Inert matrices for quantitative and semi-quantitative seeded 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 TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 243,470, filed September 13, 2021, and U.S. Provisional Patent Application No. 63 / 328,443, filed April 7, 2022, each of which is incorporated by reference in its entirety herein.

[0002] Sequence Listing The Sequence Listing has been submitted electronically in XML format and is incorporated herein by reference in its entirety. 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 ("SAA") have provided a sensitive and specific means to detect biomarker misfolded protein aggregates in tissues and body fluids at titers too low for detection by traditional immunoassay methods. 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 [with correction published in Acta Neuropathol Commun. 2021 Nov. 26; 9(1): 190]. Acta Neuropathol Commun. 2021; 9(1): 179. Published Nov. 6, 2021. doi: 10.1186 / s40478-021-01282-8, incorporated herein by reference in its entirety. SAA allows for much earlier diagnosis of pathologies involving misfolded proteins, which may prove important for disease treatment and prevention or mitigation of symptoms. See, e.g., 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, incorporated herein by reference in its entirety.

[0004] SAA relies on promoting the amplification of misfolded protein aggregates that are endogenous to the biological sample (the "seed") while avoiding self-aggregation of the assay substrate at the expense of monomeric proteins of the same type used as the assay substrate. As a negative control, SAA generally uses a biological sample from a known healthy control ("HC") donor, since there are no reliable commercially available synthetic control solutions for this purpose. The negative control is subjected to the same conditions as the biological sample of interest, including intermittent shaking and addition of substrate, buffer, and fluorescent probe. If the negative control shows no aggregation and the biological sample shows no aggregation (i.e., no amplification of the seed), the patient is considered "negative" or aggregation is "not detected." If the negative control shows no aggregation and the biological sample shows aggregation, the patient is considered "positive" or aggregation is "detected." If the negative control shows aggregation, the results must be discarded and the assay repeated, as a positive negative control indicates an assay problem related to either handling, reagent stability, or consumable quality. Thus, the negative control must not induce or allow autoaggregation of the substrate.

[0005] As a positive control, SAA generally uses a biological sample from a patient with a confirmed diagnosis of the relevant protein misfolding disorder, or a biological sample "spiked" with known amounts of synthetic misfolded protein aggregates of the same type as the biological biomarker ("synthetic seeds"). Again, human samples are used because there are no commercially available synthetic control solutions that can maintain amplification of the synthetic seeds while suppressing self-aggregation of the substrate. The positive control is subjected to the same conditions as the biological sample. If the positive control shows aggregation, the assay conditions were compatible with seed amplification, which means 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 aggregation, the results must be discarded and the assay repeated, as the lack of aggregation in the positive control indicates a problem with the assay, either with handling, reagent stability, or consumable quality.

[0006] Recent studies have shown that cerebrospinal fluid ("CSF") closely reflects brain matter with respect to the presence of misfolded proteins. See, for example, Shahnawaz, M., Mukherjee, A., Pritzkow, S. et al. Discriminating α-synuclein strains in Parkinson's disease and multiple system atrophy. Nature 578, 273-277 (2020). Thus, detection of misfolded proteins in CSF is of particular importance. Unfortunately, compositions used as inert or neutral assay matrices for positive and negative controls in research situations too often induce or allow substrate autoaggregation (in negative controls) or prevent aggregation in the presence of seeds, endogenous or synthetic (in positive controls).

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

[0008] Thus, there is a need for an inert matrix for use as a control solution that is ready to use, readily available, and abundantly supplied, and that accurately reflects the absence of misfolded protein with no, perceptibly low, or delayed form of substrate self-aggregation when used as a negative control, but that readily allows aggregation of the substrate with the seeds when used as a positive control.

[0009] Alpha-synuclein ("αS")-SAA in the CSF is the gold standard for detecting misfolded αS aggregates, but obtaining CSF requires an invasive lumbar puncture, also known as a spinal tap, to remove a sample of CSF from the subarachnoid space of the spine. Although αS-SAA in peripheral matrices (e.g., blood, saliva, skin and olfactory mucosa) is known (see, e.g., U.S. Pat. Nos. 10,989,718, 11,079,396 and 11,099,197, each of which is incorporated herein by reference in its entirety), there remains a need for reliable inert matrices for use as diluents in sample processing of such peripheral matrices. See U.S. Provisional Patent Application No. 63 / 375,126, filed September 9, 2022, which is incorporated herein by reference in its entirety.

[0010] Because one of the most challenging aspects of SAA is to identify a suitable substrate, i.e., one that does not self-aggregate but will aggregate in the presence of seeds in the biological sample environment, there is also a need for an inert matrix to use as a control solution that can be used to screen the suitability of the substrate (as well as the suitability of other reagents and consumables used in SAA).

[0011] Finally, quantification of biomarkers remains a challenge.Quantitative assays use standards to measure the concentration (M or mg / mL) of a given analyte.See, for example, U.S. Patent No. 10,215,763 and U.S. Patent Publication No. 20190137515 ("prion references"), each of which is incorporated herein by reference in its entirety. Although 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, B. R. 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 the prion references is not feasible in the context of αS-SAA in biological samples for at least two reasons. First, there is no concentration standard with the same aggregation activity as endogenous αS aggregates; synthetic seeds would aggregate much faster, resulting in a gross underestimation of endogenous αS aggregates. Furthermore, the concentration standard should match the molecular weight distribution of αS aggregates, since different species may have different seeding activities. Therefore, synthetic seeds cannot be used accurately as absolute concentration standards. Second, biological fluids are complex matrices, and the kinetics of aggregation may vary from patient to patient. For example, patients with the same concentration of αS in CSF may show different aggregation patterns due to differences in their respective CSF matrices. Therefore, an inert matrix is ​​needed to use as a diluent for serial dilution of SAA samples to estimate relative αS aggregate levels and enable a semi-quantitative version of SAA to allow comparisons between biological samples and to rank them. Summary of the Invention

[0012] In one embodiment, an inert matrix for use with 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 any of (1) human serum albumin ("HSA"), or (2) bovine serum albumin ("BSA"), BSA precursor protein, transferrin, and immunoglobulin G, and combinations thereof; and (B) a physiological saline solution comprising NaCl. When the plasma protein comprises HSA, the sCSF may further comprise a detergent, such as sarkosyl, and a buffer composition, such as (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) ("HEPES"). In one embodiment, the HEPES maintains the pH of the sCSF comprising HSA at about 7.5.

