Inactive matrix for quantitative and semi-quantitative seed amplification assays

Abstract

An inert matrix is ​​provided for use with the α-synuclein seed amplification assay "aS-SAA." When used as a negative control, the inert matrix accurately reflects the absence of misfolded αS protein, with no, perceptibly low, or delayed forms of αS substrate self-aggregation, but when used as a positive control, it readily allows aggregation of αS substrate with the seeds. The inert matrix can be used to screen the eligibility of αS-SAA reagents. The inert matrix can be used to dilute samples taken from the surrounding biological matrix. Finally, the inert matrix can be used as a diluent for serial dilutions of αS-SAA samples to allow for a semi-quantitative version of αS-SAA.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the interests of U.S. Provisional Patent Application No. 63 / 243,470, filed on 13 September 2021, and U.S. Provisional Patent Application No. 63 / 328,443, filed on 7 April 2022. Each of these applications is incorporated herein by reference in its entirety.

[0002] Sequence List The sequence listing has been submitted electronically in XML format and is incorporated in its entirety herein by reference. The XML copy created on September 9, 2022, is named Amprion-sCSFQ_ST26.xml and is 3,705 bytes in size. [Background technology]

[0003] Seed amplification assays (“SAAs”) provide a highly sensitive and specific means for detecting biomarker misfolded protein aggregates in tissues and body fluids at titers too low for detection by conventional immunoassays. See Russo MJ, Orru CD, Concha-Marambio L et al., High diagnostic performance of independent alpha-synuclein seed amplification assays for detection of early Parkinson's disease [Amendments published in Acta Neuropathol Commun. November 26, 2021;9(1):190], which is incorporated herein by reference in its entirety. See Acta Neuropathol Commun. 2021;9(1):179. Published November 6, 2021. doi:10.1186 / s40478-021-01282-8. SAAs enable much earlier diagnosis of conditions involving misfolded proteins, which may prove important for disease treatment and prevention or mitigation of symptoms. For example, see Concha-Marambio L, Farris, Carly M et al., Seed amplification assay to diagnose early Parkinson's and predict dopaminergic deficit progression. Movement Disorders. 2021;36(10):2444. July 8, 2021. doi.org / 10.1002 / mds.2871, which is incorporated herein by reference in its entirety.

[0004] SAA relies on promoting the amplification of endogenous misfolded protein aggregates in a biological sample ("seed") while avoiding self-aggregation of the assay substrate by sacrificing the same type of monomeric protein used as the assay substrate. As a negative control, since there is no reliable commercially available synthetic control solution for this purpose, SAA generally uses a biological sample from a known healthy control ("HC") donor. The negative control is subjected to the same conditions as the biological sample of interest, including intermittent shaking and the addition of substrate, buffer, and fluorescent probe. If the negative control does not show agglutination and the biological sample does not show agglutination (i.e., no amplification of the seed), the patient is considered "negative" or agglutination is considered "not detected". If the negative control does not show agglutination and the biological sample does, the patient is considered "positive" or agglutination is considered "detected". If the negative control shows agglutination, a positive negative control indicates an assay problem related to either handling, reagent stability, or consumable quality, and the result should be discarded and the assay repeated. Therefore, the negative control must neither induce nor tolerate substrate autoaggregation.

[0005] As a positive control, SAA generally uses a biological sample from a patient with a confirmed diagnosis of the associated protein misfolding disorder, or a biological sample "spiked" with a known amount of synthetic misfold protein aggregates of the same type as the biological biomarker ("synthetic seed"). Here again, human samples are used because there are no commercially available synthetic control solutions that can maintain the amplification of the synthetic seed while suppressing substrate autoaggregation. The positive control is subjected to the same conditions as the biological sample. If the positive control shows agglutination, the assay conditions are suitable for seed amplification, meaning that the reagents (including the substrate), consumables, and handling were of the necessary quality to detect the biomarker. If the positive control does not show agglutination, the lack of agglutination in the positive control indicates a problem with the assay regarding either handling, reagent stability, or consumable quality, and the results should be discarded and the assay repeated.

[0006] Recent studies have shown that cerebrospinal fluid ("CSF") closely reflects brain material in terms of the presence of misfolded proteins. See, for example, Shahnawaz, M., Mukherjee, A., and Pritzkow, S. et al., Discriminating α-synuclein strains in Parkinson's disease and multiple system atrophy. Nature 578, 273-277 (2020). Therefore, the detection of misfolded proteins in CSF is of particular importance. Unfortunately, compositions used as inactive or neutral assay matrices for positive and negative controls in the context of studies too frequently induce or allow substrate autoaggregation (in the negative control) or inhibit aggregation in the presence of seed, endogenous or synthetic (in the positive control).

[0007] Furthermore, it is impossible to rely on human CSF from healthy donors as a reproducible matrix for the clinical use of SAA for diagnostic purposes. Firstly, CSF is a highly complex biofluid that exhibits dramatic differences between individual donors. These differences are greater when comparing healthy patients with diseased patients, but patients with similar health conditions may have very different CSF compositions. In some cases, CSF samples from healthy donors may induce autoaggregation, while in other cases, CSF samples from healthy donors may partially inhibit seed aggregation. Moreover, as a practical matter, healthy human CSF samples are not available in unlimited supply.

[0008] Therefore, an inert matrix is ​​required for use as a control solution that can be used as is, is readily available and abundantly supplied, and when used as a negative control, accurately reflects the absence of misfolded proteins in a perceptually low or delayed form without substrate auto-aggregation, but when used as a positive control, it readily allows substrate aggregation with the seed.

[0009] Alpha-synuclein ("αS")-SAA of CSF is the gold standard for detecting misfolded αS aggregates, but obtaining CSF requires an invasive lumbar puncture, also known as spinal puncture, to extract a CSF sample from the subarachnoid space of the spine. While αS-SAAs of peripheral matrices (e.g., blood, saliva, skin, and olfactory mucosa) are known (see, for example, U.S. Patents 10,989,718, 11,079,396, and 11,099,197, respectively, which are incorporated herein by reference in their entirety), there is still a need for a reliable inert matrix to be used as a diluent in the sampling of such peripheral matrices. See U.S. Provisional Patent Application No. 63 / 375,126, filed 9 September 2022, which is incorporated herein by reference in its entirety.

[0010] One of the most challenging aspects of SAA is identifying eligible substrates, i.e., substrates that do not self-aggregate but aggregate in the presence of seeds in the biological sample environment. Therefore, there is also a need for an inert matrix to be used as a control solution that can be used to screen the eligibility of substrates (and other reagents and consumables used in SAA).

[0011] Finally, the quantification of biomarkers remains a challenge. Quantitative assays measure the concentration (M or mg / mL) of a given analyte using a standard. See, for example, U.S. Patent No. 10,215,763 and U.S. Patent Application Publication No. 20190137515 ("Prion References"), which are incorporated herein by whole reference. While αS-SAA is quantitative under ideal conditions (synthetic seeds in buffer) (see, for example, Shahnawaz, M. et al., Development of a Biochemical Diagnosis of Parkinson Disease by Detection of α-Synuclein Misfolded Aggregates in Cerebrospinal Fluid. JAMA Neurol. 74, 163 (2017); and Groveman, BR et al., Rapid and ultra-sensitive quantitation of disease-associated α-synuclein seeds in brain and cerebrospinal fluid by αSyn RT-QuIC. Acta Neuropathol. Commun. 6, 7 (2018)), the quantification described in prion references is not feasible in the context of αS-SAA in biological samples for at least two reasons. Firstly, there is no concentration standard with the same aggregation activity as endogenous αS aggregates; synthetic seeds aggregate much faster, leading to a significant underestimation of endogenous αS aggregates. Furthermore, since different species may possess different seeding activities, the concentration standard should be consistent with the molecular weight distribution of αS aggregates. Therefore, synthetic seeds cannot be accurately used as absolute concentration standards. Secondly, body fluids are complex matrices, and the dynamics of aggregation differ from patient to patient. For example, patients with the same concentration of αS in their CSF may exhibit different aggregation patterns due to differences in their respective CSF matrices. Therefore, an inert matrix is ​​needed to be used as a diluent for serial dilution of SAA samples to enable a semi-quantitative version of SAA that estimates relative αS aggregate levels and allows for comparison and ranking between biological samples. [Overview of the project]

[0012] In one embodiment, an inert matrix for use in αS-SAA is provided. In one embodiment, the inert matrix (hereinafter sometimes referred to as synthetic CSF or "sCSF") comprises (A) a plasma protein comprising (1) human serum albumin ("HSA"), or (2) a plasma protein selected from the group consisting of bovine serum albumin ("BSA"), BSA precursor protein, transferrin, and immunoglobulin G, and combinations thereof; and (B) an aqueous solution of physiological salts containing NaCl. If the plasma protein comprises HSA, the sCSF may further comprise a washing agent, e.g., sarcosyl, and a buffer composition, e.g., (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) ("HEPES"). In one embodiment, HEPES maintains the pH of the sCSF containing HSA at about 7.5.

