

[0105] Removing excess beads through the exit opening 51 helps ensure that only a single bead 20 drops into each well 71 when the gate 14 is moved to the positions of FIGS. 18 and 23. A transparent tray panel 42 having a color contrast between the bead 20 and the gate 14 also helps ensure the deposition of only a single bead 20. For example, the apparatus 10 can include a white gate 14 visible through the transparent panel 42 to more clearly display the darker-hued beads 20.

[0106] Once the beads are in place, incubate the incubation mixture for a sufficient time to form misfolded α-synuclein aggregates from monomeric α-synuclein protein and soluble misfolded α-syn of the biological sample. After incubating the incubation mixture to form misfolded α-synuclein aggregates, at least a portion of the incubation mixture is deaggregated before repeating the cycle.

[0107] As used herein, aggregates of misfolded α-synuclein protein refer to non-covalent associations of proteins including soluble misfolded α-synuclein protein. Aggregates of misfolded α-synuclein protein can be “deaggregated,” broken, or fragmented to release smaller fragments or aggregates, such as soluble misfolded α-synuclein protein and fragmented fibrils. The catalytic activity of an aggregate of misfolded α-synuclein protein seeds can be at least partially proportional to the number of seeds in the mixture. Thus, fragmenting the aggregates of misfolded α-synuclein protein in the mixture to release soluble misfolded α-synuclein protein and fragmented fibril seeds can lead to an increase in catalytic activity for the aggregation of monomeric α-synuclein protein.

[0108] In some embodiments, the deaggregation of the incubation mixture can include one or more types of physical milling selected from shaking, sonication, stirring, freeze / thaw, laser irradiation, autoclave incubation, high pressure, homogenization, and the like. Shaking can include periodic agitation such as orbital stirring. Periodic agitation may be performed at about 50 revolutions per minute (RPM) to 10,000 RPM. Periodic agitation may be performed at about 200 RPM to about 2000 RPM. Periodic agitation may be performed at about 600 to 800 RPM. The deaggregation of the incubation mixture may be performed once, for about 5 seconds to about 10 minutes, about 30 seconds to about 1 minute, about 45 seconds to about 1 minute, about 1 minute, etc., after each incubation cycle.

[0109] The steps of incubating and deaggregating the incubation mixture are repeated a sufficient number of times to amplify the soluble misfolded α-syn of the sample to obtain a detectable amount of misfolded α-syn aggregates. The two steps of incubating the incubation mixture and then deaggregating the incubation mixture are herein referred to as an incubation cycle. The incubation cycle may be repeated about 2 times to about 1000 times, about 5 times to about 500 times, about 50 times to about 500 times, about 150 times to about 250 times, etc. In the final cycle of the incubation cycle, it may be preferable to omit the deaggregation step before analyzing the incubation mixture.

[0110] The incubation cycle may be performed for a time such as about 1 minute to about 5 hours, about 10 minutes to about 2 hours, about 15 minutes to about 1 hour, about 25 minutes to about 45 minutes, etc. In some embodiments, incubating the incubation mixture and at least partially deaggregating misfolded α-syn aggregates includes an incubation cycle that lasts from 0.3 to 1 hour. Each incubation cycle may include independently incubating and deaggregating the incubation mixture in one or more of: an incubation of about 1 minute to about 5 hours and a deaggregation of about 5 seconds to about 10 minutes; an incubation of about 10 minutes to about 2 hours and a deaggregation of about 30 seconds to about 1 minute; an incubation of about 15 minutes to about 1 hour and a deaggregation of about 45 seconds to about 1 minute; an incubation of about 25 minutes to about 45 minutes and a deaggregation of about 45 seconds to about 1 minute; and an incubation of about 1 minute and a physical grinding of about 1 minute.

[0111] Biological sample Aspects of the methods described herein may include obtaining a biological sample from a subject. As used herein, "biological sample" means any biological sample from a subject that is suitable for analysis for the detection of misfolded α-syn protein. Suitable biological samples include, but are not limited to, body fluids such as blood-related samples (e.g., whole blood, serum, plasma, and other blood-derived samples), urine, sputum, saliva, urine, CSF, etc. Another example of a biological sample is a tissue sample. The α-syn protein can be evaluated quantitatively or qualitatively, and the detection can be determined either in vitro or ex vivo.

[0112] The method includes preparing or obtaining a biological sample from a subject. In some embodiments, the method includes obtaining a biological sample from a subject. The biological sample can be obtained by any known means including, but not limited to, finger prick, needle biopsy, swab, etc. In an exemplary method, the biological sample is a CSF sample and can be obtained, for example, by lumbar puncture where a needle is inserted into the subarachnoid space and then CSF is extracted.

[0113] In some embodiments, the method of the present invention is performed on a biological sample provided. The biological sample may be fresh or stored. The biological sample can be stored or preserved under appropriate tissue preservation conditions or has been stored or preserved. The biological sample can be a biological sample obtained explicitly for the assays described herein or a sample obtained for another purpose that can be subsampled for the assays described herein. Preferably, when the biological samples are stored, they are cooled or frozen immediately after collection to prevent sample degradation. For example, CSF samples can be stored at -80 °C in polypropylene tubes. The CSF samples can be frozen in liquid nitrogen (“snap frozen”) or by placing the sample in an environment maintained at -80 °C (e.g., a refrigerator or freezer).

[0114] The biological sample may be pretreated by dilution in an appropriate buffer solution as needed, concentrated as desired, or fractionated by any number of methods, such as, but not limited to, ultracentrifugation, fractionation by fast protein liquid chromatography (FPLC) or HPLC, or precipitation of proteins by dextran sulfate or other methods. Any of several standard buffered aqueous solutions at physiological pH, such as phosphate, Tris, etc., can be used.