[0013] In one aspect, a method of using sCSF as a negative control in αS-SAA is provided, comprising the steps of: (I) providing sCSF as disclosed herein; (II) providing a pre-incubation mixture comprising: (A) a monomeric αS substrate; (B) a buffer composition; (C) a salt composition; (D) a 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 with intermittent stirring cycles to form an incubated mixture; (V) illuminating 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 to a predetermined second fluorescence intensity, wherein a second fluorescence intensity greater than the first fluorescence intensity indicates the absence of an appreciable amount of self-aggregated αS substrate in the sCSF.

[0014] In one aspect, sCSF is provided that is suitable for determining the suitability of one or more of the reagents and consumables in αS-SAA, including the suitability of αS monomeric protein as an αS-SAA substrate and / or the suitability of an SAA buffer composition. In one aspect, as shown in FIG. 1, which does not use beads, the method includes the steps of: (A) providing an incubation mixture comprising: (1) monomeric αS protein; (2) a buffer composition; (3) a salt composition; (4) a fluorescent protein aggregation indicator; (5) sCSF as disclosed herein; and, optionally, (6) beads; (B) incubating the incubation mixture with intermittent agitation 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, where no significant increase in fluorescence indicates no self-aggregation of monomeric αS protein, which indicates the suitability of the αS monomeric protein and the suitability of the buffer composition as an αS-SAA substrate.

[0015] In one aspect, sCSF is provided that is suitable for determining the suitability of one or more of the reagents and consumables in αS-SAA, including the suitability of αS monomeric protein as an αS-SAA substrate and / or the suitability of an SAA buffer composition. In one embodiment, as shown in FIG. 2, which does not use beads, the method includes the steps of: (A) providing a recombinant synthetic αS in sCSF as disclosed herein; (B) providing a pre-incubation mixture comprising: (1) monomeric αS protein; (2) a buffer composition; (3) a salt composition; (4) a 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 with intermittent agitation cycles to form an incubated mixture; (E) illuminating 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, where a significant increase in fluorescence indicates amplification of the synthetic seed at the expense of the 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, sCSF is provided as a diluent in a semi-quantitative method for detecting the presence of misfolded αS aggregates. In one embodiment, the method includes the steps of: (A) providing (1) a first human biological sample; and (2) a second human biological sample; (B) providing a pre-incubation mixture as described herein; (C) serially diluting (1) the first biological sample by removing a portion of the first biological sample and combining the removed portion with a volume of sCSF as disclosed herein to form a diluted first biological sample; and (2) serially diluting (2) the second biological sample by removing a portion of the second biological sample and combining the removed portion with a volume of sCSF to form a diluted second biological sample; (D) repeating step (C) a predetermined number of times with the serially diluted first biological sample and the serially diluted second biological sample; and (E) subjecting each of the serially diluted first biological sample and the second biological sample to αS-SAA.

[0017] In another aspect, a semi-quantitative method for detecting the presence of misfolded αS aggregates in a plurality of human biological samples includes the steps of: (A) (1) providing a first human biological sample divided into at least two reaction vessels to form an individual first human biological sample aliquot; (2) providing a second human biological sample divided into at least two reaction vessels to form an individual second human biological sample aliquot; and (3) providing a third human biological sample divided into at least two reaction vessels to form an individual third human biological sample aliquot; (B) providing a pre-incubation mixture comprising: (1) monomeric αS protein; (2) a buffer composition; (3) a salt composition; and (4) an indicator reagent comprising a fluorophore; (C) (1) combining the first aliquot of the first biological sample with the pre-incubation mixture in one of the first plurality of reaction vessels to form a first baseline incubation mixture; (D) providing a second human biological sample divided into at least two reaction vessels to form an individual second human biological sample aliquot; (2) combining the preincubation mixture with a first aliquot of the second biological sample in one of the second plurality of reaction vessels to form a second baseline incubation mixture; and (3) combining the preincubation mixture with a first aliquot of the third biological sample in one of the third plurality of reaction vessels to form a third baseline incubation mixture; (D) incubating the baseline incubation mixture with intermittent agitation cycles to form a baseline incubated mixture; (E) irradiating the baseline incubated mixture with light of a wavelength that excites the fluorophore; (F) determining a fluorescence level during incubation, an increase in the fluorescence level indicating the presence of αS aggregates in each biological sample, and determining the time required for the fluorescence level to reach half of the maximum fluorescence for each biological sample (baseline T 50(G) (1) adding a constant amount of exogenous αS seeds to a second aliquot of the first biological sample to form a spiked first biological sample; (2) adding an equal amount of exogenous αS seeds to a second aliquot of the second biological sample to form a spiked second biological sample; (3) adding an equal amount of exogenous αS seeds to a second aliquot of the third biological sample to form a spiked third biological sample; (4) repeating steps (C)-(F) for the spiked biological sample; and (5) calculating the following equation: (a) φ(first biological sample)=baseline T 50( First biological sample) / spike T 50( (b) φ(first biological sample) = baseline T 50( Second biological sample) / spike T 50( (2nd biosample); and (c) φ(3rd biosample) = baseline T 50( Third biological sample) / Spike T 50( 3) baseline T by determining the normalized activity coefficient φ for each biological sample according to 50 (H) comparing the normalized activity coefficient for each biological sample to a clinically estimated assessment on a disease progression scale.

[0018] The present invention may be more readily understood with reference to the following figures. [Brief description of the drawings]

[0019] [Figure 1] FIG. 13 shows an exemplary depiction of "slow" (i.e., without beads) αS-SAA using sCSF as a negative control and / or in determining the suitability of monomeric αS protein as an αS substrate (specifically, to determine whether monomeric αS protein is prone to self-aggregation).

