WO2021183469A1

WO2021183469A1 - Cerebrospinal fluid assay control solution

Info

Publication number: WO2021183469A1
Application number: PCT/US2021/021441
Authority: WO
Inventors: Luis CONCHA; Carly FARRIS
Original assignee: Amprion, Inc.
Priority date: 2020-03-09
Filing date: 2021-03-09
Publication date: 2021-09-16

Abstract

A control solution is provided as a control for assaying for misfolded alpha-synuclein ("αS") proteins in human cerebrospinal fluid samples. The control solution may comprise an aqueous physiological salt solution and a plasma protein. As a negative control, the control solution decreases and/or delays self-aggregation of a substrate during protein misfolding cyclic amplification ("PMCA"). As a positive control, the control solution permits aggregation of the substrate with αS protein "seeds" during PMCA.

Description

CEREBROSPINAL FLUID ASSAY CONTROL SOLUTION

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority from U.S. Provisional Patent Application No. 62/986,921, filed on March 9, 2020, which is incorporated by reference herein in its entirety.

SEQUENCE LISTING

[0002] A Sequence Listing has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy, created on March 8, 2021, is named aCSF_ST25.txt and is 2,884 bytes in size.

BACKGROUND

[0003] Most protein molecules must fold into defined three-dimensional structures to acquire functional activity. However, protein chains can adopt a multitude of conformational states, and their biologically active conformation is often only marginally stable. Metastable proteins tend to populate misfolded species that are prone to forming toxic aggregates, including soluble oligomers and fibrillar amyloid deposits, which are linked with neurodegeneration in e.g., Parkinson’s Disease (“PD”) and many other pathologies.

[0004] Seeding Amplification Assays, including protein misfolding cyclic amplification (“PMCA”), have provided increased sensitivity for detecting misfolded protein aggregates in tissues and fluids for titers that are too low for detection by traditional immunoassay methods. Amplification assays allow for much earlier diagnosis of pathologies involving misfolded proteins, which may prove critical to treatment of the disease and prevention or mitigation of symptoms. [0005] PMCA relies on accelerated aggregation of monomeric protein substrate with misfolded protein present in a biological sample, while avoiding self-aggregation of the substrate. As a negative control, amplification assays commonly use a control solution that consists of a substrate in a misfolded protein-free solution. The control solution is subjected to the same conditions as the biological sample. Generally speaking, if the control solution shows no aggregation, and the biological sample shows no aggregation, the patient is considered “negative.” If the control solution shows no aggregation, and the biological sample shows aggregation, the patient is considered “positive.” Thus, the control solution must accurately reflect the absence of self-aggregation in the case of a negative control. As a positive control, amplification assays commonly use a control solution that consists of a substrate in a solution containing “seeds” - that is, protein aggregates. The control solution is subjected to the same conditions as the biological sample. If the control solution shows aggregation, then the substrate can be considered “active.” The control solution must function properly as a positive control and as a negative control. Otherwise, the PMCA results of the biological sample will not have meaning.

[0006] Finally, it is known that cerebrospinal fluid (“CSF”) closely reflects brain matter as it respects the presence of misfolded protein. See, e.g., Shahnawaz, M., Mukherjee, A., Pritzkow, S. et al. Discriminating a-synuclein strains in Parkinson’s disease and multiple system atrophy. Nature 578, 273-277 (2020). Thus, detection of misfolded protein in CSF is of particular importance. Unfortunately, existing artificial CSF control solutions too frequently induce or permit self-aggregation of the substrate (in a negative control) or, less commonly, prevent aggregation in the presence of seeds (in a positive control). And it is not possible to rely on human CSF as a reproducible matrix for the commercial use of PMCA for diagnostic purposes. Although simpler than blood, CSF is a very complex biofluid that presents dramatic differences between individual donors. These differences are greater when comparing healthy versus sick patients, but patients with similar health status can have very different CSF compositions. In some cases, CSF samples from healthy donors may induce self-aggregation, while in other cases, CSF samples from healthy donors may partially inhibit seeded aggregation.

[0007] Thus, a need exists for a control solution that will accurately reflect the absence of misfolded protein when used as a negative control, in the form of no, perceptively low, or delayed substrate self-aggregation, yet will readily permit aggregation of the substate with seeds when used as a positive control. As one of the most challenging aspects of PMC A is identifying high quality substrate, that is, substrate that does not have a tendency to self-aggregate but does aggregate in the presence of misfolded protein in a biological sample, a need also exists for a control solution that may be used to screen for substrate quality.