[0115] Subject The terms “individual,” “subject,” and “patient” are used interchangeably herein, regardless of whether the subject is receiving or currently undergoing any form of treatment. As used herein, the term “subject” generally refers to any vertebrate, including but not limited to mammals. Examples of mammals include primates, including monkeys and humans, equines (e.g., horses), canines (e.g., dogs), felines, various domestic animals (e.g., ungulates such as swine, pigs, goats, sheep, etc.), and captive pets (e.g., cats, hamsters, mice, and guinea pigs). The analysis of biological samples from human subjects is of particular interest.

[0116] In some embodiments, the subject may be at risk of developing PD, may have a risk of having PD, or may be under treatment for PD, and may have a risk of having a disease associated with dysregulation, misfolding, aggregation, or deposition of α-syn, such as MSA, LBD, or PAF, may have a disease associated with dysregulation, misfolding, aggregation, or deposition of α-syn, or may be under treatment for a disease associated with dysregulation, misfolding, aggregation, or deposition of α-syn. In another embodiment, the subject may be at risk of developing Alzheimer's disease, or may be at risk of developing a neurodegenerative disease associated with aggregates of misfolded protein species such as α-syn dysregulation, misfolding, aggregation, or deposition, and Aβ and tau.

[0117] Determining the presence of soluble misfolded α-syn protein The method may include determining whether a detectable amount of misfolded α-syn aggregates is present in a biological sample. Detection of misfolded α-syn aggregates indicates the presence of soluble misfolded α-syn in the biological sample. The process of detecting misfolded α-syn aggregates can be performed during or after each incubation cycle, or can be performed at the completion of a predetermined number of incubation cycles.

[0118] In some embodiments, the method includes contacting an incubation mixture with a protein aggregation indicator to determine whether a detectable amount of misfolded α-synuclein aggregates are present in the incubation mixture. The protein aggregation indicator may be characterized by presenting a display state in the presence of misfolded α-synuclein protein aggregates and a non-display state in the absence of misfolded α-synuclein protein aggregates. Determining the presence of soluble misfolded α-synuclein protein in a biological sample may include detecting the display state of an indicator of misfolded α-synuclein protein aggregates. The display state of the indicator and the non-display state of the indicator can be characterized by differences in fluorescence, light absorption, or radioactivity depending on the particular indicator. The step of determining the presence of soluble misfolded α-synuclein protein in a biological sample may include detecting differences in fluorescence, light absorption, or radioactivity depending on the particular indicator used.

[0119] In some embodiments, the method may include contacting a molar excess of the protein aggregation indicator with one or both of the incubation mixture or the detection mixture. The molar excess may be greater than the total molar amount of α-synuclein protein monomers and soluble misfolded α-synuclein protein in the incubation mixture.

[0120] In some embodiments, the protein aggregation indicator may include one or more of thioflavin T (ThT), congo red, m-I-stilbene, chrysoidine G, PIB, BF-227, X-34, TZDM, FDDNP, MeO-X-04, IMPY, NIAD-4, luminescent conjugated polythiophene, fusions with fluorescent proteins such as green fluorescent protein and yellow fluorescent protein, and derivatives thereof. A suitable protein aggregation indicator is ThT.

[0121] Soluble misfolded α-synuclein can be detected using a variety of different methods. These include the use of Western blot assays, ELISA, ThT binding assays, congo red binding assays, sedimentation assays, electron microscopy, atomic force microscopy, surface plasmon resonance, spectroscopy, etc. ELISA can include a sandwich ELISA on both sides. Spectroscopy can include one or more of quasi-elastic light scattering spectroscopy, multi-spectrum ultraviolet spectroscopy, confocal two-color fluorescence correlation spectroscopy, Fourier transform infrared spectroscopy, capillary electrophoresis with spectroscopic detection, electron spin resonance spectroscopy, nuclear magnetic resonance spectroscopy, fluorescence resonance energy transfer (FRET) spectroscopy, etc.

[0122] In some embodiments, detecting soluble misfolded α-synuclein includes contacting a detection mixture with a protease, such as proteinase K or thermolysin. Soluble misfolded α-synuclein can be detected using either a Western blot assay or ELISA, using sequence-based or anti-misfolded protein antibodies.

[0123] In some embodiments, determining the presence of soluble misfolded α-syn protein in a biological sample includes determining the amount of soluble misfolded α-syn protein in the biological sample. The amount of soluble misfolded α-syn protein in the sample can be determined in comparison to a control sample. The amount of soluble misfolded α-syn protein in the biological sample can be detected with at least about one or more sensitivities and specificities of 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%. The amount of soluble misfolded α-syn protein detected in the sample can be less than about one or more of 100 nmol, 10 nmol, 1 nmol, 100 pmol, 10 pmol, 1 pmol, 100 fmol, 10 fmol, 3 fmol, 1 fmol, 100 attomol, 10 attomol, and 1 attomol. The amount of soluble misfolded α-syn protein in the sample can be detected as a molar ratio to monomeric α-syn protein.

[0124] Soluble misfolded α-syn can be detected or measured by an analytical device, such as a kit, or a conventional laboratory device, which can be either portable or stationary. In some embodiments, the level of soluble misfolded α-syn can be compared to the level of a corresponding internal standard in one or more samples when performing an analysis to quantify the amount of soluble misfolded α-syn detected.

[0125] Once the presence and / or level of soluble misfolded α-syn is determined, it can be presented in a variety of ways. For example, the level can be graphically presented on a display as a numerical value or a bar (i.e., bar graph), or any other presentation method known to those of skill in the art. The graphical presentation can provide a visual representation of the amount of dispersion in the biological sample being evaluated.

[0126] Concentration of Soluble Misfolded α-syn In some embodiments, the method can include selectively enriching one or more soluble misfolded α-synuclein proteins of a sample, an incubation mixture, and a detection mixture. In some embodiments, the method further includes a step of enriching soluble misfolded α-synuclein in the sample before incubating the biological sample using an antibody that specifically binds to soluble misfolded α-syn. Selectively enriching soluble misfolded α-synuclein protein can include pretreating the biological sample before forming the incubation mixture. The step of selectively enriching soluble misfolded α-synuclein protein can include pretreating the incubation mixture before incubating the incubation mixture. The step of selectively enriching soluble misfolded α-synuclein protein can include contacting the incubation mixture with an antibody that specifically binds to soluble misfolded α-syn to form a captured soluble misfolded α-synuclein protein.