[0013] In one embodiment, a method is provided for using sCSF as a negative control in αS-SAA, comprising: (I) preparing sCSF as disclosed herein; (II) preparing a pre-incubation mixture comprising (A) monomeric αS substrate; (B) buffer composition; (C) salt composition; (D) fluorescent protein aggregation indicator; and optionally (E) beads; (III) combining the sCSF and the pre-incubation mixture to form an incubation mixture; (IV) incubating the incubation mixture using intermittent stirring cycles to form an incubated mixture; (V) irradiating the incubated mixture with light of a wavelength sufficient to excite the fluorescent protein aggregation indicator if the fluorescent protein aggregation indicator is bound to protein aggregates; (VI) determining a first fluorescence intensity; and (VII) comparing the first fluorescence intensity with a predetermined second fluorescence intensity, wherein a second fluorescence intensity greater than the first fluorescence intensity indicates the absence of a perceptible amount of self-aggregating αS substrate in the sCSF.

[0014] In one embodiment, an sCSF is provided that is suitable for determining the qualification of one or more reagents and consumables in αS-SAA, including the qualification of αS monomer protein as an αS-SAA substrate and / or the qualification of an SAA buffer composition. In one embodiment, as shown in Figure 1, without using beads, the method comprises the steps of: (A) preparing an incubation mixture comprising (1) monomeric αS protein; (2) buffer composition; (3) salt composition; (4) fluorescent protein aggregation indicator; (5) sCSF as disclosed herein; and optionally (6) beads; (B) incubating the incubation mixture using intermittent stirring cycles to form an incubated mixture; (C) irradiating the incubated mixture with light of a wavelength sufficient to excite the fluorescent protein aggregation indicator if the fluorescent protein aggregation indicator is bound to the protein aggregates; and (D) determining the fluorescence intensity during incubation, wherein no significant increase in fluorescence indicates no self-aggregation of monomeric αS protein, and the absence of self-aggregation of monomeric αS protein indicates the suitability of the αS monomeric protein as an αS-SAA substrate and the suitability of the buffer composition.

[0015] In one embodiment, an sCSF is provided that is suitable for determining the qualification of one or more reagents and consumables in αS-SAA, including the qualification of αS monomer protein as an αS-SAA substrate and / or the qualification of an SAA buffer composition. In one embodiment, as shown in Figure 2, without the use of beads, the method comprises: (A) preparing recombinant synthetic αS in sCSF disclosed herein; (B) preparing a pre-incubation mixture comprising (1) monomeric αS protein; (2) buffer composition; (3) salt composition; (4) fluorescent protein aggregation indicator; and optionally (5) beads; (C) combining the recombinant synthetic αS seed and the pre-incubation mixture to form an incubation mixture; (D) incubating the incubation mixture using intermittent stirring cycles to form an incubated mixture; (E) irradiating the incubated mixture with light of a wavelength sufficient to excite the fluorescent protein aggregation indicator if the fluorescent protein aggregation indicator is bound to the protein aggregates; and (F) determining the fluorescence intensity during incubation, wherein a significant increase in fluorescence indicates amplification of the synthetic seed at the expense of monomeric αS protein, which indicates the suitability of the αS monomeric protein as an αS-SAA substrate and the suitability of the buffer composition.

[0016] In one embodiment, an sCSF suitable as a diluent in a semi-quantitative method for detecting the presence of misfolded αS aggregates is provided. In one embodiment, the method includes (A) the steps of (1) preparing a first human biological sample and (2) a second human biological sample; (B) preparing a pre-incubation mixture as described herein; (C) serially diluting the first biological sample by (1) taking a portion of the first biological sample and diluting the portion taken with a certain volume of sCSF disclosed herein to form a first biological sample; and (2) serially diluting the second biological sample by taking a portion of the second biological sample and diluting the portion taken with a certain volume of sCSF to form a second biological sample; (D) repeating step (C) a predetermined number of times using the sequentially diluted first biological sample and the sequentially diluted second biological sample; and (E) subjecting each of the sequentially diluted first biological sample and the second biological sample to αS-SAA.

[0017] In another embodiment, a semi-quantitative method for detecting the presence of misfolded αS aggregates in multiple human biological samples, comprising the steps of: (A) (1) preparing a first human biological sample divided into at least two reaction vessels to form individual first human biological sample aliquots; (2) preparing a second human biological sample divided into at least two reaction vessels to form individual second human biological sample aliquots; and (3) preparing a third human biological sample divided into at least two reaction vessels to form individual third human biological sample aliquots; (B) preparing a pre-incubation mixture comprising (1) monomeric αS protein; (2) buffer composition; (3) salt composition; and (4) indicator containing fluorophores; and (C) (1) combining a first aliquot of the first biological sample and the pre-incubation mixture in one of the first multiple reaction vessels to form a first baseline incubation mixture; (2) In one of the second set of reaction vessels, combine a first aliquot of the second biological sample with a pre-incubation mixture to form a second baseline incubation mixture; and (3) In one of the third set of reaction vessels, combine a first aliquot of the third biological sample with a pre-incubation mixture to form a third baseline incubation mixture; (D) Incubate the baseline incubation mixture using intermittent stirring cycles to form a baseline incubated mixture; (E) Irradiate the baseline incubated mixture with light of a wavelength that excites fluorophores; (F) Determine the fluorescence level during incubation, where an increase in fluorescence level indicates the presence of αS aggregates in each biological sample, and the time required for the fluorescence level to reach half of the maximum fluorescence of each biological sample (baseline T 50Determining step; (G) (1) A first aliquot of a first biological sample is spiked with a certain amount of exogenous αS seeds to form a spiked first biological sample; (2) A second aliquot of a second biological sample is spiked with an equal amount of exogenous αS seeds to form a spiked second biological sample; (3) A second aliquot of a third biological sample is spiked with an equal amount of exogenous αS seeds to form a spiked third biological sample; (4) Steps (C) to (F) are repeated for the spiked biological samples; and (5) The following formula: (a) φ (first biological sample) = baseline T 50( (first biological sample) / spike T 50( (first biological sample); (b) φ (second biological sample) = baseline T 50( (second biological sample) / spike T 50( (second biological sample); and (c) φ (third biological sample) = baseline T 50( (third biological sample) / spike T 50( (third biological sample), and by determining the normalized activity coefficient φ of each biological sample, baseline T 50 Normalizing step; (H) A method is provided that includes comparing the normalized activity coefficient for each biological sample with a clinically estimated assessment on a disease progression scale.

[0018] The present invention can be more easily understood by referring to the following figures.

Brief Description of the Drawings

[0019] [Figure 1] [[ID=二十六]]Figure showing an exemplary depiction of "low speed" (i.e., without using beads) αS-SAA in the determination of the suitability of sCSF as a negative control and / or of monomeric αS protein as an αS substrate (specifically, for determining whether the monomeric αS protein has a tendency to self-aggregate).

[0020] [Figure 2]Figure showing an exemplary depiction of low-speed αS-SAA using sCSF containing recombinant synthetic αS seeds in the determination of the suitability of monomeric αS protein as an αS substrate (specifically, to determine whether the monomeric αS protein can aggregate with αS seeds).

[0021] [Figure 3] Figure showing a graph of the fluorescence intensity over time for low-speed αS-SAA of an αS substrate using Harvard Apparatus artificial CSF + 0.155 mg / mL BSA + 0.042 mg / mL transferrin as a negative control solution.

[0022] [Figure 4] Figure showing a graph of the fluorescence intensity over time for low-speed αS-SAA of an αS substrate using Harvard Apparatus artificial CSF + 0.155 mg / mL BSA + 0.042 mg / mL transferrin as a negative control solution.

[0023] [Figure 5] Figure showing a graph of the fluorescence intensity over time for low-speed αS-SAA of an αS substrate using Harvard Apparatus artificial CSF + 0.2015 mg / mL BSA as a negative control solution.

[0024] [Figure 6] Figure showing a graph of the fluorescence intensity over time for low-speed αS-SAA of an αS substrate using 20 fg of synthetic seeds in Harvard Apparatus artificial CSF + 0.155 mg / mL BSA + 0.042 mg / mL transferrin as a positive control solution.

[0025] [Figure 7] Figure showing a graph of the fluorescence intensity over time for low-speed αS-SAA of an αS substrate using 20 fg of synthetic seeds in Harvard Apparatus artificial CSF + 0.2015 mg / mL BSA as a positive control solution.

[0026] [Figure 8] This figure shows a graph of the time-dependent fluorescence intensity for "fast" (i.e., using beads) αS-SAA of eligible αS substrates using 100 mM HEPES, pH 7.5, 75 mM NaCl, 1.5 mg / mL has and 0.5% sarcosyl as negative control solutions.

[0027] [Figure 9] This figure shows a graph of the time-dependent fluorescence intensity for fast αS-SAA of ineligible αS substrates using 100 mM HEPES, pH 7.5, 75 mM NaCl, 1.5 mg / hasHSA, and 0.5% sarcosyl as negative control solutions.

[0028] [Figure 10] This figure shows a graph of the time-dependent fluorescence intensity of fast αS-SAA for αS substrates, using 100 mM HEPES, pH 7.5, 75 mM NaCl, 1.5 has mL HSA, and 20 fg of synthetic seed in 0.5% sarcosyl as a positive control solution.