SUMMARY

[0008] In one aspect, a control solution is provided as a control for assaying misfolded alpha- synuclein (“aS”) protein in CSF using PMCA. The control solution may comprise an aqueous physiological salt solution and a plasma protein having a concentration of 1.0 mg/mL or less. [0009] A method is also provided for determining the presence of soluble, misfolded aS protein in a biological sample. In one aspect, the method comprises: (I) in a first assay, (A) providing a first incubation mixture, the first incubation mixture comprising: (1) a first portion of a seed-free monomeric aS protein; (2) a first buffer composition; (3) a first salt solution; (4) a first indicator; and (5) a control solution comprising an aqueous physiological salt solution and a plasma protein having a concentration of 1.0 mg/mL or less; (B) conducting an incubation cycle two or more times on the first incubation mixture, each incubation cycle comprising: (1) incubating the first incubation mixture for a first predetermined time; and (b) shaking the incubation mixture for a second predetermined time, to form an incubated control solution; and (C) detecting via indicator fluorescence any misfolded aS protein aggregates in the incubated control solution, thereby providing a first fluorescence intensity; (II) in a second assay, (A) providing a second incubation mixture, the second incubation mixture comprising: (1) a second portion of the seed- free monomeric aS protein; (2) a second buffer composition; (3) a second salt solution; (4) a second indicator; and (5) a human CSF sample; (B) conducting an incubation cycle two or more times on the second incubation mixture, each incubation cycle comprising: (1) incubating the second incubation mixture effective to cause aggregation of the second portion of the seed-free monomeric aS substrate in the presence of any soluble, misfolded aS protein in the human CSF sample; and (b) shaking the incubation mixture effective to break up any misfolded aS protein aggregates present, to form an incubated human CSF sample; and (C) detecting via indicator fluorescence any misfolded aS protein aggregates in the incubated human CSF sample, thereby providing a second fluorescence intensity; and (III) comparing the first fluorescence intensity to the second fluorescence intensity, wherein a second fluorescence intensity that is significantly greater than the first fluorescence intensity is indicative of the presence of soluble, misfolded protein in the human CSF sample.

BRIEF DESCRIPTION OF THE FIGURES

[0010] The present invention may be more readily understood by reference to the following Figures, wherein:

[0011] Figure 1 is an example depiction of the slow PMCA process using a control solution. [0012] Figure 2 is an example depiction of the slow PMCA process using a biological sample that contains soluble, misfolded aS protein.

[0013] Figure 3 shows a graph of fluorescence intensity over time for slow PMCA of an aS substrate using Harvard perfusion fluid + 0.155mg/mL bovine serum albumin (“BSA”) + 0.042mg/mL transferrin as a negative control solution. [0014] Figure 4 shows a graph of fluorescence intensity over time for slow PMCA of an aS substrate using Harvard perfusion fluid + 0.155mg/mL BSA + 0.042mg/mL transferrin as a negative control solution.

[0015] Figure 5 shows a graph of fluorescence intensity over time for slow PMCA of an aS substrate using Harvard perfusion fluid + 0.2015mg/mL BSA as a negative control solution. [0016] Figure 6 shows a graph of fluorescence intensity over time for slow PMCA of an aS substrate in the presence of 20fg of “seeds” using Harvard perfusion fluid + 0.155mg/mL BSA + 0.042mg/mL transferrin as a positive control solution.

[0017] Figure 7 shows a graph of fluorescence intensity over time for slow PMCA of an aS substrate in the presence of 20fg of “seeds” using Harvard perfusion fluid + 0.2015mg/mL BSA as a positive control solution.

[0018] Figure 8 shows a graph of fluorescence intensity over time for fast PMCA of an aS substrate using Harvard perfusion fluid + 0.155mg/mL BSA + 0.042mg/mL transferrin as a negative control solution.

DETAILED DESCRIPTION

[0019] Control solutions are provided for use as controls for assaying human CSF samples. The control solutions may comprise an aqueous physiological salt solution and a plasma protein having a concentration of 1.0 mg/mL or less.

[0020] A method is also provided for determining the presence of soluble, misfolded aS protein in a biological sample. In one aspect, the method comprises: (I) in a first assay, depicted in Figure 1, (A) providing a first incubation mixture, the first incubation mixture comprising: (1) a first portion of a seed-free monomeric aS protein; (2) a first buffer composition; (3) a first salt solution; (4) a first indicator; and (5) a control solution comprising an aqueous physiological salt solution and a plasma protein having a concentration of 1.0 mg/mL or less; (B) conducting an incubation cycle two or more times on the first incubation mixture, each incubation cycle comprising: (1) incubating the first incubation mixture for a first predetermined time; and (b) shaking the incubation mixture for a second predetermined time, to form an incubated control solution; and (C) detecting via indicator fluorescence any misfolded aS protein aggregates in the incubated control solution, thereby providing a first fluorescence intensity; (II) in a second assay, depicted in Figure 2, (A) providing a second incubation mixture, the second incubation mixture comprising: (1) a second portion of the seed-free monomeric aS protein; (2) a second buffer composition; (3) a second salt solution; (4) a second indicator; and (5) a human CSF sample; (B) conducting an incubation cycle two or more times on the second incubation mixture, each incubation cycle comprising: (1) incubating the second incubation mixture effective to cause aggregation of the second portion of the seed-free monomeric aS substrate in the presence of any soluble, misfolded aS protein in the human CSF sample; and (b) shaking the incubation mixture effective to break up any misfolded aS protein aggregates present, to form an incubated human CSF sample; and (C) detecting via indicator fluorescence any misfolded aS protein aggregates in the incubated human CSF sample, thereby providing a second fluorescence intensity; and (III) comparing the first fluorescence intensity to the second fluorescence intensity, wherein a second fluorescence intensity that is significantly greater than the first fluorescence intensity is indicative of the presence of soluble, misfolded protein in the human CSF sample. Control Solution