[0127] Antibodies are designed for specific binding as a result of the affinity of the antibody's complementarity determining regions for the epitope of the biological analyte. An antibody "specifically binds" if the antibody preferentially binds to the target structure or a subunit thereof, but binds to or does not bind to biological molecules that are not the target structure to a substantially lower extent. In some embodiments, the antibody specifically binds to soluble misfolded α-syn with a specific affinity of 10 -8 M to 10 -11 M. In some embodiments, the antibody or antibody fragment has a specificity greater than 10 -7 M, greater than 10 -8 M, greater than 10 -9 M, greater than 10 -10 M, or 10 -11 M, 10 -8 M - 10 -11 M, 10 -9 M - 10 -10 M, and 10 -10 M - 10 -11It binds to soluble misfolded α-syn with specific affinity for M. In one embodiment, the specific activity is measured using the competitive binding assay described in Ausubel FM (1994). Current Protocols in Molecular Biology Chichester: John Wiley and Sons ("Ausubel"), which is incorporated herein by reference.

[0128] Antibodies that specifically bind to soluble misfolded α-syn may include one or more of α / β-syn N-19; α-syn C-20-R; α-syn 211; α-syn Syn 204; α-syn 2B2D1; α-syn LB 509; α-syn SPM451; α-syn 3G282; α-syn 3H2897; α / β-syn Syn 202; α / β-syn 3B6; α / β / γ-syn FL-140, etc. One or more α-syn specific antibodies may include one or more of α / β-syn N-19; α-syn C-20-R; α-syn 211; α-syn Syn 204, etc. Such antibodies can be obtained as follows: α / β-syn N-19 (catalog number SC-7012, Santa Cruz Biotech, Dallas, Tex); α-syn C-20-R (SC-7011-R); α-syn 211 (SC-12767); α-syn Syn 204 (SC-32280); α-syn 2B2D1 (SC-53955); α-syn LB 509 (SC-58480); α-syn SPM451 (SC-52979); α-syn 3G282 (SC-69978); α-syn 3H(SC-69977); α / β-syn Syn 202 (SC-32281); α / β-syn 3B6 (SC-69699); and α / β / γ-syn FL-140 (SC-10717). One or more α-syn specific antibodies may include one or more of an antibody specific for the amino acid sequence of α-syn and an antibody specific for the conformation of the soluble misfolded α-syn protein. One or more α-syn specific antibodies may be bound to a solid phase.

[0129] The solid phase can include one or more of paramagnetic particles (e.g., iron oxide) and multiwell plates. For example, an ELISA plate can be coated with an antibody used to capture α-syn from a biological sample. The antibody-coated ELISA plate may be incubated with the biological sample, unbound material may be washed away, and a PMCA reaction may be performed on the concentrated sample. The antibody can also be bound to particles (e.g., Dynabeads). The particles can be incubated with the patient sample and used to separate the α-syn-antibody complex from the remaining portion of the biological sample.

[0130] In some embodiments, methods for concentrating soluble misfolded α-syn in a biological sample can include ultracentrifugation. Soluble misfolded α-syn can be pelleted from the biological sample using high-speed centrifugation. The resulting pellet is then resuspended in buffer and analyzed using any of the methods described in this application. The centrifugation step may use surfactants such as, for example, sarcosyl, Tween-20, Triton.

[0131] Method for diagnosing diseases associated with α-syn aggregation Another aspect provides a method of diagnosing a disease associated with α-synuclein aggregation in a subject (i.e., synucleinopathy). The method includes contacting a biological sample with monomeric α-synuclein protein and one or more beads having an average diameter of about 1 mm to about 5 mm, greater than 2.3 mm to about 5 mm, greater than 3 mm to about 5 mm, about 2.38 mm, or about 2.45 mm to form an incubation mixture; incubating the incubation mixture to form misfolded α-synuclein aggregates from monomeric α-synuclein protein and soluble misfolded α-synuclein of the biological sample; at least partially deaggregating the misfolded α-synuclein aggregates; repeating the incubating and deaggregating steps a sufficient number of times to amplify the soluble misfolded α-synuclein of the sample to obtain a detectable amount of misfolded α-synuclein aggregates; and determining whether a detectable amount of misfolded α-synuclein aggregates is present in the sample, wherein detection of misfolded α-synuclein aggregates indicates that the subject has a disease associated with α-synuclein aggregation. The method may include determining or diagnosing the presence of a disease associated with α-synuclein aggregation in the subject by comparing the amount of soluble misfolded α-synuclein protein in the biological sample to a predetermined threshold amount, wherein a level higher than the threshold amount results in a diagnosis of a disease associated with α-synuclein aggregation in the subject.

[0132] For example, one method for the diagnosis of PD involves obtaining a sample from a subject clinically suspected of having PD and subjecting the sample in triplicate to the PMCA assay described herein, along with healthy controls. A positive sample is considered to be one in which the ThT fluorescence at about 490 nm after excitation at about 440 nm exceeds 5,000 relative fluorescence units (RFU). If the ThT fluorescence exceeding 5,000 RFU is not shown in repeated tests or is shown only once, the subject is considered PD negative. If the ThT fluorescence exceeding 5,000 RFU is shown twice in repeated tests, the subject is considered PD indeterminate. In the case of an indeterminate result, an attempt may be made to retest the sample in triplicate to obtain a final result. If the ThT fluorescence exceeding 5,000 RFU is shown in all three of the repeated tests, the subject is considered PD positive. In the method described herein, in more than 90% of cases, a positive PD test correlates with a sample clinically suspected of having PD. In another example, one or more algorithms can be used to associate a positive PD test with a sample clinically suspected of having PD. The algorithms can be found in U.S. Provisional Patent Application Nos. 63 / 073,420 and 63 / 073,424, each of which is incorporated herein by reference in its entirety.