[0020] [Diagram 2]FIG. 1 shows an exemplary depiction of slow αS-SAA using sCSF containing recombinant synthetic αS seeds in determining the suitability of monomeric αS protein as an αS substrate (specifically, to determine whether monomeric αS protein can aggregate with αS seeds).

[0021] [Diagram 3] FIG. 13 shows a graph of fluorescence intensity over time for the αS substrate slow αS-SAA using Harvard Apparatus artificial CSF+0.155 mg / mL BSA+0.042 mg / mL transferrin as a negative control solution.

[0022] [Figure 4] FIG. 13 shows a graph of fluorescence intensity over time for the αS substrate slow αS-SAA using Harvard Apparatus artificial CSF+0.155 mg / mL BSA+0.042 mg / mL transferrin as a negative control solution.

[0023] [Diagram 5] FIG. 13 shows a graph of fluorescence intensity over time for the αS substrate slow αS-SAA using Harvard Apparatus artificial CSF + 0.2015 mg / mL BSA as the negative control solution.

[0024] [Figure 6] FIG. 13 shows a graph of fluorescence intensity over time for the αS substrate slow αS-SAA using 20 fg 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] FIG. 13 shows a graph of fluorescence intensity over time for the αS substrate slow αS-SAA using 20 fg synthetic seeds in Harvard Apparatus artificial CSF+0.2015 mg / mL BSA as a positive control solution.

[0026] [Figure 8] FIG. 13 shows a graph of fluorescence intensity over time for the qualified αS substrate "fast" (i.e., with beads) αS-SAA using 100 mM HEPES, pH 7.5, 75 mM NaCl, 1.5 mg / mL has and 0.5% sarkosyl as a negative control solution.

[0027] [Figure 9] FIG. 13 shows a graph of fluorescence intensity over time for the unsuitable αS substrate, fast αS-SAA, using 100 mM HEPES, pH 7.5, 75 mM NaCl, 1.5 mg / has HSA and 0.5% Sarkosyl as a negative control solution.

[0028] [Figure 10] FIG. 13 shows a graph of fluorescence intensity over time for the αS substrate fast αS-SAA using 20 fg synthetic seeds in 100 mM HEPES, pH 7.5, 75 mM NaCl, 1.5 hasmL HSA and 0.5% Sarkosyl as a positive control solution.

[0029] [Figure 11] FIG. 1 shows a graph depicting the variability of aggregation inhibition for αS-SAA using CSF from different healthy control (HC) donors.

[0030] [Figure 12] FIG. 13 shows a series of graphs of fluorescence intensity over time during fast αS-SAA conditions of PD positive samples at serial dilution levels using 100 mM HEPES, pH 7.5, 75 mM NaCl, 1.5 mg / mL HSA, and 0.5% Sarkosyl as diluents compared to HC-CSF as diluent and NPH-CSF as diluent.

[0031] [Figure 13]FIG. 1 shows a series of graphs of fluorescence intensity over time in alternative fast αS-SAA conditions of PD positive samples at serial dilution levels using 100 mM HEPES, pH 7.5, 75 mM NaCl, and 1.5 mg / mL HSA as diluent.

[0032] [Figure 14] FIG. 13 shows a chart comparing the SD50 values ​​of two CSF ​​samples in fast αS-SAA or alternative fast αS-SAA conditions in NPH-CSF or sCSF as an inert matrix.

[0033] [Figure 15] FIG. 1 shows a graph depicting normalized seeding coefficient (φ) versus Hoehn-Yahr (H&Y) score for three samples from patients diagnosed with PD.

[0034] [Figure 16] 1 shows an αS-SAA aggregation curve using monomeric αS substrate corresponding to SEQ ID NO: 2 in the presence of olfactory mucosa samples from patients diagnosed with PD compared to non-synucleinopathy controls. The olfactory mucosa samples were diluted using sCSF as provided herein prior to αS-SAA. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[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 with no, perceptibly low, or delayed forms of substrate autoaggregation, but when used as a positive control, it readily allows aggregation of substrates with seeds. sCSF can be used to screen the suitability of substrates and other reagents. cCSF can be used as a diluent for pretreatment of the surrounding matrix. Finally, sCSF can be used as a diluent for serial dilutions of SAA samples to allow for a semi-quantitative version of SAA.

[0036] definition The term "about" in conjunction with a number is intended to include ±10% of that number. This is true whether "about" is modifying an individual number or a number at either or both ends of a numerical range. In other words, "about 10" means 9 to 11. Similarly, "about 10 to about 20" contemplates 9 to 22 and 11 to 18. In the absence of the term "about" or express indication of a range (e.g., ±10%), the exact number is intended. In other words, "10" means 10.

[0037] The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a bead" includes a plurality of beads.

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

[0039] "Misfolded αS aggregates" or "αS aggregates" refers to aggregates of misfolded αS protein. Aggregates may be referred to as oligomers or polymers, and aggregation may be referred to as oligomerization or polymerization.

[0040] A "misfolded αS protein" is an αS protein that lacks all or part of the structural conformation of the protein as it exists in its typical non-pathogenic normal function in a biological system. Misfolded αS protein can aggregate. Misfolded αS protein can localize to protein aggregates. Misfolded αS protein can be a non-functional protein. Misfolded αS protein can be a pathogenic conformer of the protein.

[0041] As used herein, "soluble" species, including soluble misfolded αS aggregates, may form a solution in a body fluid under physiological conditions, whereas "insoluble" species may exist in such body fluids as precipitates, fibrils, deposits, tangles or other non-dissolved forms. Species that dissolve in non-body fluids under physiological conditions but do not dissolve in body fluids are considered insoluble. For example, fibrils such as αS may be soluble in an aqueous solution of a detergent such as sodium dodecyl sulfate (SDS), but may still be insoluble in body fluids under physiological conditions and therefore be considered insoluble.

[0042] Nucleation-dependent aggregation can be characterized by a slow "lag phase" during which aggregation nuclei form and stimulate the rapid formation of further and / or larger aggregates. The lag phase can be minimized or eliminated by the addition of preformed "nuclei" or "seeds." "Seeds" or "nuclei" refer to misfolded αS protein or short fragmented fibrils that have the ability to induce further aggregation.