[0021] In one aspect, a control solution is provided as a control for assaying for misfolded ocS proteins in human CSF samples. The control solution may comprise an aqueous physiological salt solution and a plasma protein. The control solution decreases and/or delays self-aggregation of substrate during PMC A.

[0022] The aqueous physiological salt solution may be designed to mimic physiological CSF. The aqueous physiological salt solution may comprise a water-based solution comprising a salt that corresponds to at least one of the salts that are present in human CSF. In some aspects, the aqueous physiological salt solution comprises one or more of sodium, potassium, chloride, calcium, magnesium, and phosphate ions. The salts in the aqueous physiological salt solution may be provided in concentrations similar to those found in human CSF. For example, an aqueous physiological salt solution may comprise 130-160 mM NaCl; 2.7-3.9 mM KC1; 1-10 mM CaCl₂2H₂0; 0.5-10 mM MgCl₂6H₂0; 0.5-5 mM Na₂HP0₄7H₂0; and 0.1-2 mM NaH₂P0 H₂0. In some aspects, the aqueous physiological salt solution is comprised of 148 mM NaCl; 3 mM KC1; 1.4 mM CaCl₂2H₂0; 0.8 mM MgCl₂6H₂0; 0.8 mM Na₂HP0₄7H₂0; and 0.2 mM NaH₂P0₄H₂0. For example, one aqueous physiological salt solution comprises about 150 mM Na, about 3 mM K, about 1.4 mM Ca, about 0.8 mM Mg, about 1.0 mMP, and about 155 mM Cl. In some aspects, the control solution further comprises 20-25 mM sodium carbonate and/or 0.2- 1.5 mM glucose or sucrose. In some aspects, the control solution further comprises 0.2 to 1.0 mg/mL sucrose.

[0023] Methods for preparing aqueous physiological salt solutions are known in the art, and components of aqueous physiological salt solutions are also commercially available. For example, in some aspects, the aqueous physiological salt solution comprises Harvard Apparatus perfusion fluid, which is commercially available from Harvard Apparatus, Holliston, Massachusetts.

[0024] The control solution may have a pH that is lower than about 8, lower than about 7.5, from about 5 to 8, from about 5.5 to about 7.5, from about 6 to about 7.5, from about 6 to about 7, and about 6.5.

[0025] The control solution also comprises one or more plasma proteins. Plasma proteins are proteins normally found in blood plasma. Human CSF contains some of the proteins found in blood plasma, although in much lower concentrations. Human CSF contains approximately 0.3% plasma proteins or approximately 15 to 40 mg/dL of total plasma protein. Accordingly, in some aspects, the total plasma protein, which represents the combined amount of various plasma proteins in the solution, has a concentration ranging from 0.01 mg/mL to 1.0 mg/mL, from 0.02 mg/mL to 0.8 mg/mL, from 0.02 mg/mL to 0.4 mg/mL, or from 0.05 mg/mL to 0.4 mg/mL.

[0026] Examples of plasma proteins comprise albumins (e.g., human serum albumin or BSA), fibrinogen, albumin precursor protein (e.g., BSA precursor protein), transthyretin, gamma globulins (e.g., immunoglobulin G), lipoproteins (e.g., high density or low density lipoprotein), complement proteins, prothrombin, and transferrin. In some aspects, the plasma protein is selected from the group consisting of BSA, BSA precursor protein, transferrin, and Immunoglobulin G, and combinations thereof.

[0027] In some aspects, the plasma protein consists of or consists essentially of 0.1 to 0.3 mg/mL BSA. In other aspects, the plasma protein consists of or consists essentially of 0.1 to 0.2 mg/mL BSA and 0.02 to 0.06 mg/mL transferrin. In a further aspect, the plasma protein consists of or consists essentially of 0.4 to 0.5 mg/mL BSA, 0.01 to 0.03 mg/mL BSA precursor protein, and 0.005 to 0.02 mg/mL Immunoglobulin G, and the control solution also comprises 0.2 to 1.0 mg/mL sucrose.