[0133] Protein misfolding disorders (PMDs) include Alzheimer's disease (AD), PD, type 2 diabetes, Huntington's disease, amyotrophic lateral sclerosis, systemic amyloidosis, prion diseases, and the like. PMDs also include diseases associated with α-syn aggregation. Mis-folded aggregates of different proteins can form and accumulate. The mis-folded aggregates can induce, among other effects, cellular dysfunction and tissue damage. In some embodiments, the disease associated with α-syn aggregation is PD, and in further embodiments, the disease associated with α-syn aggregation is LBD, MSA, or PAF.

[0134] The method may include diagnosing PD in a subject based on the detection of soluble misfolded α-synuclein protein in a biological sample. α-Synuclein misfolding and aggregation have been shown to be associated with the etiology of PD. Sahay et al., Curr Protein Pept Sci., 18(7):656-676 (2017). The diagnosis of PD may also include determining the level of soluble misfolded α-synuclein protein in a biological sample as compared to a control sample taken from a control subject, relative to that present in healthy subjects. The method may include determining or diagnosing the presence of a disease associated with α-synuclein aggregation in a subject according to the presence of soluble misfolded α-synuclein protein in a biological sample. The method may include determining or diagnosing the presence of PD, MSA, LBD, or PAF, either alone or in a subject, according to the presence of soluble misfolded α-synuclein protein in a biological sample.

[0135] In some embodiments, the sample may be taken from a subject who does not exhibit clinical signs of PD. In other embodiments, the biological sample may be taken from a subject who exhibits clinical signs of PD. The most recognizable symptom of PD is movement-related dysfunction. However, additional symptoms include autonomic dysfunction, neuropsychiatric disorders (changes in mood, cognition, behavior, or thinking), sensory dysfunction (particularly changes in smell), and sleep disorders.

[0136] In some aspects, the method includes treating a subject diagnosed with a disease associated with α-synuclein aggregation with an α-synuclein modulating therapy. Several novel therapies targeting α-synuclein homeostasis are currently under development through various mechanisms. α-synuclein modulating therapies can include, for example, inhibiting the production of α-synuclein which inhibits the aggregation of α-synuclein using appropriate inhibitors, active or passive immunotherapy approaches, and the like. Therapeutic approaches targeting α-synuclein homeostasis can include active immunization such as PD01A+ or PD03A+, or passive immunization such as PRX002, BIIB054, ATV:aSyn, ABBV-0805 or MEDI1341. α-synuclein modulating therapies can also include methods of stimulating the patient's immune response to remove α-synuclein aggregates. Using the methods described herein for detecting the presence of soluble misfolded α-synuclein, it is possible to determine which patients may be treated with an α-synuclein modulating therapy. Currently, PD is incurable, but various drugs such as levodopa, dopamine agonists and monoamine oxidase B inhibitors are useful for treating the motor symptoms of PD.

[0137] In some aspects, the method is used to monitor the treatment of synucleinopathy in a subject. Biological samples can be taken from a subject under α-synuclein modulating therapy at different times over a period of time. The method can include determining or diagnosing whether a subject responds to an α-synuclein modulating therapy due to changes in soluble misfolded α-synuclein protein over a period of time or is non-responsive to an α-synuclein modulating therapy due to the homeostasis of soluble misfolded α-synuclein protein over a period of time. The method can include treating a subject determined to respond to an α-synuclein modulating therapy with an α-synuclein modulating therapy.

[0138] Kit Another aspect provides a kit for determining the presence of soluble misfolded α-syn in a biological sample. The kit includes a known amount of monomeric α-syn protein; a known amount of a protein aggregation indicator; a container for incubating the incubation mixture; a buffer composition; one or more beads having an average diameter of about 1 mm to about 5 mm, greater than 2.3 mm to about 5 mm, greater than 3 mm to about 5 mm, about 2.38 mm, or about 2.45 mm; and instructions for guiding the user in performing the method for detecting soluble misfolded α-syn described herein. The kit should also include a package for holding the components of the kit.

[0139] The kit includes a package having one or more containers that hold the reagents as one or more separate compositions or, optionally, as a mixture that allows compatibility of the reagents. The kit may further include a buffer solution, a labeling agent, a control, and any other materials necessary to perform the detection of α-syn. The kit can also include a tool for obtaining a sample from a subject, such as a hypodermic needle for performing a lumbar puncture.

[0140] The kit can also include instructions for performing a method for inducing treatment of synucleinopathy in a subject using the kit. In some aspects, the synucleinopathy is PD. The instructions included in the kit can be attached to the packaging material or included as an insert. The instructions are typically in writing or printed, but are not limited thereto. Any medium capable of storing such instructions and communicating them to an end user is contemplated by the present disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic disks, tapes, cartridges, chips), optical media (e.g., CD-ROM), etc. As used herein, the term "instructions" can include the address of an Internet site that provides the instructions.

[0141] The instructions are to contact a biological sample with a known amount of monomeric α-syn protein and one or more beads in a container to form an incubation mixture; incubate the incubation mixture to form misfolded α-syn aggregates from the monomeric α-syn protein and soluble misfolded α-syn of the biological sample; disaggregate at least a portion of the misfolded α-syn aggregates; repeat the steps of incubation and disaggregation a sufficient number of times to amplify the soluble misfolded α-syn of the biological sample to obtain a detectable amount of misfolded α-syn aggregates; and contact the incubation mixture with a known amount of protein aggregation indicator to instruct the user to determine whether a detectable amount of misfolded α-syn aggregates is present in the biological sample. Detection of misfolded α-syn aggregates indicates the presence of soluble misfolded α-syn in the biological sample.