[0043] Aggregates of misfolded αS protein can be "disaggregated", i.e., disrupted or broken down to release smaller fragments and aggregates, e.g., fragmented fibrils and smaller misfolded aS aggregates. The catalytic activity of a collection of misfolded αS aggregate seeds can be at least partially proportional to the number of seeds in the mixture. Thus, disrupting misfolded αS aggregates to release smaller misfolded αS aggregates and fragmented fibrils as seeds can result in increased catalytic activity for further aggregation.

[0044] The phrases "monomeric αS protein" and "monomeric αS substrate" are used interchangeably and refer to one or more seed-free αS protein molecules in their native, non-pathogenic configuration that do not have catalytic activity for seed-associated aggregation.

[0045] Reference to the term "each" does not mean "all, without exception." For example, if incubation cycles are referenced and "each incubation cycle" is said to include a particular step, then if 10 incubation cycles are performed and one of the incubation cycles includes a particular step, then that incubation cycle is intended to satisfy the restriction.

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

[0047] 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.

[0048] sCSF In one embodiment, the sCSF can include physiological saline solution and plasma proteins.

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

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

[0051] In some embodiments, the sCSF can be a buffered solution. In one embodiment, the buffer comprises HEPES. In some embodiments, the buffer comprises 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 between 1 mM HEPES and 1 M HEPES, including any value or range between any two of these concentrations.

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

[0053] In one embodiment, the sCSF is in a buffered solution, the buffer comprises HEPES and has a pH of about 7.5.

[0054] sCSF also contains one or more plasma proteins. Plasma proteins are proteins that are normally found in plasma. Human CSF contains some of the proteins found in plasma, but at a much lower concentration. Human CSF contains approximately 0.3% plasma proteins or approximately 15-40 mg / dL of total plasma proteins. Thus, in some embodiments, total plasma proteins, which represents the total amount of the various plasma proteins in solution, have 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 globulins (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 protein is selected from the group consisting of HSA, BSA, BSA precursor protein, transferrin, and immunoglobulin G, and combinations thereof.

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

[0057] In some embodiments, the plasma protein is between 0.01 mg / mL and 1.5 mg / mL, between 0.02 mg / mL and 0.8 mg / mL, between 0.02 mg / mL and 0.4 mg / mL, between 0.05 mg / mL and 0.4 mg / mL, about 1.5 mg / mL, about 2.0 mg / mL, about 2.5 mg / mL, about 3.0 mg / mL, about 3.5 mg / mL, about 4.0 mg / mL, about 4.5 mg / mL, about 5.0 mg / mL, about 5.5 mg / mL, about 6.0 mg / mL, about 6.5 mg / mL, about 7.0 mg / mL, about 7.5 mg / mL, about 8.0 mg / mL, about 9.0 mg / mL, about 10.0 mg / mL, about 11.0 mg / mL, about 12.0 mg / mL, about 13.0 mg / mL, about 14.0 mg / mL, about 15.0 mg / mL, about 16.0 mg / mL, about 17.0 mg / mL, about 18.0 mg / mL, about 19.0 mg / mL, about 20.0 mg / mL, about 21.0 mg / mL, about 22.0 mg / mL, about 23.0 mg / mL, about 24.0 mg / mL, about 25.0 mg / mL, about 26.0 mg / mL, about 27.0 mg / mL, about 28.0 mg / mL, about 29.0 mg / mL, about 30.0 mg / mL, about 31.0 mg / mL, about 32.0 mg / mL, about 33.0 mg / mL, about 34.0 mg / mL, about 35.0 mg / mL, about 36.0 mg / mL, about 37.0 mg / mL, about 38.0 mg / mL, about 39.0 The composition may consist of, or consist essentially of, HSA at a concentration of 0.01 mg / mL to 15 mg / mL, including about 8.5 mg / mL, about 9.0 mg / mL, about 9.5 mg / mL, about 10.0 mg / mL, about 10.5 mg / mL, about 11.0 mg / mL, about 11.5 mg / mL, about 12.0 mg / mL, about 12.5 mg / mL, about 13.0 mg / mL, about 13.5 mg / mL, about 14.0 mg / mL, about 14.5 mg / mL, about 15.0 mg / mL, or any value or range between any two of those concentrations.

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

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

[0060] In some embodiments, sCSF can essentially consist of 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 sarkosyl), particularly when acting as a control solution for 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 its entirety. In an embodiment in which the assay does not include sarkosyl, sCSF essentially consists of HEPES, NaCl solution, HSA, and sarkosyl. In one embodiment, sCSF essentially consists of 100 mM HEPES, pH 7.5, 75 mM NaCl, HSA, and 0.5% sarkosyl.

[0061] In some embodiments, sCSF is used as a component of a positive control. The positive control can be used to provide the expected results when αS aggregates are present in the biological sample (i.e., a positive result). The positive control further comprises a seed, whether endogenous or synthetic. In some embodiments, sCSF is a component of a negative control. The negative control can be used to provide the expected results when αS aggregates are not present in the biological sample (i.e., a negative result). The negative control further comprises a monomeric αS substrate and all components of the pre-incubation mixture. In further embodiments, sCSF is a comparative control that comprises 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 can be used as a diluent in semi-quantitative αS-SAA.

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

[0063] In another embodiment, synthetic seeds can be used to generate a calibration curve in sCSF. The kinetic parameters of such a curve can be useful for determining the concentration of endogenous seeds in a biological sample.

[0064] In yet another embodiment, sCSF can be used as a diluent in SAA of tissues such as skin or olfactory mucosa.Certain tissues can interfere with SAA.When seed concentration in tissue is high, sCSF can be used to dilute the interfering surface of tissue, but still retain enough seed in SAA to undergo detectable amplification.