[0029] [Figure 11] This figure shows a graph illustrating the variability of aggregation inhibition against αS-SAA when using CSF derived from different healthy control (HC) donors.

[0030] [Figure 12] This figure shows a series of graphs of the fluorescence intensity over time of PD-positive samples under rapid αS-SAA conditions at serial dilution levels using 100 mM HEPES, pH 7.5, 75 mM NaCl, 1.5 mg / mL HSA, and 0.5% sarcosyl as diluents, compared to HC-CSF and NPH-CSF as diluents.

[0031] [Figure 13]This figure shows a series of graphs of the fluorescence intensity over time of PD-positive samples under alternative rapid αS-SAA conditions at serial dilution levels using 100 mM HEPES, pH 7.5, 75 mM NaCl, and 1.5 mg / mL HSA as diluents.

[0032] [Figure 14] This figure shows a chart comparing the SD50 values ​​of two CSF ​​samples under fast αS-SAA or alternative fast αS-SAA conditions in NPH-CSF or sCSF as an inert matrix.

[0033] [Figure 15] This figure shows a graph of normalized seeding coefficients (φ) versus Horn-Yahr (H&Y) scores for three samples from patients diagnosed with PD.

[0034] [Figure 16] This figure shows the αS-SAA aggregation curve using the monomeric αS substrate corresponding to Sequence ID No. 2, in the presence of olfactory mucosal samples from patients diagnosed with PD, compared to a non-synucleinopathy control. The olfactory mucosal samples were diluted with sCSF provided herein before αS-SAA. [Modes for carrying out the invention]

[0035] An inert matrix or sCSF for AS SAA is provided. When used as a negative control, sCSF accurately reflects the absence of misfolded proteins in a form that is not substrate auto-aggregating, is perceptually low, or is delayed; however, when used as a positive control, it readily allows substrate aggregation with the seed. sCSF can be used to screen the qualification of substrates and other reagents. cCSF can be used as a diluent for pretreatment of peripheral matrices. Finally, sCSF can be used as a diluent for serial dilution of SAA samples to enable a semi-quantitative version of SAA.

[0036] definition The term "approximately" when used with a number is intended to include ±10% of that number. This is true whether "approximately" modifies a number independently or modifies a number at either or both ends of a range. In other words, "approximately 10" means 9 to 11. Similarly, "approximately 10 to approximately 20" intends 9 to 22 and 11 to 18. If there is no explicit indication of the term "approximately" or range (e.g., ±10%), the exact number is intended. In other words, "10" means 10.

[0037] The singular forms "a," "an," and "the" refer to multiple objects unless explicitly indicated otherwise in the context. Therefore, for example, a reference to "a bead" refers to multiple beads.

[0038] If a range of values ​​is provided, it includes (unless the context clearly indicates otherwise) the intervening values ​​between the upper and lower limits of that range, up to one-tenth of the lower limit unit, and any other stated values ​​or intervening values ​​within the range of those stated values. The upper and lower limits of these smaller ranges may be independently included in smaller ranges and may also be included, subject to any specifically excluded limits within the stated range. If a stated range includes one or both limits, it also includes the range excluding one or both of the included limits.

[0039] "Misfolded αS aggregates" or "αS aggregates" refer to aggregates of misfolded αS proteins. Aggregates are sometimes called oligomers or polymers, and aggregation is sometimes called oligomerization or polymerization.

[0040] A "misfolded αS protein" is an αS protein that lacks all or part of its structural conformation, as is typical for its non-pathogenic normal function within a biological system. Misfolded αS proteins can aggregate. Misfolded αS proteins can localize to protein aggregates. Misfolded αS proteins can be non-functional proteins. Misfolded αS proteins can be pathogenic conformers of proteins.

[0041] As used herein, “soluble” species, including soluble misfolded αS aggregates, may form solutions in body fluids under physiological conditions, while “insoluble” species may exist in such body fluids as precipitates, protofibrils, deposits, entanglements, or other insoluble forms. Species that dissolve in non-body fluids under physiological conditions but do not dissolve in body fluids are considered insoluble. For example, protofibrils such as αS may dissolve in aqueous solutions of surfactants such as sodium dodecyl sulfate (SDS), but may still be insoluble in body fluids under physiological conditions and are therefore considered insoluble.

[0042] Nucleation-dependent aggregation may be characterized by a slow “delay phase” in which aggregation nuclei form, stimulating the rapid formation of further and / or larger aggregates. The delay phase may be minimized or eliminated by the addition of pre-formed “nuclei” or “seeds.” “Seeds” or “nuclei” refer to misfolded αS proteins or short, fragmented fibrils that have the ability to induce further aggregation.

[0043] Misfold αS protein aggregates can "deaggregate," i.e., break down or destroy, releasing smaller fragments and aggregates, such as fragmented protofibrils and smaller misfold αS aggregates. The catalytic activity of collecting misfold αS aggregate seeds may be at least partially proportional to the number of seeds in the mixture. Therefore, destroying misfold αS aggregates to release smaller misfold αS aggregates and fragmented protofibrils as seeds may result in increased catalytic activity for further aggregation.

[0044] The terms “monomer αS protein” and “monomer αS substrate” are used interchangeably and refer to αS protein molecules that do not contain one or more seeds of their natural, non-pathogenic composition, and which do not possess catalytic activity for seed-related aggregation.

[0045] When referring to the term "each," it does not mean "all, without exception." For example, if incubation cycles are mentioned and it is said that "each incubation cycle" includes a certain step, then if there are 10 incubation cycles and one of them includes that particular step, then that incubation cycle is intended to satisfy the restriction.

[0046] The transitional phrase "consisting essentially of" limits the scope of the claims to the specified materials or steps and "that do not substantially affect the basic and novel features of the claimed invention." MPEP §2111.03(III).

[0047] Unless otherwise defined, all technical and scientific terms have the same meaning as they are generally understood by those skilled in the art to which this invention pertains.

[0048] sCSF In one embodiment, sCSF may contain an aqueous solution of physiological salts and plasma proteins.

[0049] A physiological saline solution can be designed to mimic physiological CSF. The physiological saline solution can include an aqueous solution containing salts corresponding to at least one of the salts present in human CSF. In some embodiments, the physiological saline solution includes one or more of sodium, potassium, chloride, calcium, magnesium, and phosphate ions. The salts in the physiological saline solution can be provided at concentrations similar to those found in human CSF. For example, the physiological saline solution can include 130-160 mM of NaCl; 2.7-3.9 mM of KCl; 1-10 mM of CaCl2 . 2H2O; 0.5-10 mM of MgCl2 . 6H2O; 0.5-5 mM of Na2HPO4 . 7H2O; and 0.1-2 mM of NaH2PO4 . H2O. In some embodiments, the physiological saline solution consists of 148 mM of NaCl; 3 mM of KCl; 1.4 mM of CaCl2 . 2H2O; 0.8 mM of MgCl2 . 6H2O; 0.8 mM of Na2HPO4 . 7H2O; and 0.2 mM of NaH2PO4 . H2O. For example, a physiological saline solution can include about 150 mM of Na, about 3 mM of K, about 1.4 mM of Ca, about 0.8 mM of Mg, about 1.0 mM of P, and about 155 mM of Cl. In some embodiments, sCSF further includes 20-25 mM of sodium carbonate and / or 0.2-1.5 mM of glucose or sucrose. In some embodiments, sCSF further includes 0.2-1.0 mg / mL of sucrose. In some embodiments, the physiological saline solution is optional. In some embodiments, the physiological saline solution consists essentially of an NaCl solution having a concentration of up to about (i.e., within ±10%) 150 mM of NaCl, including up to about 75 mM of NaCl.

[0050] Methods for preparing physiological saline aqueous solutions are known in the art, and the components of physiological saline aqueous solutions are commercially available. For example, in some embodiments, the physiological saline aqueous solution includes Harvard Apparatus artificial CSF, which is commercially available from Harvard Apparatus, Holliston, Massachusetts. In some embodiments, the physiological saline aqueous solution includes perfusion fluid, which is commercially available from M Dialysis Inc. In some independent embodiments, Harvard Apparatus artificial and / or perfusion fluid is specifically excluded from use in the present invention.

[0051] In some embodiments, sCSF may be a buffered solution. In one embodiment, the buffer contains HEPES. In some embodiments, the buffer contains HEPES between 1 mM and 1 M, including about 50 mM HEPES, about 100 mM HEPES, about 150 mM HEPES, about 200 mM HEPES, about 250 mM HEPES, about 500 mM HEPES, about 1 M HEPES, or any value or range between any two of these concentrations.

[0052] sCSF may have a pH less than approximately 8, approximately 7.5, approximately 5-8, approximately 5.5-7.5, approximately 6-7.5, approximately 6-7, approximately 6.5, or any value or range between any two of those pH values.

[0053] In one embodiment, sCSF is a buffered solution, the buffer contains HEPES, and the pH is approximately 7.5.

[0054] sCSF also contains one or more plasma proteins. Plasma proteins are proteins normally found in plasma. Human CSF contains some of the proteins found in plasma, albeit at much lower concentrations. Human CSF contains approximately 0.3% plasma protein or approximately 15–40 mg / dL of total plasma protein. Therefore, in some embodiments, total plasma protein, which represents the sum of various plasma proteins in the solution, has a concentration in the range of 0.01 mg / mL to 15 mg / mL.