[0142] In various embodiments, the kit may include a known amount of monomeric folded α-syn protein and a known amount of indicator for misfolded α-syn protein. The kit may include one or more of a bead dispenser, a multi-well plate containing a plurality of wells, a microfluidic plate, a shaker, an incubator, and a fluorescence measurement device, included as one or more individual plates or devices, or as a combined device. For example, a shaking microplate reader can be used to perform cycles of incubation and shaking and automatically measure ThT fluorescence during the course of the experiment (e.g., FLUOstar OPTIMA, BMG LABTECH Inc., Cary, N.C.).

[0143] Examples are included to more clearly illustrate specific embodiments of the invention and their associated advantages. However, there are a wide variety of other aspects within the scope of the invention, and these should not be limited to the specific examples provided herein.

[0144] Example Example 1: Rapid α-syn PMCA (αS-PMCA) for Detecting α-syn Seeds in the CSF of PD Patients Examples were carried out to show conditions that facilitate the rapid detection of α-syn from samples obtained from a subject while avoiding or reducing the self-aggregation of monomeric proteins. The results of these examples are shown in FIGS. 2-9.

[0145] Unless otherwise specified, the αS-PMCA reaction conditions used to generate the data shown in FIGS. 2-9 were as follows: The PMCA mixture (100 mM PIPES-NaOH pH 6.5 [Sigma, catalog number 80635-50G], 500 mM NaCl [Lonza, catalog number 51202], 10 μM ThT [Sigma, catalog number T3516-25G], and 0.3 mg / ml (19.6 μM) of SEQ ID NO: 2 (rec-αS)) and 40 μL of CSF in a total volume of 160 μL. Si3N4 beads were used in the reaction to accelerate aggregation. The diameter of the beads was 2.38 mm.

[0146] The assay was assembled in a 96-well ELISA plate [Corning, catalog number 3916]. One bead was added to each well, and the plate was covered with a pierceable optical film [Excel Scientific, catalog number XP-100] to avoid cross-contamination while preparing the assay. After all the PMCA reactants were pipetted into the plate, the pierceable optical film was removed, and the plate was covered with a fluorescence-compatible film (Applied Biosystems, catalog number 4311971).

[0147] The plate was transferred to a FLUOstar (registered trademark) Omega microplate reader [BMG], a device that shakes, incubates, and automatically reads fluorescence. The plate was shaken orbitally at 800 rpm for 1 minute, read (440~10 excitation and 490~10 emission), and incubated at 37 °C. The plate was shaken for 1 minute every 29 minutes, which constituted a 30-minute αS-PMCA cycle. 200 cycles were performed for the high-speed assay containing beads (100 hours, about 4 days) and compared with 600 cycles (300 hours, about 12 days) of the low-speed assay without beads.

[0148] The beads used in the experiment were made from a very high-density pure Si3N4 material by uniform compression (3.25 g / cm 3 ). The beads are of high precision "grade 3", meaning they are pure Si3N4 ceramic balls that are exactly circular in sphericity to within 3 / 1,000,000 inches.

[0149] Abcam synthetic seeds aggregated very rapidly in PMCA, and they were used as αS aggregate models to evaluate the acceleration of seeded aggregation in aCSF. See Figures 2A - 2E. Seeds were added to 40 μL of aCSF to mimic CSF samples containing endogenous αS seeds (e.g., from human PD-CSF). Given the rapid aggregation of the synthetic seeds, a small acceleration in their aggregation indicates a substantial acceleration in human CSF carrying αS seeds.

[0150] Figure 2A shows a 1 mg / mL substrate without beads. The seeded aggregation of 20 fg of Abcam seeds started after 10 hours and there was no self-aggregation (the seed-free control showed no aggregation). Inclusion of beads larger than 0.4 mm in the PMCA reaction induced self-aggregation (Figures 2B, 2C, and 2D). The larger the beads, the faster the aggregation of Ab seeds (20 fg). The 0.4 mm beads did not seem to have an effect on promoting seeded aggregation (Figure 2A vs. Figure 2E). The 0.4 mm beads were very difficult to handle and were not used further, but they did not seem to have a harmful effect on PMCA (i.e., there was no self-aggregation).

[0151] Figure 3A shows a 1 mg / mL substrate without beads. The addition of 1.5 mm beads induced self-aggregation of the bead-free control (Figure 3B). To reduce self-aggregation, the concentration of rec-αS was decreased to 0.5 and 0.1 mg / mL. When 0.5 mg / ml and 1.5 mm beads were used (Figure 3C), there was a decrease in self-aggregation compared to 1 mg / ml, but aggregation still started 10 hours earlier at 20 fg. When 0.1 mg / ml was used (Figure 3D), there was no self-aggregation, but there was no acceleration of 20 fg aggregation either, or it was insufficient compared to the bead-free case (Figure 3A). Furthermore, when 0.1 mg / ml was used, the fluorescence difference between the positive and the control (about 50,000 RFU) (Figure 3D) was smaller than when 0.5 mg / ml was used (about 150,000 RFU) (Figure 3A).

[0152] To further accelerate the αS-PMCA assay, the effect of increasing the temperature to 42 °C was evaluated. When the beads were omitted (Figure 4A), self-aggregation started at 50 hours and seeded aggregation at 20 fg started at about 15 hours. After adding one 0.8 mm bead (Figure 4B), the 20 fg aggregation became faster, but self-aggregation was also accelerated to 40 hours. Self-aggregation was dramatically reduced by decreasing the substrate to 0.1 mg / ml and using larger beads (Figure 4C).

[0153] Previous results suggested that the effect on αS-PMCA becomes more significant as the beads increase in size. Therefore, amplification was evaluated using human control CSF as the matrix. For each of the seeded and seedless controls, 20 fg of Abcam αS seeds diluted in water were added to the CSF (Figure 5A). Under these conditions, beads of different sizes were evaluated at 37 °C. At 1 mg / ml substrate, only the 2.38 mm beads (Figure 5C) showed an acceleration of seeded aggregation compared to the beadless control (Figure 5A), while the 0.8 mm beads showed no effect (Figure 5B). Self-aggregation was reduced by the use of human CSF, as demonstrated by the fact that all seedless controls were flat (Figure 5A–Figure 5C). At 0.1 mg / ml, it was confirmed that larger beads accelerated seeded aggregation and increased the aggregation gradient (Figure 5D–Figure 5F). However, the signal was lower compared to 1 mg / ml.