[0028] In some aspects, the control solution consists essentially of Harvard perfusion fluid, 0.155 mg/mL BSA, and 0.042mg/mL transferrin. About 0.155 mg/mL BSA is considered to be the physiological concentration of BSA in human CSF (or “IX”). About 0.042mg/mL Transferrin is considered to be three times (“3X”) the physiological concentration of transferrin in human CSF. In another aspect, the control solution consists essentially of Harvard perfusion fluid and 0.2015mg/mL BSA (or “1.3X” the physiological concentration of BSA in human CSF).

[0029] In some aspects, the control solution is a positive control. A positive control can be used to generate the result expected when soluble, misfolded protein is present in the sample (i.e., a positive result). The positive control comprises a “seed” protein (i.e., soluble, misfolded ocS protein). In some aspects, the control solution is a negative control. A negative control can be used to generate the result expected when soluble, misfolded protein is not present in the sample (i.e., a negative result). A negative control does not need to comprise any additional material but will often comprise additional water to provide a comparable volume (water, alone, is ineffective as a control solution). In further aspects, the control solution is a comparative control, which comprises a known amount of seed. A comparative control may be used as a benchmark for determining the amount of misfolded protein aggregates that have been formed using PMCA.

Biological Samples

[0030] Aspects of the methods described herein may include the step of obtaining a biological sample from the subject. A “biological sample” is meant to include any biological sample from a subject that is suitable for analysis for detection of misfolded aS aggregates. CSF is a suitable biological sample. A CSF sample may be obtained, for example, by lumbar puncture, in which a needle is inserted into the subarachnoid space and CSF is extracted.

[0031] In some aspects, the methods of the invention are carried out on a biological sample that is provided. A biological sample may be fresh or stored. Biological samples may be or have been stored or banked under suitable tissue storage conditions. The biological sample may be a biological sample expressly obtained for the assays as described herein or a sample obtained for another purpose that can be sub-sampled for the assays as described herein. Preferably, if stored, biological samples are either chilled or frozen shortly after collection to prevent deterioration of the sample. For example, CSF samples may be stored in polypropylene tubes at -80 °C. CSF samples may be frozen in liquid nitrogen or by placing the samples in an environment kept at -80 °C, such as a cold-room or freezer.

[0032] The biological sample may be pretreated as necessary by dilution in an appropriate buffer solution, concentrated if desired, or fractionated by any number of methods, including but not limited to, ultracentrifugation, fractionation by fast performance liquid chromatography (FPLC) or HPLC, or precipitation of proteins with dextran sulfate or other methods. Any of a number of standard aqueous buffer solutions at physiological pH, such as phosphate, Tris, or the like, can be used.

Monomeric aS Substrate

[0033] As used herein, “aS” may refer to full-length, 140 amino acid alpha-synuclein protein, e g., “aS-140.” Other isoforms or fragments may include “aS-126,” alpha-synuclein- 126, which lacks residues 41-54, e.g., due to loss of exon 3; and “aS-112” alpha-synuclein- 112, which lacks residue 103-130, e.g., due to loss of exon 5. Various aS isoforms may include, e.g., aS-140, aS-

126, and aS-112. [0034] In one aspect, the monomeric aS substrate comprises, consists essentially of, or consists of wild type or recombinant human ocS protein having 140 amino acids, having a molecular mass of 14,460 Da, and being represented by the sequence:

SEQ ID NO. 1:

MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH

GVATVAEKTK EQVTNVGGAV VTGYTAVAQK TVEGAGSIAA ATGFVKKDQL

GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA

[0035] Also included are slightly modified forms of otS, such as those including a tag for purification. Thus, in one aspect, the monomeric aS substrate comprises, consists of, or consists essentially of a recombinant aS protein comprising six additional histidine amino acids (i.e., a polyHis purification tag) on the C-terminus of SEQ ID NO. 1, resulting in a molecular mass of 15,283 Da and being represented by the sequence:

SEQ ID NO. 2:

MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH

GVATVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL

GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA HHHHHH

[0036] In some aspects, the monomeric aS substrate may be any of the monomeric aS substrates disclosed in US20210063416A1, which is incorporated by reference herein in its entirety.

[0037] In some aspects, the monomeric aS substrate may be expressed and purified 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 by reference herein in its entirety. [0038] In some aspects, the monomeric aS substrate may be expressed and purified as described in U.S. Provisional Patent Application No. 63/026,394, which is incorporated by reference herein in its entirety.