[0065] Biological samples "Biological sample" is meant to include any biological sample from a subject suitable for analysis to detect misfolded αS aggregates. Suitable biological samples may include, for example, fluids or fluids represented by amniotic fluid, bile, blood, plasma, CSF, earwax, skin, exudate, feces, gastric juice, lymph, milk, mucus, mucous membranes, including nasal mucosa, including olfactory mucosa, ascites, pleural fluid, pus, saliva, sebum, semen, sweat, synovial fluid, tears, and urine. When a bodily fluid is removed from the body and, if applicable, processed and / or prepared for use in the methods and kits described herein, it is referred to as a "biological sample" or simply a "sample". When a sample, such as a CSF sample, a skin sample, an olfactory mucosa sample, a blood or plasma sample, or a 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 SAA, unless the context clearly dictates otherwise. The methods and kits described herein are performed and used in vitro.

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

[0067] In one embodiment, the monomeric αS substrate comprises, consists essentially of, or consists of a wild-type or recombinant human αS protein having 140 amino acids and a molecular mass of 14,460 Da and represented by the following sequence: SEQ ID NO:1:

number

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

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

number

[0070] In some embodiments, the monomeric αS substrate may be any of the monomeric αS substrates disclosed in U.S. Patent No. 11,079,396 and conservative variants thereof. In some embodiments, the monomeric αS substrate or conservative variants thereof specifically excludes the monomeric αS substrate consisting of SEQ ID NO:1.

[0071] In some embodiments, monomeric αS substrates can 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), the entirety of which is incorporated herein by reference.

[0072] In some aspects, monomeric αS substrates can be expressed and prepared as described in US Pat. No. 11,254,718, the entirety of which is incorporated herein by reference.

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

[0074] The pre-incubation mixture may contain various concentrations of monomeric αS substrate as a function of the total volume of the pre-incubation mixture before performing the incubation cycle. In some embodiments, the pre-incubation mixture may contain a concentration or concentration range of monomeric αS substrate of between about 500 nM and about 500 μM; between about 1 μM and about 200 μM; between about 5 μM and about 100 μM; between about 10 μM and about 50 μM; between about 50 μM and about 75 μM; about 65 μM (i.e., about 1 mg / ml); 65 μM; between about 10 μM and 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 preincubation mixture comprises a monomeric αS substrate at a concentration of about 0.3 mg / ml as a function of the total volume of the preincubation mixture before the incubation cycle is performed.

[0075] buffer composition The pre-incubation mixture may include various buffer compositions. The buffer composition may be effective to maintain the pH of the reaction 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 one embodiment, the buffer composition may be effective to maintain the pH of the reaction mixture at about 6.5. In some embodiments, the pre-incubation mixture includes one or more of the following buffers: Tris-HCL, MES, PIPES, MOPS, BES, TES and HEPES. In some embodiments, the buffer includes PIPES at a concentration of about 100 mM, about 200 mM, about 300 mM, about 400 mM, about 500 mM, about 600 mM, or about 700 mM. In one embodiment, the buffer comprises PIPES at a concentration of about 100 mM.

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

[0077] Indicator In some embodiments, the pre-incubation mixture comprises an indicator for determining whether a detectable amount of misfolded αS aggregates is present in the reaction mixture. The indicator can be characterized as exhibiting an indicating state in the presence of a detectable amount of misfolded αS aggregates and exhibiting a non-indicating state in the absence of a detectable amount of misfolded αS aggregates. Determining the presence of misfolded αS aggregates in the biological sample can include detecting an indicating state of the indicator for misfolded αS aggregates. The indicating state of the indicator and the non-indicating state of the indicator can be characterized by a difference in fluorescence. Thus, determining the presence of misfolded αS aggregates in the biological sample can include detecting a difference in fluorescence. In some embodiments, a molar excess of the indicator can be used, e.g., a molar excess greater than the total molar amount of monomeric αS substrate and misfolded αS aggregates in the reaction mixture.

[0078] In some embodiments, the indicator comprises a fluorophore. In some embodiments, the indicator may comprise one or more of thioflavin-T (ThT), Congo Red, mI-stilbene, chrysamine G, PIB, BF-227, X-34, TZDM, FDDNP, IMPY, NIAD-4, luminescent conjugated polythiophenes, fusions with fluorescent proteins such as green fluorescent protein and yellow fluorescent protein, derivatives thereof, and the like. A suitable indicator is ThT. In one embodiment in which the indicator comprises ThT, the concentration of ThT in the pre-incubation mixture is between about 5 μM and about 10 μM. In one embodiment in which the indicator comprises ThT, the concentration of ThT in the pre-incubation mixture is 10 μM.

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

[0080] Incubation conditions The reaction mixture can be held in a suitable sized container, such as a multi-well plate having multiple wells. For example, the multi-well plate can include 96 wells. The wells of the multi-well plate can have a volume 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 multi-well plate contains one or more beads.

[0081] The temperature of the reaction mixture in each incubation cycle, in °C, can be independently 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, e.g., 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 performed at about normal physiological temperature for a warm-blooded animal. In further embodiments, incubation of the reaction mixture is performed 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 comprises incubating the reaction mixture at a temperature of about 42°C.

[0082] In some embodiments, disaggregating 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, homogenization, and the like. Shaking may include periodic stirring, such as orbital stirring. Periodic stirring may be performed at between about 50 revolutions per minute (RPM) and 10,000 RPM. Periodic stirring may be performed at between about 200 RPM and about 2000 RPM. Periodic stirring may be performed at about 500 RPM or about 600-800 RPM. In one embodiment, shaking includes orbital stirring at about 800 RPM. Disaggregation of the incubation mixture may be performed after each incubation cycle for between about 5 seconds and about 10 minutes, between about 30 seconds and about 1 minute, between about 45 seconds and about 1 minute, about 1 minute, and the like.