[0154] Two different substrate concentrations (0.1 and 0.3 mg / ml) were used in combination with higher temperature, larger beads, and higher shaking. PMCA was performed using aCSF with Si3N4 beads at one bead per well and recombinant Abcam α-syn seeds at 42 °C, 800 rpm, 1 minute on and 29 minutes off. Seeded aggregation in aCSF was faster when using 0.3 mg / ml (Figure 6D–Figure 6F) than 0.1 mg / ml (Figure 6A–Figure 6C).

[0155] The experiment was repeated using 0.3 mg / ml substrate, 42 °C, 800 rpm, and 2.38 mm beads with aCSF. The 20 fg seeds were positive up to 10 hours when spiked into aCSF (Figure 7A), while the seedless control showed a slight increase in fluorescence after 40 hours. These αS-PMCA conditions were also evaluated using human control CSF. The 20 fg seeds began to aggregate at 10 hours, while the control CSF without seeds remained flat for almost 100 hours (Figure 7B).

[0156] The next step was to evaluate the human endogenous αS seeds in the PD CSF samples using Si3N4 beads of 1.5 mm, 2 mm, and 2.38 mm (Figure 8A). The PD samples aggregated with all three beads up to 60 hours. As a comparison, PD samples that aggregated faster under the low-speed assay conditions (defined below) took 100 - 150 hours, and the average of the PD samples aggregated in 150 - 250 hours (Figure 8C).

[0157] Self-aggregation was evaluated using different control CSF samples (Figure 8B). Similar results (Figure 7B) to the previous control CSF were obtained. The 2.38 mm beads showed a very low self-aggregation signal after 80 hours.

[0158] By returning the temperature to 37 °C, the control samples remained flat, while the PD samples were positive up to 50 hours (Figure 9A and Figure 9B). When the assay was performed at 42 °C, the time to reach 50% of the maximum aggregation (T50) of the PD CSF samples was 35 hours, while the T50 at 37 °C was 41 hours. Therefore, the delay in the aggregation of the PD-CSF samples caused by the elimination of self-aggregation by lowering the temperature was only 6 hours.

[0159] Example 2: Comparison of Results Obtained Using "Low-Speed" and "High-Speed" PMCA Experiments were conducted to compare the results obtained using low-speed (SA) and high-speed (FA) PMCA assays. For clarity, SA and FA are defined below.

Table 1

[0160] 139 non-specified CSF samples, six healthy control pooled CSF samples, and six PD pooled CSF samples were analyzed in parallel using the SA and FA αS-PMCA assays. For this direct comparison, the same substrate, the same consumable lot, and the same reagents were used. PMCA was performed in triplicate for both cases. There were 59 PD samples, of which FA detected 51 as positive and SA detected 36. Of the 60 control samples, SA identified 56 as negative and FA identified 52 as negative (57 using the algorithm described in US Provisional Patent Application No. 63 / 073,424).

[0161] Some of the samples showed variability with respect to T50 when using SA (Figure 10B), while the three replicate tests in FA appeared to be very consistent (Figure 10A). One sample that was barely positive with SA (Figure 11B) was quickly detected as positive with FA (Figure 11A). Furthermore, based on the signal and the complete curve, this sample can be more reliably identified as PD in FA than in SA. One PD sample that was counted as negative with SA (Figure 12B) was actually correctly identified as strongly positive with FA (Figure 12A). Such samples strongly suggest that FA has a lower detection limit or overall higher sensitivity compared to SA. Figure 13B shows the variability of SA, showing curves that do not reach a plateau or draw atypical aggregation curves. On the other hand, FA (Figure 13A) shows reproducible complete typical aggregation curves in all three replicate tests of the PD CSF analyzed.

[0162] Data analysis (139 samples) of the projects described in Figures 9-13 was obtained as shown in Table I. The FA algorithm is described in US Provisional Patent Application No. 63 / 073,424.

Table 2

[0163] Example 3: Fast α-syn PMCA (αS-PMCA) for α-syn seed detection using blocked beads

[0164] Assays are currently being developed that use a reasonable concentration of substrate to generate a complete aggregation curve to extract kinetic parameters, are faster than slower PMCA assays, and can be scaled up with respect to measurement and workflow. The method uses 0.3 mg / ml (19.6 μM) of recombinant α-S, runs for 150 hours, records the entire aggregation curve (complete gradient and plateau), and shakes for 1 minute every 30 minutes, which means that one instrument can run 8 - 9 plates at a time. An important feature is that it includes blocked 2.38 mm Si3N4 beads. The beads were initially obtained from BC Precision, a bead supplier. Beads of several sizes were compared, and it was found that the fastest aggregation occurred with 2.38 mm beads, as described in Example 1. Specifically, the beads were Grade 5, 3 / 32 inch / 2.38125 mm Si3N4 ceramic balls weighing 0.00081863 ounces / 0.0232 g. Forty-five CSF samples were evaluated using these 2.38 m Si3N4 beads from BC Precision (BC beads), and some runs showed expected results while some others did not (Table 1B, column 1). Beads with lot number B21 were used in most experiments, and it was found that almost 50% of the control samples showed self-aggregation and 16.7% of the samples were indeterminate. Another lot number B37 was used to evaluate a few samples, and the inappropriateness of these beads was confirmed. Of the three samples analyzed, two were positive and one was indeterminate (Table 1B, column 2). Without intending to be bound by theory, it is thought that the beads increase the shaking efficiency and accelerate the growth of α-S aggregates present in PD-CSF, so the PD samples aggregate faster. In the case of control donors, α-S aggregates are not present in the CSF, which means that the beads accelerate de novo seed nucleation. Since shaking accelerates the fragmentation of already formed seeds, de novo seed formation can be explained by the surface interaction between the beads and the reaction components. To solve the problem of self-aggregation, the surface of the beads was blocked using an aqueous solution of BSA, as described in Example 4.