[0055] Examples of plasma proteins include albumin (e.g., HSA and BSA), fibrinogen, albumin precursor protein (e.g., BSA precursor protein), transthyretin, gamma globulin (e.g., immunoglobulin G), apolipoproteins (ApoA1, ApoE, ApoJ, ApoD, etc.), lipoproteins (e.g., high-density or low-density lipoproteins), complement proteins, prothrombin, and transferrin. In some embodiments, the plasma proteins are selected from the group consisting of HSA, BSA, BSA precursor protein, transferrin, and immunoglobulin G, as well as combinations thereof.

[0056] In some embodiments, the plasma protein consists of or is essentially composed of 0.1–0.3 mg / mL of BSA. In other embodiments, the plasma protein consists of or is essentially composed of 0.1–0.2 mg / mL of BSA and 0.02–0.06 mg / mL of transferrin. In further embodiments, the plasma protein consists of or is essentially composed of 0.4–0.5 mg / mL of BSA, 0.01–0.03 mg / mL of BSA precursor protein, and 0.005–0.02 mg / mL of immunoglobulin G, and the sCSF also includes 0.2–1.0 mg / mL of sucrose.

[0057] In some embodiments, plasma proteins are present in concentrations of 0.01 mg / mL to 1.5 mg / mL, 0.02 mg / mL to 0.8 mg / mL, 0.02 mg / mL to 0.4 mg / mL, 0.05 mg / mL to 0.4 mg / mL, approximately 1.5 mg / mL, approximately 2.0 mg / mL, approximately 2.5 mg / mL, approximately 3.0 mg / mL, approximately 3.5 mg / mL, approximately 4.0 mg / mL, approximately 4.5 mg / mL, approximately 5.0 mg / mL, approximately 5.5 mg / mL, approximately 6.0 mg / mL, approximately 6.5 mg / mL, approximately 7.0 mg / mL, approximately 7.5 mg / mL, and approximately 8.0 mg Consists of or essentially consists of HSA concentrations from 0.01 mg / mL to 15 mg / mL, including approximately 8.5 mg / mL, approximately 9.0 mg / mL, approximately 9.5 mg / mL, approximately 10.0 mg / mL, approximately 10.5 mg / mL, approximately 11.0 mg / mL, approximately 11.5 mg / mL, approximately 12.0 mg / mL, approximately 12.5 mg / mL, approximately 13.0 mg / mL, approximately 13.5 mg / mL, approximately 14.0 mg / mL, approximately 14.5 mg / mL, approximately 15.0 mg / mL, or any value or range between any two of these concentrations.

[0058] In some embodiments, the sCSF comprises a detergent or surfactant. In one embodiment, the detergent is sodium lauroyl sarcosinate, also known as sarcosyl. In another embodiment, the detergent is sodium dodecyl sulfate. In another embodiment, the detergent is about 0.1% sarcosyl, about 0.2% sarcosyl, about 0.3% sarcosyl, about 0.4% sarcosyl, about 0.5% sarcosyl, about 0.6% sarcosyl, about 0.7% sarcosyl, about 0.8% sarcosyl, about 0.9% sarcosyl, about 1.0% sarcosyl, or any value or range between any two of these concentrations.

[0059] In one embodiment, particularly when acting as a control solution for a slow-acting αS-SAA, as disclosed in whole in U.S. Patent No. 10,989,718, which is incorporated herein by reference, the sCSF essentially consists of Harvard Apparatus artificial CSF, 0.155 mg / mL of BSA, and 0.042 mg / mL of transferrin. Approximately 0.155 mg / mL of BSA is considered to be the physiological concentration (or "1X") of BSA in human CSF. Approximately 0.042 mg / mL of transferrin is considered to be three times ("3X") the physiological concentration (or "3X") of transferrin in human CSF. In another embodiment, the sCSF essentially consists of Harvard Apparatus artificial CSF and 0.2015 mg / mL of BSA (or "1.3X" of the physiological concentration (or "1.3X") of BSA in human CSF.

[0060] In certain embodiments, particularly when acting as a control solution for a fast αS-SAA, as disclosed in U.S. Patent No. 11,079,396 and U.S. Provisional Patent Application No. 63 / 375,126, each of which is incorporated herein by reference in whole, the sCSF may essentially consist of a HEPES, HSA, and NaCl solution (in these embodiments of U.S. Patent No. 11,079,396 and U.S. Provisional Patent Application No. 63 / 375,126, the assay includes sarcosyl). In one embodiment where the assay does not include sarcosyl, the sCSF essentially consists of a HEPES, NaCl solution, HSA, and sarcosyl. In one embodiment, the sCSF essentially consists of 100 mM HEPES, pH 7.5, 75 mM NaCl, HSA, and 0.5% sarcosyl.

[0061] In some embodiments, sCSF is used as a positive control component. The positive control can be used to obtain the expected result (i.e., a positive result) if αS aggregates are present in the biological sample. The positive control further includes seed, whether endogenous or synthetic. In some embodiments, sCSF is a negative control component. The negative control can be used to obtain the expected result (i.e., a negative result) if αS aggregates are not present in the biological sample. The negative control further includes all components of the monomeric αS substrate and pre-incubation mixture. In further embodiments, sCSF is a comparative control containing a known amount of seed. The comparative control can be used as a benchmark to determine the amount of αS aggregates formed using αS-SAA. In further embodiments, sCSF may be used as a diluent in semi-quantitative αS-SAA.

[0062] In one embodiment, sCSF may contain multiple system atrophy ("MSA") or PD or Lewy body dementia ("LBD") synthetic seeds as a disease-specific positive control, and the results may be used to compare and determine whether a human sample originates from a patient with MSA, a patient with PD, a patient with LBD, or a patient exhibiting two or more of these conditions.

[0063] In another embodiment, a calibration curve in sCSF can be created using synthetic seeds. The dynamic parameters of such a curve may be useful in determining the concentration of endogenous seeds in a biological sample.

[0064] In yet another embodiment, sCSF may be used as a diluent in SAAs of tissues such as skin or olfactory mucosa. Certain tissues may interfere with SAAs. If the seed concentration in the tissue is high, sCSF can be used to dilute the interfering surface of the tissue, while still retaining enough seeds in the SAA to receive detectable amplification.

[0065] Biological samples "Biological sample" means any biological sample of a subject suitable for analysis to detect misfolded αS aggregates. Suitable biological samples may include, for example, fluids or liquids represented from amniotic fluid, bile, blood, plasma, CSF, earwax, skin, exudate, feces, gastric juice, lymph, milk, mucus, mucous membranes, nasal mucosa including olfactory mucosa, ascites, pleural fluid, pus, saliva, sebum, semen, sweat, synovial fluid, tears, and urine. Body fluids are referred to as "biological sample" or simply "sample" when they are taken from a body and, if applicable, processed and / or prepared for use in the methods and kits described herein. When a sample, e.g., CSF sample, skin sample, olfactory mucosa sample, blood or plasma sample, or saliva sample is referred to as "prepared" in the claims, the intended meaning is that the sample is provided in a processed and / or prepared form ready for use in SAA, unless the context clearly indicates otherwise. The methods and kits described herein are performed and used in vitro.

[0066] As used herein, "αS" may refer to the full-length 140-amino acid alpha-synuclein protein, e.g., "αS-140". Other isoforms or fragments may include, for example, "αS-126", α-synuclein-126, lacking residues 41-54 due to the loss of exon 3; and, for example, "αS-112", α-synuclein-112, lacking residues 103-130 due to the loss of exon 5.

[0067] In one embodiment, the monomeric αS substrate comprises, essentially consists of, or comprises a wild-type or recombinant human αS protein having 140 amino acids, a molecular weight of 14,460 Da, and represented by the following sequence: Sequence ID 1:

number

[0068] In some embodiments, the monomeric αS substrate contains, essentially consists of, or comprises a conserved variant of SEQ ID NO: 1. The conserved variant may be a peptide or amino acid sequence that has similar biochemical properties and deviates from SEQ ID NO: 1 only in one or more amino acid substitutions that have minimal or beneficial effects on the activity of the resulting protein in αS-SAA. The conserved variant must function substantially similarly to the basic component, i.e., SEQ ID NO: 1. For example, the conserved variant of SEQ ID NO: 1 aggregates with a misfolded αS protein and forms aggregates under similar reaction conditions with substantially similar reaction kinetics. The conserved variant may, for example, have 1, 2, 3, 4, 5, 6, 7 (5%) and up to 14 (10%) substitutions in its amino acid sequence.

[0069] In some embodiments, the monomeric αS substrate comprises a recombinant αS protein with a molecular mass of 15,283 Da and containing six additional histidine amino acids (i.e., a polyHis purified tag) at the C-terminus of SEQ ID NO: 1, represented by the following sequence: Sequence ID 2:

number

[0070] In some embodiments, the monomeric αS substrate may be any of the monomeric αS substrates and their conserved variants disclosed in U.S. Patent No. 11,079,396. In some embodiments, the monomeric αS substrate or its conserved variant is specifically excluded from the monomeric αS substrate comprising Sequence ID No. 1.