[0039] The incubation mixture may include various concentrations of the monomeric aS substrate as a function of the total volume of the incubation mixture prior to conducting an incubation cycle. In some aspects, the incubation mixture may include the monomeric aS substrate in a concentration, or in a concentration range, of one or more of: between about 500 nM and about 500 mM; between about 1 mM and about 200 mM; between about 5 mM to about 100 mM; between about 10 mM and about 50 mM; between about 50 mM and about 75 mM; about 65 mM (i.e., about 1 mg/ml); 65 mM; between about 10 mM and about 30 mM; greater than 10 mM and less than 30 mM; about 20 mM; about 19.6 mM (i.e., about 0.3 mg/ml); or 19.6 mM.

Buffer Compositions

[0040] The incubation mixture may include various buffer compositions. The buffer composition may be effective to maintain the pH of the incubation mixture in a range from about pH 5 to about pH 9, from about pH 6 to about pH 8, from about pH 6 to about pH 7, from about pH 7 to about pH 8, about pH 7, about pH 7.4, from about pH 6.2 to about pH 6.5, including pH 6.3, 6.4, and 6.5. In some aspects, the incubation mixture comprises one or more of the buffers Tris-HCL, MES, PIPES, MOPS, BES, TES, and HEPES. In some aspects, the incubation buffer comprises PIPES in a concentration of about 100 mM, about 500 mM, about 600 mM, or about 700 mM.

Salt Solutions

[0041] In some aspects, the incubation mixture comprises salt in a given concentration. The salt may, for example, enhance signal to noise ratio in fluorescence detection. In one aspect, the salt comprises NaCl. Other suitable salts may include KC1. In one aspect, the salt, e.g., NaCl, may be present in 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 aspect, the salt, e.g., NaCl, is present in a concentration of about 500 mM.

Indicators

[0042] In some aspects, the method includes the step of contacting the incubation mixture with a protein aggregation indicator to determine if a detectable amount of misfolded aS aggregate is present in the incubation mixture. The protein aggregation indicator can be characterized by exhibiting an indicating state in the presence of misfolded aS aggregate and a non-indicating state in the absence of misfolded aS aggregate. Determining the presence of the soluble, misfolded aS protein in a biological sample may include detecting the indicating state of the indicator of misfolded aS aggregate. The indicating state of the indicator and the non-indicating state of the indicator may be characterized by a difference in fluorescence. Thus, the step of determining the presence of the soluble, misfolded aS protein in a biological sample may include detecting the difference in fluorescence. In some aspects, a molar excess of the indicator may be used, the molar excess being, for example, greater than a total molar amount of the monomeric aS substrate and the soluble, misfolded aS protein in the incubation mixture.

[0043] In some aspects, the protein aggregation indicator may include one or more of: Thioflavin-T (“ThT”), Congo Red, m-I-Stilbene, Chrysamine G, PIB, BF-227, X-34, TZDM, FDDNP, IMPY, NIAD-4, luminescent conjugated polythiophenes, a fusion with a fluorescent protein such as green fluorescent protein and yellow fluorescent protein, derivatives thereof, and the like. A suitable protein aggregation indicator is ThT. Incubation Conditions

[0044] The incubation mixture may be held within a suitably sized container, such as a multi well plate having a plurality of wells. For example, the multi-well plate may include 96 wells. The wells of the multi -well plate may have a volume of from 100 pL to 1000 pL, from 150 pL to 750 pL, or from 200 pL to 350 pL. In some aspects of the invention, each well of the multi-well plate includes a single bead.

[0045] A variety of temperatures are suitable for carrying out the incubation cycles. The temperature of the incubation mixture, in each incubation cycle, at a temperature in °C, can independently be 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 preceding 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 aspects, the incubation is carried out at about normal physiological temperatures for a warm-blooded animal. In further aspects, incubating the incubation mixture is conducted at a temperature between about 35 °C and about 40 °C or between about 37 °C and about 42 °C.

[0046] As used herein, a “misfolded protein” is a protein that no longer contains all or part of the structural conformation of the protein as it exists when involved in its typical, nonpathogenic normal function within a biological system. A misfolded protein may aggregate and may exist in or as an aggregate. A misfolded protein may localize in a protein aggregate. A misfolded protein may be a non-functional protein. A misfolded protein may be a pathogenic conformer of the protein.

[0047] Misfolded aS aggregates refer to non-covalent associations of protein including soluble, misfolded aS protein. Misfolded aS aggregates may be “de-aggregated,” broken up, or dismpted to release smaller fragments or aggregates, e g., soluble, misfolded aS protein and fragmented fibrils. The seeding activity of a collection of misfolded otS aggregate seeds may scale, at least in part, with the number of seeds in a mixture. Accordingly, disruption of misfolded aS aggregates in a mixture to release soluble, misfolded a-S protein and fragmented fibrils seeds may lead to an increase in catalytic activity for aggregation of monomeric aS substrate.