[0165]

Table 1A

Table 1B

[0166] Example 4: BC beads blocked with BSA in water 133 samples were analyzed using BC beads blocked with BSA in water, reaching a sensitivity of 89.5% and a specificity of 94.3 (Table 1B, column 7). This represents a substantial improvement compared to unblocked B21 beads (Table 1B, column 4), with a 44.3% improvement in specificity. Reproducibility was evaluated by using other lots of the same beads (B33, B36, B37) under the same assay conditions. Since the main problem was self-aggregation, only control samples were analyzed. Blocked B37 (Table 1C, column 10) showed a substantial improvement compared to unblocked B37 beads (Table 1B, column 5). Blocked B3P BC beads showed very similar results (Table 1C, column 9). However, the performance of lot B33 was very low (Table 1C, column 8) because 66% of the control samples showed self-aggregation and were thus classified as either positive (false positive) or indeterminate.

[0167]

Table 1C

[0168] The beads were blocked with a BSA solution in PIPES (1% BSA in 100 mM PIPES, pH 6.50) (Table 1C; Table 1D). This new blocking procedure significantly reduced self-aggregation when using B37 beads (Table 1D, column 13). These B37 beads had already functioned to an acceptable level with blocking with BSA in water (Table 1C, column 10), but when blocked with BSA in PIPES, the number of indeterminate samples decreased from 11.1% to 0%. B33 beads did not function even when blocked with BSA in PIPES. This indicates that there are some unique characteristics of B33 that could not be immobilized by blocking.

[0169] The bead manufacturer, Camellia Nakashima (TN), manufactures high-quality Si3N4 beads with sufficient quality documentation and strict specifications. Grade 3 beads, which are rounder and smoother than BC precision's Grade 5 beads, were used. TN also specifies that the quality of the Si3N4 raw material complies with ASTM. Since BSA-PIPES blocking effectively reduced the self-aggregation of B37 (compared to no blocking or blocking in water), it was used to block TN beads for the αS-PMCA assay (Table 1D, column 14, Table 1E columns 15 - 17). B41, B46, and B47 are different purchases from the same manufacturing lot. 610 samples were analyzed, achieving high sensitivity and specificity, and the indeterminate results were only 8%. Two additional manufacturing lots, B44 and B45 - B48 - B49 (these three are three purchases from the same manufacturing lot), were evaluated. When only control samples were used for the evaluation, these two additional manufacturing lots did not show self-aggregation, consistent with B41 - B46 - B47.

Table 1D

Table 1E

[0170] Example 5: Comparison of the use of blocked Si3N4 beads and unblocked borosilicate glass beads The αS-PMCA reaction conditions used to generate the data shown in FIGS. 14A, 14B, 15A, and 15B were as follows: 160 μL of PMCA mixture (100 mM PIPES-NaOH pH 6.5 [Sigma, catalog number 80635-50G], 500 mM NaCl [Lonza, catalog number 51202], 10 μM ThT [Sigma, catalog number T3516-25G], and 0.3 mg / ml (19.6 μM) of SEQ ID NO: 2 (rec-αS), and 40 μL of CSF were included.

[0171] When using Si3N4 beads, the beads were blocked with 1% BSA in 100 mM PIPES buffer with pH 6.5 before contacting the beads with the incubation mixture. The blocked beads were washed twice with 100 mM PIPES with pH 6.5. The diameter of the beads was 2.38 mm.

[0172] When using borosilicate glass beads, the beads were not blocked. The diameter of the beads was 2.45 mm.

[0173] FIGS. 14A-14B show the results of an example using two different bead types for PD CSF samples (PD) and three controls. Thus, exemplary results using 2.38 mm Si3N4 beads blocked with BSA are shown. The PD sample was positive for about 75 hours, while all three controls were negative. FIG. 14B is a diagram showing the results of an example using unblocked 2.45 mm borosilicate glass beads. The PD sample was positive for about 170 hours, while all three controls were negative.

[0174] Figures 15A - 15B show the results of an example using two different types of beads for two PD CSF samples from different donors (PD1 and PD2). The graphs show the average of three replicates per patient. When using either blocked 2.38 mm Si3N4 beads (Figure 15A) or unblocked 2.45 mm borosilicate glass beads (Figure 15B), both PD samples were positive. With Si3N4 beads, PD2 started aggregating at about 40 hours and PD1 at about 50 hours. The aggregation curves were very reproducible and the plateaus showed very consistent fluorescence values. When using 2.45 mm borosilicate glass beads, aggregation was delayed for PD2 and PD1 at 10 hours and 25 hours, respectively.

[0175] Example 5: Addition of Sarkosyl to FA Conditions The αS - PMCA reaction conditions used to generate the data shown in Figures 24A and 24B were as follows: 160 μL of PMCA mixture (100 mM PIPES - NaOH pH 6.5 [Sigma, catalog number 80635 - 50G], 500 mM NaCl [Lonza, catalog number 51202], 10 μM ThT [Sigma, catalog number T3516 - 25G], and 0.3 mg / ml (19.6 μM) of SEQ ID NO: 2 (rec - αS), and 40 μL of CSF, were included with a 0.1% w / v sarkosyl concentration in 1X PBS.

[0176] Figure 24A shows the results of an example where a 0.1% w / v sarkosyl concentration in 1X PBS was included in an exemplary high - speed assay (using 2.38 mm Si3N4 beads blocked with BSA, one bead per well, and using recombinant α - syn seeds at 37°C, 800 rpm, 1 minute on and 29 minutes off). A) shows the fluorescence representing the aggregation of PMCA performed on CSF obtained from subjects with PD, and B) shows the results obtained using control CSF samples. Figure 24A showed positive results within one day and significant acceleration.