[0071] In some embodiments, the monomeric αS substrate may be expressed and prepared as described in Shahnawaz, M. et al., Development of a Biochemical Diagnosis of Parkinson's Disease by Detection of alpha-Synuclein Misfolded Aggregates in Cerebrospinal Fluid. JAMA Neurol 74, 163-172 (2017), which is incorporated herein by reference in whole.

[0072] In some embodiments, the monomeric αS substrate may be expressed and prepared as described in U.S. Patent No. 11,254,718, which is incorporated herein by reference in whole.

[0073] In some embodiments, the method may include preparing a labeled monomeric αS substrate. The labeled monomeric αS substrate may be considered a conserved variant. The first labeled monomeric αS substrate may include one or more of the following: a covalently incorporated radioactive amino acid, a covalently incorporated isotopically labeled amino acid, a covalently incorporated fluorophore, etc. Therefore, detection of misfolded αS aggregates may include detecting the labeled monomeric αS substrate incorporated into the amplified portion of the misfolded αS aggregates.

[0074] The pre-incubation mixture may contain monomeric αS substrates at various concentrations as a function of the total volume of the pre-incubation mixture before the incubation cycle. In some embodiments, the pre-incubation mixture may contain monomeric αS substrates at concentrations or concentration ranges 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 50 μM to about 75 μM; about 65 μM (i.e., about 1 mg / ml); 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 (i.e., about 0.3 mg / ml); or 19.6 μM. In one embodiment, the pre-incubation mixture contains monomeric αS substrate at a concentration of approximately 0.3 mg / ml as a function of the total volume of the pre-incubation mixture before the incubation cycle.

[0075] buffer composition The pre-incubation mixture may contain various buffer compositions. The buffer compositions may be effective in maintaining the pH of the reaction mixture within the ranges of approximately pH 5 to approximately pH 9, approximately pH 6 to approximately pH 8, approximately pH 6 to approximately pH 7, approximately pH 7 to approximately pH 8, approximately pH 7, approximately pH 7.4, and approximately pH 6.2 to approximately pH 6.5 (including pH 6.3, 6.4, and 6.5). In one embodiment, the buffer composition may be effective in maintaining the pH of the reaction mixture at approximately 6.5. In some embodiments, the pre-incubation mixture contains one or more buffers from Tris-HCl, MES, PIPES, MOPS, BES, TES, and HEPES. In some embodiments, the buffer contains PIPES at concentrations of approximately 100 mM, approximately 200 mM, approximately 300 mM, approximately 400 mM, approximately 500 mM, approximately 600 mM, or approximately 700 mM. In one embodiment, the buffer solution contains PIPES at a concentration of approximately 100 mM.

[0076] Salt solution In some embodiments, the pre-incubation mixture contains a salt at a given concentration. The salt can enhance, for example, the signal-to-noise ratio in fluorescence detection. In one embodiment, the salt is NaCl. Other suitable salts include KCl. In one embodiment, the salt, e.g., NaCl, may be present in the pre-incubation mixture at concentrations 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, e.g., NaCl, is present at a concentration of about 500 mM.

[0077] Indicator In some embodiments, the pre-incubation mixture includes an indicator for determining whether a detectable amount of misfolded αS aggregates is present in the reaction mixture. The indicator may be characterized by showing an indicating state in the presence of a detectable amount of misfolded αS aggregates and a non-indicating state in the absence of a detectable amount of misfolded αS aggregates. Determining the presence of misfolded αS aggregates in a biological sample may include detecting the indicating state of the indicator for misfolded αS aggregates. The indicating and non-indicating states of the indicator may be characterized by a difference in fluorescence. Therefore, the step of determining the presence of misfolded αS aggregates in a biological sample may include detecting a difference in fluorescence. In some embodiments, a molar excess of the indicator may be used, where the molar excess is greater than, for example, the total molar amount of monomeric αS substrate and misfolded αS aggregates in the reaction mixture.

[0078] In some embodiments, the indicator contains a fluorophore. In some embodiments, the indicator may contain one or more of the following: thioflavin-T (ThT), Congo Red, mI-stilbene, Chrysamine G, PIB, BF-227, X-34, TZDM, FDDNP, IMPY, NIAD-4, luminescence-conjugated polythiophene, fusions with fluorescent proteins such as green fluorescent protein and yellow fluorescent protein, and derivatives thereof. A suitable indicator is ThT. In one embodiment where the indicator contains ThT, the ThT concentration in the pre-incubation mixture is between approximately 5 μM and approximately 10 μM. In one embodiment where the indicator contains ThT, the ThT concentration in the pre-incubation mixture is 10 μM.

[0079] Sarcosyl In some embodiments, the pre-incubation mixture contains sarcosyl. In some embodiments, sarcosyl is present at a concentration of 0.01% w / v to about 1.0% w / v. In some embodiments, sarcosyl is present at a concentration of 0.05% w / v to about 0.2% w / v. In some embodiments, sarcosyl is present at a concentration of about 0.1% w / v.

[0080] Incubation conditions The reaction mixture can be held in a container of appropriate size, such as a multiwell plate having multiple wells. For example, a multiwell plate may contain 96 wells. The wells of the multiwell plate may have volumes of 100 μL to 1000 μL, 150 μL to 750 μL, or 200 μL to 350 μL. In some embodiments, at least one well of the multiwell plate contains one or more beads.

[0081] The temperature of the reaction mixture in each incubation cycle may be, independently in degrees Celsius (°C), 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 in the range between any two of the aforementioned values, for example, between about 15°C and about 50°C, or between about 25°C and about 45°C, or between about 30°C and about 42°C. In some embodiments, incubation is carried out at a temperature approximately normal for warm-blooded animals. In further embodiments, incubation of the reaction mixture is carried out at a temperature between about 35°C and about 45°C or between about 37°C and about 42°C. In one embodiment, the method includes incubating the reaction mixture at a temperature of approximately 42°C.

[0082] In some embodiments, deagglomeration of the incubation mixture may include subjecting the incubation mixture to physical disruption such as shaking, sonication, stirring, freeze / thaw, laser irradiation, autoclave incubation, high pressure, and homogenization. Shaking may include periodic stirring such as orbital stirring. Periodic stirring may be performed between approximately 50 revolutions per minute (RPM) and 10,000 RPM. Periodic stirring may be performed between approximately 200 RPM and 2,000 RPM. Periodic stirring may be performed at approximately 500 RPM or approximately 600-800 RPM. In one embodiment, shaking includes orbital stirring at approximately 800 RPM. Deagglomeration of the incubation mixture may be performed after each incubation cycle, such as between approximately 5 seconds and 10 minutes, between approximately 30 seconds and 1 minute, between approximately 45 seconds and 1 minute, and for approximately 1 minute.

[0083] The steps of incubating and deaggregating the reaction mixture are repeated as needed to amplify misfolded α-S aggregates in the biological sample and obtain a detectable amount of misfolded α-S aggregates. Incubating the reaction mixture and deaggregating the reaction mixture constitute an incubation cycle. The incubation cycle may be repeated for periods of 1 to approximately 1000 times, 2 to approximately 500 times, approximately 50 to approximately 500 times, approximately 150 to approximately 250 times, etc. In one embodiment, it may be advantageous to omit the deaggregation step before performing the detection step in the final round of the incubation cycle.

[0084] Incubation cycles may be performed for durations such as approximately 1 minute to approximately 5 hours, approximately 10 minutes to approximately 2 hours, approximately 15 minutes to approximately 1 hour, and approximately 25 minutes to approximately 45 minutes. In some embodiments, incubating the reaction mixture and deaggregating at least a portion of the misfolded αS aggregates includes incubation cycles lasting approximately 0.1 to 1 hour. Each incubation cycle may include incubation for approximately 1 minute to approximately 5 hours and deaggregation for approximately 5 seconds to approximately 10 minutes; incubation for approximately 10 minutes to approximately 2 hours and deaggregation for approximately 30 seconds to approximately 1 minute; incubation for approximately 14 minutes to approximately 1 hour and deaggregation for approximately 45 seconds to approximately 1 minute; incubation for approximately 25 minutes to approximately 45 minutes and deaggregation for approximately 45 seconds to approximately 1 minute; and incubation for approximately 1 minute and deaggregation for approximately 1 minute, in which case the reaction mixture may be incubated independently and deaggregated. In one embodiment, each incubation cycle includes incubation for approximately 14 minutes and de-aggregation for approximately 1 minute.

[0085] beads In some embodiments, the pre-incubation mixture may contain one or more beads. The beads are small, typically spherical objects, such as high-density beads with a low-friction surface, commonly used as bearing beads. Including beads in the reaction mixture increases the rate of misfolded αS aggregate formation from monomeric αS substrates and soluble misfolded αS proteins of the biological sample. These beads differ in composition and function from antibody-coated magnetic or paramagnetic beads or particles (e.g., Dynabeads) used in the concentration and / or immunodepletion steps described elsewhere in this specification.

[0086] The beads can be composed of various chemically inert materials. For example, in some embodiments, the beads are made of silica, glass, borosilicate glass, or Si3N4.