[0048] In several aspects, de-aggregating the incubation mixture may include one or more types of physical disruption selected from: shaking, sonication, stirring, freezing/thawing, laser irradiation, autoclave incubation, high pressure, homogenization, and the like. Shaking may include cyclic agitation, such as orbital agitation or double orbital agitation. The cyclic agitation may be conducted between about 50 rotations per minute (RPM) and 10,000 RPM. The cyclic agitation may be conducted between about 200 RPM and about 2000 RPM. The cyclic agitation may be conducted at about 500 RPM or about 600-800 RPM. De-aggregation of the incubation mixture may be conducted 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, for about 1 minute, and the like.

[0049] The steps of incubating and de-aggregating the incubation mixture are repeated a number of times sufficient to amplify the soluble, misfolded aS protein of the sample to provide a detectable amount of misfolded a-S aggregate. The two steps of incubating the incubation mixture and de-aggregating the incubation mixture are referred to herein as the incubation cycle. The incubation cycle may be repeated between about two times and about 1000 times, between about five times and about 500 times, between about 50 times and about 500 times, between about 150 times and about 250 times, and the like. In one aspect, for the final round of the incubation cycle, it may be advantageous to omit the de-aggregation step before performing the detecting step.

[0050] The incubation cycle may be carried out for a time 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, and the like. In some aspects, incubating the incubation mixture and de-aggregating at least a portion of the misfolded aS aggregate comprise an incubation cycle lasting from 0.3 to 1 hours. Each incubation cycle may include independently incubating and de-aggregating the incubation mixture for one or more of: incubating between about 1 minute and about 5 hours and de-aggregating between about 5 seconds and about 10 minutes; incubating between about 10 minutes and about 2 hours and de-aggregating between about 30 sec and about 1 minute; incubating between about 15 minutes and about 1 hour and deaggregating between about 45 seconds and about 1 minute; incubating between about 25 minutes and about 45 minutes and de-aggregating between about 45 seconds and about 1 minute; incubating for about 33 minutes and de-aggregating for about 1 minute; and incubating for about 1 minute and de-aggregating for about 1 minute.

Detection

[0051] The method comprises repeating the steps of incubating and de-aggregating the incubation mixture a number of times necessary to amplify sufficient soluble, misfolded aS protein present in the biological sample to provide an amplified incubation mixture having a detectable amount of misfolded aS aggregate. The incubation mixture may then be contacted with a fluorescent aggregation indicator, and the level of fluorescence of the amplified incubation mixture may be determined. [0052] The method includes the step of contacting the incubation mixture with a fluorescent aggregation indicator to provide a fluorescent response that can determine if a subject having a neurological disorder has, e.g., PD, MSA, a spectrum of aspects of both, or some other synucleinopathy. The fluorescent aggregation indicator can be characterized by exhibiting an indicating state in the presence of misfolded aS aggregate and a non-indicating state in the absence of misfolded aS aggregate.

[0053] A suitable fluorescent aggregation indicator is ThT, which is also known as Basic yellow 1. When ThT is added to samples containing b-sheet-rich deposits, such as the cross-b- sheet quaternary structure 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.

[0054] ThT fluorescence is typically measured by fluorescence spectroscopy using a filter fluorometer or spectrofluorometer. In some aspects, the ThT fluorescence emission intensity may be compared to the level of a corresponding control sample. A biological sample fluorescence intensity that is significantly greater than the control fluorescence intensity is indicative of the presence of soluble, misfolded protein in the biological sample. By “significantly greater” or “significant increase in fluorescence” is meant an increase in fluorescence of at least the mean relative fluorescence plus two times the standard deviation of the mean relative fluorescence of the negative control or baseline fluorescence at a suitable time point, typically between 50 and 150 hours, but up to about 300 hours. [0055] Once the ThT fluorescence level has been determined, it can be displayed in a variety of ways. For example, the levels can be displayed graphically on a display as numeric values, proportional bars (i.e., a bar graph), or any other display method known to those skilled in the art. [0056] The present invention is illustrated by the following examples. However, the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.

EXAMPLES

Example 1 : Control solutions used as negative controls during “slow assay” aS PMCA conditions [0057] Control solutions were used as negative controls under “slow assay” aS PMCA conditions. General conditions for the “slow assay” aS-PMCA are described in US20160077111 Al, which is incorporated herein by reference in its entirety. The specific PMCA assay conditions used to generate the results herein are as follows:

[0058] For negative controls, an incubation mixture was provided in a 96 well plate, the incubation mixture comprising: (1) 1 mg/ml seed-free aS represented by SEQ ID NO: 2; (2) a buffer composition comprising 0.1M PIPES at a pH of 6.5; (3) a salt solution comprising 500 mM NaCl; (4) an indicator comprising 10 mM ThT; and (5) 40 pL of a control solution, for a total volume of 200 pL.