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

[0084] The incubation cycles may be performed for a time period between about 1 minute and about 5 hours, between about 10 minutes and about 2 hours, between about 15 minutes and about 1 hour, between about 25 minutes and about 45 minutes, etc. In some embodiments, incubating the reaction mixture and disaggregating at least a portion of the misfolded αS aggregates comprises an incubation cycle lasting about 0.1 to 1 hour. Each incubation cycle may include independently incubating and disaggregating the reaction mixture with one or more of: incubation between about 1 minute and about 5 hours and disaggregation between about 5 seconds and about 10 minutes; incubation between about 10 minutes and about 2 hours and disaggregation between about 30 seconds and about 1 minute; incubation between about 14 minutes and about 1 hour and disaggregation between about 45 seconds and about 1 minute; incubation between about 25 minutes and about 45 minutes and disaggregation between about 45 seconds and about 1 minute; and incubation for about 1 minute and disaggregation for about 1 minute. In one embodiment, each incubation cycle comprises about 14 minutes of incubation and about 1 minute of disaggregation.

[0085] beads In some embodiments, the pre-incubation mixture may include one or more beads. Beads are small, typically spherical objects, such as high-density beads with low-friction surfaces that are commonly used as bearing beads. The inclusion of beads in the reaction mixture increases the rate of formation of misfolded αS aggregates from monomeric αS substrate and soluble misfolded αS protein of biological samples. These beads are different in composition and function from the antibody-coated magnetic or paramagnetic beads or particles (e.g., Dynabeads) used in the enrichment and / or immunodepletion steps described elsewhere herein.

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

[0087] In some embodiments, the beads comprise, consist essentially of, or consist of Si3N4. In some embodiments, the beads comprise, consist essentially of, 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.

[0088] In some embodiments, the beads included 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, while 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 in the range of 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, while 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 comprise, consist essentially 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 comprise, consist essentially of, or consist of Si3N4, have an average diameter of 3.175 mm, and are unblocked. In some embodiments, the beads comprise, consist essentially of, or consist of borosilicate glass, have an average diameter of 2.45 mm, and are unblocked. 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 size distribution is defined such that greater than 90% of the beads are found between 80-120% of the average bead diameter or between 90-110% of the average bead diameter.

[0089] The number of beads included 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 includes a plurality of 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 a protein. Blocking the surface of a bead with a protein refers to providing a coating or layer on all or a substantial portion of the surface of the bead. Any suitable biocompatible protein can be used to coat the surface of the bead. A suitable protein for use in blocking the surface of the bead is albumin, such as BSA. Other suitable blocking proteins can include casein or milk powder. The bead or beads can be blocked by immersing the bead or beads in a solution containing the protein. The solution can be an aqueous and / or buffered solution, such as PIPES, Tris-HCl, MES, MOPS, BES, TES, and HEPES.

[0091] The incubation mixture is held in a suitable sized container, 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 multi-well plate that includes multiple wells. For example, the multi-well plate can include 96 wells. In one embodiment, the container can be a black bottom 96-well plate (Costar 3916), for example, when the beads are Si3N4 beads. In one embodiment, the container can be a bottom-read Greiner CBP plate, for example, when the beads are Si3N4 beads. In one embodiment, the container can be a clear bottom 96-well plate (Costar 3603), for example, when the beads are borosilicate glass beads.

[0092] detection The detection includes repeating the steps of incubating and disaggregating the reaction mixture as necessary to amplify sufficient misfolded αS aggregates present in the biological sample to obtain an amplified incubation mixture having a detectable amount of misfolded αS aggregates. The incubation mixture is contacted with an indicator reagent, and the level of fluorescence of the amplified reaction mixture can be determined.

[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 structures of amyloid fibrils, ThT fluoresces strongly with excitation and emission maxima at about 435 nm (or about 440 nm, depending on the fluorometer or spectrofluorometer) and about 485 nm (or about 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, ThT fluorescence emission intensity can be compared with the level of a corresponding control sample when performing 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 can be displayed graphically 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] The increase in fluorescence level indicates the presence of αS aggregates in the biological sample.In some embodiments, the significant increase in fluorescence level indicates the presence of αS aggregates in the biological sample.In some embodiments, "significant increase" is an increase in the fluorescence level of the incubated mixture at maximum fluorescence that is at least two times the standard deviation of the fluorescence of the incubated mixture at maximum fluorescence compared to the fluorescence level of the incubated mixture at any time during the lag phase, indicating the presence of αS aggregates in the biological sample.

[0096] Neuropathies and Synucleinopathies αS aggregation can be related to protein misfolding disorders (PMDs), such as PD, LBD and MSA.However, existing technology does not clearly show whether this aggregation phenomenon is the cause of these diseases; it is only speculated that these misfolded αS aggregates can cause cell dysfunction and tissue damage, among other effects.That is, the method and kit described herein do not directly determine whether an individual has a certain disease based on whether the individual has misfolded αS aggregates.

[0097] The information obtained by the method and kit described herein for determining whether misfolded αS aggregates exist in a sample is merely an intermediate result or a kind of reference information, and the result cannot be used to directly draw a conclusion that an individual has a certain disease.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 for aiding in the diagnosis of PD, LBD, MSA, or the spectrum of each embodiment in subjects with neurological disorders.Neurological disorders are any disorders of the nervous system.Examples of neurological disorders include movement 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 synucleinopathy.Synucleinopathy is a neurodegenerative disease characterized by abnormal accumulation of αS aggregates in the cells of the nervous system, such as neurons, nerve fibers, and glial cells.In some embodiments, synucleinopathy has symptoms related to PD, LBD or MSA, including, for example, cognitive impairment, sleep disorder and gastrointestinal dysfunction.

[0100] In some embodiments, the sample can be taken from a subject that does not show clinical signs of PD, LBD or MSA.In other embodiments, the biological sample can be taken from a subject that shows 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 a subject diagnosed with PD with a treatment for PD and / or its symptoms. Deep brain stimulation can be used to reduce motor symptoms associated with PD. Drugs useful for treating motor symptoms of PD include levodopa, dopamine agonists and monoamine oxidase B inhibitors. However, additional treatments for PD continue to be developed. See Radhakrishnan DM, Goyal V, Neurol India., 66(Suppl):S26-S35(2018) and Iarkov et al., Front Aging Neurosci., 12:4(2020).