[0087] In some embodiments, the beads contain, essentially consist of, or comprise Si3N4. In some embodiments, the beads contain, essentially consist of, or comprise borosilicate glass. In one embodiment, zirconium / silica beads are excluded. In one embodiment, glass beads other than borosilicate glass beads are excluded.

[0088] In some embodiments, the beads in the incubation mixture may have an average diameter greater than 0.5 mm. In some embodiments, the beads have an average diameter greater than 0.5 mm to about 10 mm. In some embodiments, the beads have an average diameter greater than 0.5 mm to about 5 mm. In further embodiments, the beads have an average diameter in the range of greater than 0.5 mm to about 3.5 mm. In some embodiments, the beads have an average diameter of about 1.0 to about 10 mm, but in additional embodiments, the beads have an average diameter of about 1.0 mm to about 5 mm. In further embodiments, the beads have an average diameter greater than 1.0 mm to about 3.5 mm. In some embodiments, the beads have an average diameter of 2.38 mm to about 10 mm, but in additional embodiments, the beads have an average diameter of 2.38 mm to about 5 mm. In further embodiments, 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 embodiments, the beads may have 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, about 2.45 mm, or about 3.175 mm. In some embodiments, the beads contain, are essentially made of, or consist of Si3N4, have an average diameter of 2.38 mm, and are blocked with bovine serum albumin (BSA). In some embodiments, the beads contain, are essentially made of, or consist of Si3N4, have an average diameter of 3.175 mm, and are not blocked. In some embodiments, the beads contain, are essentially made of, or consist of borosilicate glass, have an average diameter of 2.45 mm, and are not blocked. In some embodiments, beads having an average diameter of 2.3 mm or less are excluded from the present invention. In some embodiments, glass beads having an average diameter of 2.3 mm or less are excluded from the present invention. In some embodiments, beads having an average diameter of 3 mm or less are excluded from the present invention. The bead diameter distribution is defined as the case where more than 90% of the beads are found to be between 80% and 120% of the average bead diameter or between 90% and 110% of the average bead diameter.

[0089] The number of beads in the pre-incubation mixture can vary. In some embodiments, the pre-incubation mixture consists of one bead. In some embodiments, the pre-incubation mixture consists of two beads. In some embodiments, the pre-incubation mixture contains multiple beads. In one embodiment, the pre-incubation mixture consists of two unblocked 1 / 8-inch (3.175 mm) Si3N4 beads.

[0090] In some embodiments, the surface of one or more beads is "blocked" with protein. Blocking the surface of a bead with protein refers to providing a coating or layer over all or a significant portion of the surface of the bead. Any suitable biocompatible protein can be used to coat the surface of the beads. Suitable proteins for use in blocking the surface of beads include albumin, such as BSA. Other suitable blocking proteins may include casein or milk powder. One or more beads can be blocked by immersing them in a protein-containing solution. The solution may be an aqueous solution and / or buffered solution such as PIPES, Tris-HCl, MES, MOPS, BES, TES, and HEPES.

[0091] The incubation mixture is held in a container of appropriate size, such as a test tube. Suitable sterile incubation containers are known to those skilled in the art. In some embodiments, the incubation mixture is contained in a multiwell plate containing multiple wells. For example, a multiwell plate may contain 96 wells. In one embodiment, for example, if the beads are Si3N4 beads, the container may be a black-bottomed 96-well plate (Costar 3916). In one embodiment, for example, if the beads are Si3N4 beads, the container may be a bottom-readable Greiner CBP plate. In one embodiment, for example, if the beads are borosilicate glass beads, the container may be a clear-bottomed 96-well plate (Costar 3603).

[0092] detection Detection involves amplifying a sufficient amount of misfolded αS aggregates present in the biological sample by repeating the steps of incubating and deaggregating the reaction mixture as needed, to obtain an amplified incubation mixture having a detectable amount of misfolded αS aggregates. The incubation mixture can be contacted with an indicator to determine the fluorescence level of the amplified reaction mixture.

[0093] A suitable indicator is ThT, also known as Basic Yellow 1. When ThT is added to a sample containing β-sheet rich deposits, such as the cross-β-sheet quaternary structure of amyloid fibrils, ThT strongly fluoresces, with excitation and emission maxima at approximately 435 nm (or approximately 440 nm depending on the fluorometer or spectrofluorometer) and approximately 485 nm (or approximately 490 nm depending on the fluorometer or spectrofluorometer), respectively.

[0094] ThT fluorescence is typically measured by fluorescence spectroscopy using a filter fluorometer or spectrofluorometer. In some embodiments, the ThT fluorescence intensity may be compared to the level of a corresponding control sample when performing an analysis to quantify the amount of misfolded αS aggregates in a biological sample. Once the ThT fluorescence level is determined, it can be displayed in various ways. For example, the level may be graphically displayed on a display as a numerical value, a proportional bar (i.e., a bar graph), or any other display method known to those skilled in the art.

[0095] An increase in fluorescence level indicates the presence of αS aggregates in the biological sample. In some embodiments, a significant increase in fluorescence level indicates the presence of αS aggregates in the biological sample. In some embodiments, a "significant increase" is an increase in the fluorescence level of the incubated mixture at maximum fluorescence of at least twice the standard deviation of the fluorescence of the incubated mixture at maximum fluorescence compared to the fluorescence level of the incubated mixture at any point during the delay period, indicating the presence of αS aggregates in the biological sample.

[0096] Neurological disorders and synucleinopathies αS aggregation may be associated with protein misfolding disorders (PMDs), such as PD, LBD, and MSA. However, existing technologies do not clearly demonstrate whether this aggregation phenomenon is the cause of these diseases; it is merely speculated that these misfolded αS aggregates may cause cellular dysfunction and tissue damage, among other effects. In other words, the methods and kits described herein do not directly determine whether an individual has a particular disease based on whether or not the individual has misfolded αS aggregates.

[0097] The information obtained by the methods and kits described herein for determining whether misfolded αS aggregates are present in a sample is merely an intermediate result or a form of reference information, and it is not possible to directly conclude that an individual has a particular disease based on that result. Therefore, in at least one embodiment, what is claimed in this application is not a method for diagnosing a disease.

[0098] In another embodiment, a method is provided to assist in the diagnosis of PD, LBD, MSA, or a spectrum of each embodiment in subjects with neurological disorders. Neurological disorders are any disorders of the nervous system. Examples of neurological disorders include motor disorders such as PD, autonomic nervous system disorders such as MSA, and neuropsychiatric disorders such as LBD.

[0099] In some embodiments, the neurological disorder is a synucleinopathy. Synucleinopathy is a neurodegenerative disease characterized by the abnormal accumulation of αS aggregates in cells of the nervous system, such as neurons, nerve fibers, and glial cells. In some embodiments, the synucleinopathy has symptoms associated with PD, LBD, or MSA, including, for example, cognitive impairment, sleep disorders, and gastrointestinal dysfunction.

[0100] In some embodiments, samples may be collected from subjects that do not show clinical signs of PD, LBD, or MSA. In other embodiments, biological samples may be collected from subjects that show clinical signs of PD, MSA, LBD, or any combination thereof. The most recognizable symptom of PD is motor-related dysfunction.

[0101] In some embodiments, the method includes treating subjects diagnosed with PD with treatments for PD and / or its symptoms. Deep brain stimulation can be used to alleviate motor symptoms associated with PD. Drugs useful for treating motor symptoms of PD include levodopa, dopamine agonists, and monoamine oxidase B inhibitors. However, further treatments for PD continue to be developed. See Radhakrishnan DM, Goyal V, Neurol India., 66(Appendix):S26~S35 (2018) and Iarkov et al., Front Aging Neurosci., 12:4 (2020).

[0102] Supplementary diagnostic tests In some embodiments, the method may further include additional tests to confirm the αS-SAA-based indication, for example, to further differentiate between patient-derived misfolded αS aggregates indicated by αS-SAA when PD is present and patient-derived misfolded αS aggregates indicated by αS-SAA when MSA or LBD is present. Examples of additional tests include the use of a ligand with high affinity for one of the misfolded αS aggregates of PD, MSA, or LBD, the creation of a profile of protease-resistant fragments from the misfolded αS aggregates, and the evaluation of the structure of the detected misfolded αS aggregates using CD, FTIR, or cryo-ET.

[0103] kit Another embodiment provides a kit for detecting the presence of misfolded αS aggregates in a biological sample. The kit comprises a known amount of monomeric αS substrate; a known amount of indicator; a buffer composition; and optionally 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, about 2.45 mm, or about 3.175 mm; and optionally sarcosyl. The kit also comprises sCSF as described herein. The kit may include instructions for the user to perform the method for detecting misfolded αS aggregates as described herein, as well as instructions for testing the suitability of the monomeric αS substrate and αS-SAA buffer, for diluting a biological sample derived from the surrounding matrix, and for serial diluting the biological sample for semi-quantitative αS-SAA. The kit should also include packaging for holding the components of the kit.

[0104] A kit typically includes a package having one or more containers for holding reagents, either as one or more separate compositions or, optionally, as a mixture, where reagent compatibility permits. The kit may further include buffers, labeling agents, controls, and any other materials necessary for detecting misfolded αS aggregates. The kit may also include tools for obtaining samples from the subject, such as swabs or other body fluid collection devices.