[0059] Incubation cycles were performed on the incubation mixture, each incubation cycle comprising: (1) incubating the first incubation mixture for 29 min; and (b) orbitally shaking the incubation mixture for 1 minute at 700 rpm, using an Omega FLUOstar at a constant temperature of 37 °C for 300 total hours, to form an incubated control solution. ThT fluorescence was measured in the plates every 30 minutes at 490 nm after excitation at 440 nm.

[0060] Four different control solutions were used: 1. Condition “1B3T”: Harvard perfusion fluid + 0.155mg/mL BSA (IX) + 0.042mg/mL Transferrin (3X)

2. Condition “1.3B”: Harvard perfusion fluid + 0.2015mg/mL BSA (1.3X)

3. Condition “3BlIgG”: Harvard perfusion fluid + 0.465mg/mL BSA (3X) + 0.012mg/mL IgG (lX)

4. Condition “H”: Harvard perfusion fluid

[0061] Although all of the substrates tested corresponded to SEQ ID NO: 2, the substrates were prepared at different times and some by different expression and/or purification conditions. [0062] The results are shown in Table 1, where “Neg/Total Neg Controls” means the number of trials that showed no substrate self-aggregation compared to the total number of trials; and “Pos/Total Neg Controls” means the number of trials that showed substrate self-aggregation compared to the total number of trials.

Table 1

* Substrate rejected as inadequate

[0063] Figure 3 shows a graph of fluorescence intensity (in kRFUs) over time for slow PMCA of AMP-13 using 1B3T as the negative control solution.

[0064] Figure 4 shows a graph of fluorescence intensity (in kRFUs) over time for slow PMCA of AMP-14 using 1B3T as the negative control solution.

[0065] Figure 5 shows a graph of fluorescence intensity (in kRFUs) over time for slow PMCA of AMP-6 using 1.3B as the negative control solution.

Example 2: Control solutions used as positive controls during “slow assay” aS PMCA conditions [0066] Control solutions were used as positive controls during “slow assay” ocS PMCA conditions. For positive controls, the incubation mixture further comprised 20 fg of synthetic aS aggregates (purchased from Abeam) comprising wild-type human recombinant protein as represented by SEQ ID NO: 1.

[0067] The results are shown in Table 2, where “Pos/Total Pos Controls” means the number of trials that show aggregation of substrate with seeds compared to the total number of trials; and “Neg/Total Pos Controls” means the number of trials that showed no aggregation of substrate with seeds compared to the total number of trials. Table 2

* Substrate rejected as inadequate

[0068] Figure 6 shows a graph of fluorescence intensity (in kRFUs) over time for slow PMCA of AMP-13 in the presence of 20fg of seeds using 1B3T as the positive control solution.

[0069] Figure 7 shows a graph of fluorescence intensity (in kRFUs) over time for slow PMCA of AMP-6 in the presence of 20fg of seeds using 1.3B as the positive control solution. Comparative Example 1: Control solutions used as negative controls during “fast assay” aS PMCA conditions

[0070] Control solutions were used as negative controls under “fast assay” aS PMCA conditions. General conditions for the “fast assay” aS-PMCA are described in US20210063416A1. Specifically, the PMCA assay was conducted using 0.3 mg/ml seed-free aS represented by SEQ ID NO: 2, orbitally shaken at 800 rpm, in the presence of a 2.38 mm silicon nitride bead.

[0071] Figure 8 shows a graph of fluorescence intensity (in kRFUs) over time for fast PMCA of AMP-13 using 1B3T as the negative control solution.

[0072] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. [0073] Where a range of values is provided, each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, 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 in the invention.

[0074] The term “about” in conjunction with a number is intended to include ±10% of the number. This is true whether “about” is modifying a stand-alone number or modifying a number at either or both ends of a range of numbers. In other words, “about 10” means from 9 to 11. Likewise, “about 10 to about 20” contemplates 9 to 22 and 11 to 18. In the absence of the term “about,” the exact number is intended. In other words, “10” means 10.

[0075] The singular forms “a”, “and”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a sample” also includes a plurality of such samples and reference to “a monomeric aS substrate” includes reference to one or more such molecule, and so forth.

[0076] When reference is made to the term “each,” it is not meant to mean “each and every, without exception.” For example, if reference is made to an incubation cycle, and “each incubation cycle” is said to involve certain steps, if the incubation cycle is conducted 10 times, and two or more of the incubation cycles involve the certain steps, then that subset of two or incubation cycles is intended to meet the limitation. [0077] The transitional phrase “consisting essentially of’ limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention. MPEP § 2111.03(111).

[0078] The complete disclosure of all patents, patent applications, and publications, and electronically available material cited herein are incorporated by reference, whether or not the specific citation herein so states. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.