[0102] Supplemental Diagnostic Tests In some embodiments, the method may further include additional tests to confirm the indication based on αS-SAA, for example, to further distinguish between misfolded αS aggregates from patients whose αS-SAA indicates that they have PD and those whose αS-SAA indicates that they have MSA or LBD. Examples of additional tests include using a ligand with high affinity for one of the misfolded αS aggregates of PD or MSA or LBD, creating a profile of protease-resistant fragments from the misfolded αS aggregates, and evaluating 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 includes a known amount of monomeric αS substrate; a known amount of an indicator; a buffer composition; optionally, one or more beads having 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, about 2.45 mm, or about 3.175 mm; and optionally, sarkosyl. The kit also includes sCSF as described herein. The kit may include instructions directing a user to perform the method of 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 the biological sample from the surrounding matrix, and for serially diluting the biological sample for semi-quantitative αS-SAA. The kit should also include packaging for holding the components of the kit.

[0104] The kit generally includes a package having one or more containers that hold the reagents, either as one or more separate compositions, or, optionally, as a mixture, if the compatibility of the reagents allows.The kit may further include buffers, labeling agents, controls, and any other materials required to perform the detection of misfolded αS aggregates.The kit may also include tools for obtaining samples from a subject, such as swabs or other bodily fluid collection devices.

[0105] The kit may also include instructions for performing a method of using the kit to guide the treatment of synucleinopathy in a subject. The instructions included in the kit may be attached to a packaging material or may be included as a package insert. The instructions are typically, but are not limited to, written or printed. Any medium capable of storing such instructions and transmitting 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), and the like. As used herein, the term "instructions" may include the address of an internet site that provides the instructions.

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

[0107] example The present invention is illustrated by the following examples, however the specific examples, materials, amounts 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 the "slow assay" αS-SAA condition A control solution was used as a negative control under "slow assay" αS-SAA conditions. The general conditions for the "slow assay" αS-SAA are described in U.S. Pat. No. 10,989,718. The specific αS-SAA conditions used to obtain the results herein are as follows:

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

[0110] Incubation cycles were performed on the incubation mixtures, each of which included (1) incubating the first incubation mixture for 29 min; and (b) orbitally shaking the incubation mixture at 700 rpm for 1 min using an Omega FLUOstar at a constant temperature of 37° C. for a total of 300 h to form an incubated control solution. ThT fluorescence was measured in the plate every 30 min at 490 nm after excitation at 440 nm.

[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] All of the substrates tested were intended to correspond to SEQ ID NO:2, however the substrates were prepared at different times, some with different expression and / or purification conditions.

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

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

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

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

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

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

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

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

[0121] Example 3: sCSF used as a negative control during the "fast assay" αS-SAA condition sCSF was used as a negative control under "fast assay" αS-SAA conditions. The general conditions for the "fast assay" αS-SAA are described in US Pat. No. 11,079,396. Specifically, αS-SAA was performed using 0.3 mg / ml of unseeded αS represented by SEQ ID NO:2 (suitably prepared according to the protocol disclosed in US Pat. 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 fluorescence intensity over time for the αS substrate fast αS-SAA using 100 mM HEPES, pH 7.5, 75 mM NaCl, 1.5 mg / mL HSA and 0.5% Sarkosyl as a negative control solution. In Figure 8, no autoaggregation of the αS substrate is observed. Of 96 wells, only 3 (3.1%) showed autoaggregation. In a comparative experiment, the percentage of wells with autoaggregation using healthy human CSF as a control solution produced 5% autoaggregation.

[0123] Example 4: sCSF for use in screening substrates using "rapid assay" αS-SAA conditions Substrates were screened under "fast assay" αS-SAA conditions using sCSF (essentially as a negative control). In this experiment, αS substrate was purified in a manner that deviated from the teachings of U.S. Pat. No. 11,254,718. Figure 9 shows a graph of fluorescence intensity over time for fast αS-SAA of the ineligible αS substrate using 100 mM HEPES, pH 7.5, 75 mM NaCl, 1.5 mg / mL HSA and 0.5% Sarkosyl as a control solution. As shown in Figure 9, the αS substrate exhibited self-aggregation.

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

[0125] Figure 10 shows a graph of fluorescence intensity over time for the αS substrate fast αS-SAA in the presence of 20 fg seeds using 100 mM HEPES, pH 7.5, 75 mM NaCl, 1.5 mg / mL HSA and 0.5% Sarkosyl 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, determine the dilution at which 50% of αS-SAA reactions are positive (SD 50 The relative amount of αS was determined by estimating the positive titer of the αS. This estimation depends purely on the number of positive wells, so kinetic changes due to a particular CSF matrix are not relevant. When evaluating increasing dilutions of CSF from PD and DLB patients, the positivity of technical replicates decreases. However, when diluting using CSF from different HC donors, the number of positive wells can vary. In other words, CSF from different patients has different effects on aggregation. This variable effect is demonstrable with rec-seed, which aggregates very reproducibly when spiked into buffer. However, when spiked into CSF ​​from different patients, aggregation varies 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 dilutions, as the titer changes depending on the HC-CSF used. For reproducible titration based on serial dilutions, 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 fixed 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. An aliquot of the mixture is taken for αS-SAA. The mixed sample is diluted again to obtain higher dilutions with even lower concentrations of seeds. This procedure is repeated until the highest desired dilution is reached, i.e. the dilution with the lowest concentration of seeds. Here, 1:3 serial dilutions up to 1:81 (meaning each dilution contains 66.6% dilution) were performed. By analyzing the samples in this way, the relative seed numbers can be determined, since samples containing more seeds show seeding activity at higher dilutions than samples with lower concentrations of seeds.

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

[0129] Figure 12 shows a series of graphs of fluorescence intensity over time during the "fast assay" αS-SAA conditions at serial dilution levels. Figure 12 demonstrates that sCSF consisting essentially of 100 mM HEPES, pH 7.5, 75 mM NaCl, 1.5 mg / mL HSA, and 0.5% sarkosyl reproduced results obtained using CSF from normal pressure hydrocephalus ("NPH") patients, and the results changed when CSF from HCs was used. Both negative NPH and HCs are negative in this assay. This highlights the need for a stable and reproducible matrix to allow semi-quantification. The "SD50," the dilution required for 50% of the wells to be positive, can be calculated to estimate the amount of seeds. However, while it is possible to estimate the number of seeds with a standard deviation, the definition of "seed" is not clear. Thus, this method provides a semi-quantitative alternative to compare seeding activity between samples, rather than quantification of misfolded αS. Furthermore, sCSF inhibits amplification less than HC-CSF, allowing a larger window to be distinguished between samples.