[0105] The kit may also include instructions for using the kit to guide a method for treating synucleinopathy in a subject. Instructions included in the kit may be attached to the packaging material or included as accompanying documentation. Instructions are typically, but not limited to, written or printed materials. Any medium capable of storing such instructions and communicating them to end users is contemplated by this disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic disks, tapes, cartridges, chips), optical media (e.g., CD-ROMs), and the like. As used herein, the term “instructions” may include the address of an internet site providing the instructions.

[0106] The kit may include one or more of the following: a bead dispenser; a multiwell plate containing multiple wells; a microfluidic plate; a shaker; an incubation device; and a fluorescence analyzer, either as one or more individual plates or devices, or as a combined device. For example, a shaking microplate reader can be used to perform incubation and shaking cycles and automatically measure ThT fluorescence emission during the experiment (e.g., FLUOstar OPTIMA, BMG LABTECH Inc., Cary, NC, or Buehler Shaker TIMIX 5 shaker).

[0107] example The present invention will be illustrated by the following examples. However, specific examples, materials, quantities, and procedures should be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.

[0108] Example 1: sCSF used as a negative control during a "slow assay" under αS-SAA conditions. The control solution was used as a negative control under "slow assay" αS-SAA conditions. General conditions for "slow assay" αS-SAA are described in U.S. Patent No. 10,989,718. The specific αS-SAA conditions used to obtain the results herein are as follows:

[0109] For the negative control, the incubation mixture was prepared in a 96-well plate, and the incubation mixture for a total volume of 200 μL contained (1) a seedless αS substrate represented by SEQ ID NO: 2 at 1 mg / ml; (2) a buffer composition containing 100 mM PIPES at pH 6.5; (3) a salt solution containing 500 mM NaCl; (4) an indicator containing 10 μM ThT; and (5) 40 μL of a control solution.

[0110] Incubation cycles were performed on the incubation mixture, with each incubation cycle comprising: (1) incubating the first incubation mixture for 29 minutes; and (b) orbital shaking the incubation mixture at 700 rpm for 1 minute for a total of 300 hours at a constant temperature of 37°C using an Omega FLUOstar to form an incubated control solution. After excitation at 440 nm, ThT fluorescence was measured in the plate at 490 nm every 30 minutes.

[0111] Four different control solutions were used: 1. Condition "1B3T": Harvard Apparatus artificial CSF + 0.155 mg / mL BSA (1x) + 0.042 mg / mL transferrin (3x) 2. Condition "1.3B": Harvard Apparatus artificial CSF + 0.2015mg / mL BSA (1.3X) 3. Conditions “3B1IgG”: Harvard Apparatus artificial CSF+0.465mg / mL BSA(3X)+0.012mg / mL IgG(1X) 4. Condition “H”: Harvard Apparatus Artificial CSF

[0112] Although all the substrates tested were intended to correspond to Sequence ID No. 2, the substrates were prepared at different times, and some under different expression and / or purification conditions.

[0113] The results are shown in Table 1. "Neg / Total Neg Control" refers to the number of trials that did not show substrate autoaggregation compared to the total number of trials. "Pos / Total Neg Control" refers to the number of trials that showed substrate autoaggregation compared to the total number of trials. [Table 1]

[0114] Figure 3 shows a graph of the time-dependent fluorescence intensity (kRFU) for slow-rate αS-SAA of AMP-13 using 1B3T as the negative control solution.

[0115] Figure 4 shows a graph of the time-dependent fluorescence intensity (kRFU) for slow-rate αS-SAA of AMP-14 using 1B3T as the negative control solution.

[0116] Figure 5 shows a graph of the time-dependent fluorescence intensity (kRFU) for slow-rate αS-SAA of AMP-6 using 1.3B as the negative control solution.

[0117] Example 2: sCSF used as a positive control during "slow assay" αS-SAA conditions The control solution was used as a positive control during the "slow assay" αS-SAA conditions. For the positive control, the incubation mixture further contained 20 fg of synthetic αS aggregates (purchased from Abcam) containing wild-type human recombinant protein represented by SEQ ID NO: 1.

[0118] The results are shown in Table 2. "Pos / Total Pos Control" refers to the number of trials that showed substrate aggregation with the seed, compared to the total number of trials. "Neg / Total Pos Control" refers to the number of trials that did not show substrate aggregation with the seed, compared to the total number of trials. [Table 2]

[0119] Figure 6 shows a graph of the time-dependent fluorescence intensity (kRFU) of slow-reacting αS-SAA for AMP-13 in the presence of 20 fg of seed in 1B3T as a positive control solution.

[0120] Figure 7 shows a graph of the time-dependent fluorescence intensity (kRFU) for AMP-6 slow-rate αS-SAA in the presence of 20 fg of seed in 1.3B as a positive control solution.

[0121] Example 3: sCSF used as a negative control during a "rapid assay" under αS-SAA conditions. sCSF was used as a negative control under “rapid assay” αS-SAA conditions. General conditions for “rapid assay” αS-SAA are described in U.S. Patent No. 11,079,396. Specifically, αS-SAA was performed using seedless αS represented by Sequence ID No. 2 at 0.3 mg / ml (which was appropriately prepared according to the protocol disclosed in U.S. Patent No. 11,254,718) in the presence of 2.38 mm silicon nitride beads, with orbital shaking at 800 rpm.

[0122] Figure 8 shows a representative graph of the time-dependent fluorescence intensity of the αS substrate for fast αS-SAA using 100 mM HEPES, pH 7.5, 75 mM NaCl, 1.5 mg / mL HSA, and 0.5% sarcosyl as negative control solutions. In Figure 8, no autoaggregation of the αS substrate was observed. Only 3 out of 96 wells (3.1%) showed autoaggregation. In a comparative experiment using healthy human CSF as the control solution, 5% of the wells showed autoaggregation.

[0123] Example 4: sCSF for use in screening substrates using the "high-speed assay" αS-SAA condition. Substrates were screened under “fast assay” αS-SAA conditions using sCSF (essentially as a negative control). In this experiment, αS substrates were purified in a manner deviating from the teachings of U.S. Patent No. 11,254,718. Figure 9 shows a graph of fluorescence intensity over time for fast αS-SAA of ineligible αS substrates using 100 mM HEPES, pH 7.5, 75 mM NaCl, 1.5 mg / mL HSA and 0.5% sarcosyl as control solutions. As shown in Figure 9, the αS substrates exhibited autoaggregation.

[0124] Example 5: sCSF used as a positive control during "rapid assay" αS-SAA conditions sCSF was used as a positive control during the "rapid assay" αS-SAA conditions. For the positive control, the incubation mixture further contained 20 fg of synthetic αS aggregates (purchased from Abcam) containing wild-type human recombinant protein represented by SEQ ID NO: 1.

[0125] Figure 10 shows a graph of the fluorescence intensity over time for fast αS-SAA of the αS substrate in the presence of 20 fg of seed, using 100 mM HEPES, pH 7.5, 75 mM NaCl, 1.5 mg / mL HSA, and 0.5% sarcosyl as a positive control solution. Figure 10 shows the expected aggregation. Indeed, 100% of the wells showed the expected aggregation.

[0126] Example 6: HC-derived CSF used as a diluent Using serial dilutions, 50% of αS-SAA reactions are positive (SD). 50 The relative amount of αS was determined by estimating ). This estimation is purely dependent on the number of positive wells and therefore does not involve kinetic changes due to specific CSF matrices. When evaluating increasing dilutions of CSF from PD and DLB patients, the positivity of technical replicates decreases. However, when diluting with CSF from different HC donors, the number of positive wells may vary. In other words, CSF from different patients has a different effect on aggregation. This variability effect can be demonstrated with rec-seed, which aggregates very reproducibly when spiked in buffer. However, when spiked with CSF from different patients, aggregation changes based on the CSF sample. See Figure 11. When the experiment was repeated, the effect of each CSF on rec-seed was reproducible. Therefore, HC-CSF cannot be used as a diluent for serial dilution because its titer changes depending on the HC-CSF used. For reproducible titrations based on serial dilution, sCSF as described herein is required.

[0127] Example 7: sCSF used as a diluent for semi-quantitative αS-SAA Serial dilution refers to mixing a certain volume of a CSF sample containing seeds with another solution that does not contain seeds (e.g., sCSF). Serial dilution reduces the concentration of seeds in the mixed sample. Aliquots of the mixture are taken for αS-SAA. The mixed sample is diluted again to obtain higher dilutions containing even lower concentrations of seeds. This procedure is repeated until the best desired dilution is reached, i.e., a dilution with the lowest concentration of seeds. Here, 1:3 serial dilutions up to 1:81 (meaning each dilution contains a 66.6% dilution) were performed. By analyzing the samples in this way, the relative number of seeds can be determined, as samples containing more seeds exhibit seeding activity at higher dilutions than samples containing lower concentrations of seeds.

[0128] For this procedure to be practical, the diluent must be abundant. For this comparison to be meaningful, all samples must be diluted with the same diluent. Therefore, healthy control human CSF is not viable in terms of both availability and the variety of proteins and other biological profiles across different CSF samples from different patients. The sCSF described herein enables the commercial and large-scale use of serial dilutions for αS-SAA.