Claims

CLAIMS What is claimed is:

1. A control solution for assaying for misfolded alpha-synuclein (“otS”) proteins, the control solution comprising: an aqueous physiological salt solution; and a plasma protein having a concentration of 1.0 mg/mL or less.

2. The control solution of claim 1, wherein the plasma protein is selected from the group consisting of bovine serum albumin (“BSA”), BSA precursor protein, transferrin, and Immunoglobulin G, and combinations thereof.

3. The control solution of claim 1, wherein the plasma protein has a concentration ranging from 0.02 mg/mL to 0.4 mg/mL.

4. The control solution of claim 1, wherein the plasma protein consists essentially of 0.1 to 0.3 mg/mL BSA.

5. The control solution of claim 1, wherein the plasma protein consists essentially of 0.1 to 0.2 mg/mL BSA and 0.02 to 0.06 mg/mL transferrin.

6. The control solution of claim 1, wherein the plasma protein consists essentially of 0.1 to 0.2 mg/mL BSA, 0.02 to 0.06 mg/mL transferrin, 0.01 to 0.03 mg/mL BSA precursor protein, and 0.005 to 0.02 mg/mL Immunoglobulin G.

7. The control solution of claim 1, wherein the control solution further comprises 0.2 to 1.0 mg/mL sucrose.

8. The control solution of claim 1, wherein the control solution has a pH from 6 to 7.5.

9. The control solution of claim 1, wherein the aqueous physiological salt solution comprises sodium, potassium, chloride, calcium, magnesium, and phosphate ions.

10. The control solution of claim 1, wherein the artificial cerebrospinal fluid comprises Harvard Apparatus perfusion fluid.

11. A method for determining the presence of misfolded alpha-synuclein (“aS”) protein in a human cerebrospinal fluid (CSF) sample, the method comprising: (I) in a first assay, (A) providing a first incubation mixture, the first incubation mixture comprising: (1) a first portion of a seed- free monomeric aS protein; (2) a first buffer composition; (3) a first salt solution; (4) a first indicator; and (5) a control solution comprising an aqueous physiological salt solution and a plasma protein having a concentration of 1.0 mg/mL or less; (B) conducting an incubation cycle two or more times on the first incubation mixture, each incubation cycle comprising: (1) incubating the first incubation mixture for a first predetermined time; and (b) shaking the incubation mixture for a second predetermined time, to form an incubated control solution; and (C) detecting via indicator fluorescence any misfolded aS protein aggregates in the incubated control solution, thereby providing a first fluorescence intensity; (II) in a second assay, (A) providing a second incubation mixture, the second incubation mixture comprising: (1) a second portion of the seed- free monomeric aS protein; (2) a second buffer composition; (3) a second salt solution; (4) a second indicator; and (5) a human CSF sample; (B) conducting an incubation cycle two or more times on the second incubation mixture, each incubation cycle comprising: (1) incubating the second incubation mixture effective to cause aggregation of the second portion of the seed-free monomeric aS substrate in the presence of any soluble, misfolded aS protein in the human CSF sample; and (b) shaking the incubation mixture effective to break up any misfolded aS protein aggregates present, to form an incubated human CSF sample; and (C) detecting via indicator fluorescence any misfolded aS protein aggregates in the incubated human CSF sample, thereby providing a second fluorescence intensity; and (III) comparing the first fluorescence intensity to the second fluorescence intensity, wherein a second fluorescence intensity that is significantly greater than the first fluorescence intensity is indicative of the presence of soluble, misfolded protein in the human CSF sample.

12. The method of claim 11, wherein the plasma protein is selected from the group consisting of bovine serum albumin (“BSA”), BSA precursor protein, transferrin, and Immunoglobulin G, and combinations thereof.

13. The method of claim 11, wherein the plasma protein has a concentration ranging from 0.02 mg/mL to 0.4 mg/mL.

14. The method of claim 11, wherein the plasma protein consists essentially of 0.1 to 0.3 mg/mL BSA.

15. The method of claim 11, wherein the plasma protein consists essentially of 0.1 to 0.2 mg/mL BSA and 0.02 to 0.06 mg/mL transferrin.

16. The method of claim 11, wherein the plasma protein consists essentially of 0.1 to 0.2 mg/mL BSA, 0.02 to 0.06 mg/mL transferrin, 0.01 to 0.03 mg/mL BSA precursor protein, and 0.005 to 0.02 mg/mL Immunoglobulin G.

17. The method of claim 11, wherein the control solution further comprises 0.2 to 1.0 mg/mL sucrose.

18. The method of claim 11, wherein the control solution has a pH from 6 to 7.5.

19. The method of claim 11, wherein the aqueous physiological salt solution comprises sodium, potassium, chloride, calcium, magnesium, and phosphate ions.

20. The method of claim 11, wherein the artificial cerebrospinal fluid comprises Harvard Apparatus perfusion fluid.