[0130] FIG. 13 shows a series of graphs of fluorescence intensity over time in alternative "fast assay" αS-SAA conditions (specifically, the assay itself contains sarkosyl, as described in U.S. Pat. No. 11,079,396) of PD-positive samples at serial dilution levels using 100 mM HEPES, pH 7.5, 75 mM NaCl, and 1.5 mg / mL HSA as diluent. The three circles under each graph represent the three replicates analyzed per dilution. The grey circles represent replicates that showed detectable seed amplification, and the white circles represent replicates that showed no detectable seed amplification. This notation is also used in FIG. 14.

[0131] With further reference to FIG. 14, two CSF ​​samples that showed strong (2603) and weak (2978) amplification patterns in the fast assay conditions were serially diluted 3-fold into NPH-CSF and sCSF using the three substrates and two versions of the fast assay. SD 50was calculated for each sample using the Spearmen-Karber model. With AMP-A, there was a substantial difference in seeding activity between these two CSF ​​samples, as the estimated number of 2603 seeds was higher than that of 2978 seeds (67.5 > 10.8). A similar pattern was observed with AMP50. SD 50 The results shown in Figure 14 are highly reproducible, since the estimates of usually show a variation of several orders of magnitude. Using the same AMP50 substrate, the dilution into sCSF was remarkably similar to the dilution into NPH-CSF. The alternative rapid assay version shows a lower limit of detection (higher analytical sensitivity), which is also observed here, since higher dilutions of the 2603 and 2978 CSF samples showed positive replicates. Overall, all conditions shown in Figure 14 were able to estimate a higher seed number in the 2603 case than in the 2978 case. (N / T: not tested. SE: standard error.)

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

[0133] Example 9: Detection of misfolded αS aggregates in the olfactory mucosa 5.1 Collection and preprocessing The patient was subjected to local anesthesia (nasal spray with lidocaine) 10 minutes before the procedure. Through a rigid fiberscope, the olfactory mucosa (between the nasal septum and the middle turbinate) was identified. 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 scratched to collect the sample. The swab was removed from the nose and placed into a 15 mL conical tube containing 3 mL of physiological solution (saline buffer). Using disposable scissors, the swab was cut to fit within the 15 mL conical tube. The tube was vortexed for 1 minute. Using disposable tweezers, 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 tweezers, 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. 3 mL from each of the 15 mL tubes (total of 9 mL) was pooled into a single 15 mL tube, which was centrifuged at 800×g for 20 minutes at 4° C. 8 mL of supernatant saline was discarded. The pellet and 1 mL of saline were stored at −80° C.

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

[0135] 5.3 Sample processing for αS-SAA. OM / PBS samples were thawed and a 1:20 dilution was made by pipetting 4 μL of sample into 76 μL of sCSF (Amprion, Cat# S2022). A 1:400 (12 ng / 40 μL) dilution was prepared by pipetting 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 preincubation mixture. The preincubation mixture contained 100 mM PIPES pH 6.5, 500 mM NaCl, 10 μM ThT, 0.1% Sarkosyl, and two Si3N4 beads (1 / 8 inch, grade 5). The plates are orbitally shaken for 1 min followed by incubation for 14 min at 42 °C for a total cycle of 15 min. When using the robotic arm associated with the Omega shaker / reader (8 plates at a time), agitation was set to 600 RPM and the standalone Omegas (1 plate at a time) was set to 800 RPM. Fluorescence readings were taken at 440-10 nm (excitation) and 490-10 nm (emission). Figure 16 shows αS-SAA aggregation curves from patients diagnosed with PD compared to non-synucleinopathy controls.

[0137] The complete disclosures of all patents, patent applications, and publications cited herein, as well as electronically available materials, are incorporated by reference, regardless of whether a specific citation in this specification states so. The above detailed description and examples are given for clarity of understanding only. No unnecessary limitations should be understood therefrom. The present invention is not limited to the exact details shown and described, and variations obvious to one skilled in the art will be included within the invention defined by the claims.

Claims

1. (A) human serum albumin (HSA); (B) an aqueous solution of NaCl, and (C) (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES) 1. A composition consisting essentially of: wherein said composition is inactive towards monomeric alpha-synuclein protein and a fluorescent protein aggregation indicator.

2. The composition described in claim 1, wherein the HSA is present at a concentration of approximately 1.5 mg / mL.

3. The composition described in claim 1, wherein the HSA is present at a concentration of approximately 15 mg / mL.

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

5. The composition of claim 1, wherein the HEPES maintains a pH of the composition of about 8.

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

7. (1) about 1.5 mg / mL human serum albumin, (2) 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, having a pH of about 8; (3) Approximately 75 mM NaCl, and optionally (4) about 0.5% sarkosyl 1. A composition consisting essentially of: wherein said composition is inactive towards monomeric alpha-synuclein protein and a fluorescent protein aggregation indicator.

8. (A) HSA, (B) an aqueous solution of NaCl, and (C) HEPES The composition of claim 1 consisting of:

9. (A) human serum albumin (HSA), (B) NaCl aqueous solution, (C) 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and (D) Sarkosyl 1. A composition consisting essentially of: wherein said composition is inactive towards monomeric alpha-synuclein protein and a fluorescent protein aggregation indicator.

10. The composition described in claim 9, wherein the HSA is present at a concentration of approximately 1.5 mg / mL.

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

12. The composition of claim 9, wherein the HEPES maintains the pH of the composition at about 8.

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

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

15. (A) HSA, (B) NaCl aqueous solution, (C) HEPES, and (D) Sarkosyl The composition of claim 9 consisting of:

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