[0177] The complete disclosure of all patents, patent applications, and publications, and electronically available materials cited in this specification are incorporated by reference. The above detailed description and examples are given only to clarify the understanding. No unnecessary limitations should be understood therefrom. The present invention is not limited to the exact details shown and described, and variations obvious to those skilled in the art are included within the invention as defined by the claims.

Sequence Listing Free-Text

[0178] Sequence Listing 1-19 <223> Synthesis

Claims

1. A method for determining the presence of soluble misfolded α-synuclein (α-syn) protein in a biological sample, comprising: (A) contacting the biological sample with a pre-incubation mixture comprising: (1) monomeric α-syn protein; (2) a buffer composition; (3) a salt; and (4) an indicator to form an incubation mixture; (B) performing an incubation cycle on the incubation mixture, wherein the incubation cycle comprises: each incubation cycle comprising: (i) incubating the incubation mixture in the presence of the soluble misfolded α-syn protein for a time and under conditions effective to cause at least partial misfolding and / or aggregation of the monomeric α-syn protein; and (ii) physically disrupting the incubation mixture wherein: (1) the incubation mixture is subjected to two or more times the amount effective to form an amplified portion of misfolded α-syn protein from the monomeric α-syn protein; (2) Si including the coating of bovine serum albumin (BSA) ３ N ４ carried out in the presence of beads; and (C) determining whether a detectable amount of misfolded α-syn aggregates is present in the biological sample, wherein detection of misfolded α-syn aggregates indicates the presence of soluble misfolded α-syn protein in the biological sample.

2. said Si ３ N ４ The method according to claim 1, wherein the beads have a diameter of from about 1 mm to about 5 mm.

3. said Si ３ N ４ The method according to claim 1, wherein the beads have a diameter of about 2.38 mm.

4. The method of claim 1, wherein the biological sample comprises human cerebrospinal fluid (CSF).

5. The method of claim 1, wherein the monomeric α-syn protein is present at a concentration of about 10 μM to about 30 μM.

6. The method of claim 1, wherein the monomeric α-syn protein comprises SEQ ID NO:

2.

7. The method of claim 6, wherein the monomeric α-syn protein is present at a concentration of about 19.6 μM.

8. The method of claim 1, wherein the buffer composition has a pH of about 6.2 to about 6.

5.

9. The method of claim 1, wherein the buffer composition comprises PIPES.

10. The method of claim 1, wherein the salt comprises NaCl.

11. The method of claim 1, wherein the salt comprises NaCl at a concentration of about 500 mM to about 700 mM.

12. The method of claim 1, wherein the indicator comprises thioflavin T (ThT).

13. The method according to claim 12, wherein the detection comprises measuring ThT fluorescence.

14. The method according to claim 1, wherein the physical grinding comprises shaking.

15. A method for determining the presence of soluble misfolded α-syn protein in human CSF, comprising: (A) contacting the human CSF with a pre-incubation mixture comprising: (1) monomeric α-syn protein at a concentration of about 10 μM to about 30 μM; (2) a buffer composition having a pH of about 6.2 to about 6.5; (3) NaCl at a concentration of about 500 mM to about 700 mM; and (4) ThT to form an incubation mixture; (B) subjecting the incubation mixture to an incubation cycle, wherein the incubation cycle comprises: each incubation cycle comprising: (i) incubating an incubation mixture effective to cause at least partial misfolding and / or aggregation of the monomeric α-syn protein in the presence of the soluble misfolded α-syn protein; and (ii) shaking the incubation mixture and (1) two or more times with respect to the incubation mixture effective to form an amplified portion of misfolded α-syn protein from the monomeric α-syn protein; (2) Si having a diameter greater than 2.3 mm and including a coating of BVA ３ N ４ carried out in the presence of beads; and (C) determining whether a detectable amount of misfolded α-syn aggregates is present in the biological sample, wherein detection of the misfolded α-syn aggregates indicates the presence of soluble misfolded α-syn protein in the biological sample, and the detection comprises measuring ThT fluorescence.

16. The method according to claim 15, wherein the monomeric α-syn protein comprises SEQ ID NO:

2.

17. The method according to claim 16, wherein the monomeric α-syn protein is present at a concentration of about 19.6 μM.

18. A method for determining the presence of soluble misfolded α-syn protein in a biological sample, comprising: (A) contacting the biological sample with a pre-incubation mixture comprising: (1) monomeric α-syn protein comprising SEQ ID NO: 2; (2) a buffer composition; (3) a salt; and (4) an indicator to form an incubation mixture. (B) performing an incubation cycle on the incubation mixture, wherein the incubation cycle comprises: each incubation cycle comprising: (i) incubating the incubation mixture in the presence of the soluble misfolded α-syn protein for a time effective to cause at least partial misfolding and / or aggregation of the monomeric α-syn protein; and (ii) physically grinding the incubation mixture ; being performed more than once on the incubation mixture effective to form an amplified portion of misfolded α-syn protein from the monomeric α-syn protein; (2) being performed in the presence of borosilicate glass beads having a diameter greater than 2.3 mm; and (C) determining whether a detectable amount of misfolded α-syn aggregates are present in the biological sample, wherein detection of misfolded α-syn aggregates indicates the presence of soluble misfolded α-syn protein in the biological sample. **Claim 19** The method of claim 18, wherein the biological sample is human CSF. **Claim 20** (i) the monomeric α-S protein is present at a concentration of about 10 μM to about 30 μM; (ii) the buffer composition comprises PIPES and has a pH of about 6.2 to about 6.5; (iii) the salt comprises NaCl at a concentration of about 500 mM to about 700 mM; (iv) the borosilicate glass beads have a diameter of 2.45 mm; (v) the indicator comprises ThT at a concentration of about 5 μM to about 10 μM; and (vi) the detection comprises measuring ThT fluorescence The method of claim 18.

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