[0129] Figure 12 shows a series of graphs of fluorescence intensity over time under “fast assay” αS-SAA conditions at serial dilution levels. Figure 12 demonstrates that sCSF, essentially consisting of 100 mM HEPES, pH 7.5, 75 mM NaCl, 1.5 mg / mL HSA, and 0.5% sarcosyl, reproduced the results obtained using CSF from normal pressure hydrocephalus ("NPH") patients, and that the results changed when HC-derived CSF was used. In this assay, both negative NPH and HC are negative. This highlights the need for a stable and reproducible matrix to enable semi-quantification. The amount of seed can be estimated by calculating the “SD50,” which is the dilution required for 50% of the wells to be positive. However, while it is possible to estimate the number of seeds by standard deviation, the definition of “seed” is not clear. Therefore, this method provides a semi-quantitative alternative for comparing seeding activity between samples rather than quantifying misfolded αS. Furthermore, sCSF does not inhibit amplification as much as HC-CSF, which allows for the differentiation of a larger spectrum between samples.

[0130] Figure 13 shows a series of graphs of fluorescence intensity over time in an alternative “fast assay” αS-SAA condition (specifically, the assay itself contains sarcosyl, as described in U.S. Patent No. 11,079,396) for PD-positive samples at serial dilution levels using 100 mM HEPES, pH 7.5, 75 mM NaCl, and 1.5 mg / mL HSA as diluents. The three circles below each graph represent the three replicates analyzed for each dilution. Gray circles represent replicates showing detectable seed amplification, and white circles represent replicates showing no detectable seed amplification. This notation is also used in Figure 14.

[0131] Referring further to Figure 14, two CSF ​​samples that showed strong (2603) and weak (2978) amplification patterns under rapid assay conditions were serially diluted 3-fold to NPH-CSF and sCSF using three substrates and two versions of the rapid assay. 50This was calculated for each sample using the Spearmen-Karber model. Using AMP-A, the estimated number of seeds was 2603, which was greater than the estimated number of seeds for 2978 (67.5 > 10.8), indicating a substantial difference in seeding activity between these two CSF ​​samples. A similar pattern was observed with AMP50. SD 50 Since the estimations typically show variations of several orders of magnitude, the results shown in Figure 14 are highly reproducible. Using the same AMP50 substrate, dilution to sCSF was remarkably similar to dilution to NPH-CSF. The alternative rapid assay version exhibited a lower limit of detection (higher analytical sensitivity), which is also observed here, as higher dilutions of 2603 and 2978 CSF samples showed positive repeats. Overall, all conditions shown in Figure 14 were able to estimate a higher seed count for 2603 than for 2978. (N / T: Not tested. SE: Standard error.)

[0132] Example 8: Half-Q αS-SAA-kinetics normalization rec-seed T 50 This varied in a reproducible manner depending on the CSF donor (Figure 11), revealing inhibitory or amyloidogenic CSF components. Therefore, three PD-CSF samples were analyzed using neat and spiked rec-seed. Normalized seeding coefficients (φ) were calculated and their association with H&Y scores was assessed. Notably, φ was strongly associated with H&Y scores. See Figure 15. Analysis of larger cohorts of samples and additional clinical parameters, including the Unified Parkinson's Disease Assessment Scale (UPDRS), the Scale for Outcomes in Parkinson's-Autonomic Dysfunction (SCOPA-AUT), the Montreal Cognitive Function Assessment (MoCA) test, and the Dopamine Transporter Specific Binding Ratio (DaTscan SBR), should be evaluated.

[0133] Example 9: Detection of misfolded αS aggregates in the olfactory mucosa 5.1 Collection and Preprocessing Ten minutes before the procedure, the patient was given local anesthesia (nasal spray with lidocaine). The olfactory mucosa (between the nasal septum and the middle turbinate) was identified using a rigid fiberscope. While holding the fiberscope in place, a cotton swab was inserted into the nostril, and once it reached the olfactory mucosa (OM), the wall of the nostril was gently scraped to collect a sample. The swab was removed from the nose and placed in a 15 mL conical tube containing 3 mL of physiological solution (saline buffer). Using disposable scissors, the swab was cut so that the vapors would be contained within the 15 mL conical tube. The tube was vortexed for 1 minute. Using disposable forceps, the swab was transferred to a second 15 mL conical tube containing 3 mL of physiological solution (saline buffer) and vortexed for 1 minute. Using the same disposable forceps, the swab was transferred to a third 15 mL conical tube containing 3 mL of physiological solution and vortexed for 1 minute. The swab was discarded. Three mL (9 mL total) was pooled from each of the 15 mL tubes into a single 15 mL tube, which was then centrifuged at 800 × g for 20 minutes at 4°C. Eight mL of the supernatant saline was discarded. The pellet and one mL of saline were stored at -80°C.

[0134] 5.2 Sample preparation. αS-SAA sample preparation. OM samples were collected from the pellet using bacterial inoculation loops holding approximately 2 μg of sample. Three loops were collected (6 μg) and resuspended in 50 μL of 1×PBS (Sigma, catalog no. P5493-1L) by extensive vortexing and up-and-down pipetting. The final resuspension was divided equally into three single-use aliquots, each containing 16.7 μL (2 μg of OM sample per aliquot). The samples were rapidly frozen and stored at -80°C until use.

[0135] 5.3 Sample preparation for αS-SAA. The OM / PBS sample was thawed, and 4 μL of the sample was pipetteed into 76 μL of sCSF (Amprion, catalog number S2022) to perform a 1:20 dilution. A 1:400 (12 ng / 40 μL) dilution was prepared by pipettering 24 μL of the 1:20 dilution into 456 μL of sCSF.

[0136] 5.4 αS-SAA. The reaction mixture contained 40 μL of OM sample (12 ng and / or 24 ng) and 60 μL of pre-incubation mixture. The pre-incubation mixture contained 100 mM PIPES pH 6.5, 500 mM NaCl, 10 μM ThT, 0.1% sarcosyl, and two Si3N4 beads (1 / 8 inch, grade 5). Plates were incubated at 42°C for a total of 15 minutes with 1 minute of orbital shaking, followed by 14 minutes of incubation. When using a robotic arm associated with an Omega shaker / reader (8 plates at a time), stirring was set to 600 RPM, and for standalone Omegas (1 plate at a time), it was set to 800 RPM. Fluorescence readings were acquired at 440-10 nm (excitation) and 490-10 nm (emission). Figure 16 shows αS-SAA aggregation curves from patients diagnosed with PD compared to a non-synucleinopathy control.

[0137] All patents, patent applications, and publications cited herein, as well as the complete disclosure of electronically available materials, are incorporated by reference, whether or not a specific citation herein states so. The above detailed descriptions and examples are given solely for the purpose of clarifying understanding. No unnecessary limitations should be derived therefrom. The present invention is not limited to the exact details illustrated and described, and modifications that are obvious to those skilled in the art are included within the present invention as defined by the claims.

Claims

1. (A) Human serum albumin (HSA), (B) NaCl aqueous solution, and (C) (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES) A composition comprising a monomer alpha-synuclein protein and a fluorescent protein aggregation indicator, wherein the composition is inactive to monomer alpha-synuclein protein and a fluorescent protein aggregation indicator.

2. The composition according to claim 1, wherein the HSA is present at a concentration of about 1.5 mg / mL.

3. The composition according to claim 1, wherein the HSA is present at a concentration of about 15 mg / mL.

4. The composition according to claim 1, wherein the HEPES is present at a concentration of about 100 mM.

5. The composition according to claim 1, wherein the HEPES maintains the pH of the composition at approximately 8.

6. The composition according to claim 1, wherein the aqueous NaCl solution has a concentration of about 75 mM.

7. (1) Human serum albumin at approximately 1.5 mg / mL, (2) (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, which has a pH of approximately 8 (3) Approximately 75 mM NaCl, and (4) Approximately 0.5% sarcosyl A composition comprising a monomer alpha-synuclein protein and a fluorescent protein aggregation indicator, wherein the composition is inactive to monomer alpha-synuclein protein and a fluorescent protein aggregation indicator.

8. (A) HSA, (B) NaCl aqueous solution, and (C) HEPES The composition according to claim 1, comprising:

9. (A) Human serum albumin (HSA), (B) NaCl aqueous solution, (C) 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and (D) Sarcosyl A composition comprising a monomer alpha-synuclein protein and a fluorescent protein aggregation indicator, wherein the composition is inactive to monomer alpha-synuclein protein and a fluorescent protein aggregation indicator.

10. The composition according to claim 9, wherein the HSA is present at a concentration of about 1.5 mg / mL.

11. The composition according to claim 9, wherein the HEPES is present at a concentration of about 100 mM.

12. The composition according to claim 9, wherein the HEPES maintains the pH of the composition at approximately 8.

13. The composition according to claim 9, wherein the aqueous NaCl solution has a concentration of about 75 mM.

14. The composition according to claim 9, wherein the sarcosyl is present at a concentration of about 0.5%.

15. (A) HSA, (B) NaCl aqueous solution, (C) HEPES, and (D) Sarcosyl The composition according to claim 9, comprising the above.

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