Label-free alpha-synuclein aggregate detection assay

Abstract

Provided herein is a method comprising: binding α-synuclein to a biolayer interferometry (BLI) microprobe, contacting the BLI microprobe with a biological sample, contacting the BLI microprobe with α-synuclein, and measuring an optical signal from the BLI microprobe, wherein the optical signal indicates whether α-synuclein aggregates have formed on the microprobe.

Description

[0001] LABEL-FREE ALPHA-SYNUCLEIN AGGREGATE DETECTION ASSAY

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] Pursuant to 35 U.S.C. § 119 (e), this application claims priority to the filing date of United States Provisional Patent Application Serial No. 63 / 674,640 filed July 23, 2024, the disclosure of which application is incorporated herein by reference in its entirety.

[0004] INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED AS A SEQUENCE LISTING XML FILE

[0005] A Sequence Listing is provided herewith as a Sequence Listing XML, "STAN-2212WO_SEQLIST" created on July 9, 2025 and having a size of 3,996 bytes. The contents of the Sequence Listing XML are incorporated by reference herein in their entirety.

[0006] BACKGROUND

[0007] Alpha-synuclein (a-synuclein) is a protein that is abundant in presynaptic terminals of neurons in the central nervous system. Although its exact physiological function is not well understood, a-synuclein is believed to play a role in synaptic function and neurotransmitter release.1a-Synuclein has been most commonly associated with the pathology of neurodegenerative diseases, particularly Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). Several lines of evidence suggest that a-synuclein plays a central role in the pathogenesis of these disorders, a- Synuclein forms abnormal aggregates known as Lewy bodies in these diseases, which are a hallmark of the pathology.2Mutations in the a-synuclein gene have been linked to rare familial forms of PD, indicating that the protein is directly involved in the disease process.3In addition to its role in the formation of Lewy bodies, a-synuclein may also contribute to neurodegeneration through a variety of other mechanisms. For example, it has been shown to impair proteasomal and lysosomal function, which could lead to the accumulation of toxic proteins in neurons. It has also been shown to disrupt mitochondrial function, induce oxidative stress, and promote inflammation, all of which could lead to neuronal dysfunction.4 Based on the pathology of neurodegenerative diseases, ex vivo seeding of aggregation of a-synuclein from biofluids, such as cerebrospinal fluid (CSF), is a promising biomarker for the diagnosis of PD and DLB.5-6So far a few methods have been developed to detect seeding of ex vivo a-synuclein aggregation in CSF, such as protein misfolding cyclic amplification (PMCA) and real-time quaking-induced conversion (RT-QuIC).7PMCA and RT- QuIC are analytical methods that amplify small a-synuclein aggregates in CSF by using them as seeds to convert free a-synuclein into large a-synuclein aggregates.8-10This amplification process can increase the sensitivity of a-synuclein aggregate detection in CSF, making them a promising clinical laboratory tool for early diagnosis and monitoring progression of neurodegenerative diseases.

[0008] However, the current assays to detect a-synuclein seeded aggregates have a significant limitation - the use of fluorescent dye thioflavin T (ThT) to detect the aggregates that form ex vivo. It is required to add a high concentration of ThT to the reaction systems so that ThT can be incorporated into the formed a-synuclein aggregates. The incorporated ThT molecules can then generate fluorescent response to indirectly report a positive result. The involvement of ThT in the aggregation assay may change the properties of a-synuclein aggregates and make them different from the naturally formed a-synuclein aggregates in patients' specimens. It is unknown if ThT could to some extent promote or inhibit the a- synuclein aggregation. In addition, when screening drug candidates to inhibit the formation of a-synuclein aggregates, the presence of ThT may have impact on the aggregation process to obscure the effects of drug candidates. Thus, an a-synuclein aggregate assay without using any additive is in demand.

[0009] SUMMARY

[0010] Provided herein is a method comprising: binding a-synuclein to a biolayer interferometry (BLI) microprobe, contacting the BLI microprobe with a biological sample, contacting the BLI microprobe with a-synuclein, and measuring an optical signal from the BLI microprobe, wherein the optical signal indicates whether a-synuclein aggregates have formed on the microprobe.

[0011] The BLI microprobe may be placed contact with the same by dipping the BLI microprobe into the sample, and the optical signal may be measured by while the BLI micropobe is in the sample. No additives need to be added to the sample. Such a "dip-and- measure" allows one to measure a-synuclein aggregates directly, without the need for any further additions or indirect detection during the a-synuclein aggregation process. Thus, the label-free a-synuclein aggregate assay may comprise: (1) capturing small a-synuclein aggregates (seeds) on a BLI microprobe from a patient specimen, (2) place the BLI microprobe in an a-synuclein solution to form large a-synuclein aggregates, (3) direct optical measurement of aggregates.

[0012] Depending on how the method is performed, the method may have certain advantages in comparison to other methods. For example, no materials need to be added to the specimens or a-synuclein solution, thereby removing possible interference to the study of a-synuclein aggregation. This allows for screening drug candidates and testing the effect of post-translational modifications of free a-synuclein (phosphorylation, glycosylation, etc.) on the aggregation process. In addition, the a-synuclein aggregates can be isolated and manipulated for further measurement. Finally, the kinetics of a-synuclein aggregate formation can be observable.

[0013] These and other features of the present teachings are set forth herein.

[0014] BRIEF DESCRIPTION OF THE FIGURES

[0015] The skilled artisan will understand that the drawings, described below, are for illustration purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

[0016] Fig. 1. Experiment steps of the label-free a-synuclein aggregate detection assay.

[0017] Figs. 2A and 2B. (2A) Sensorgrams of 2 positive samples from PD patients in the label-free a-synuclein aggregate detection assay. (2B) Zoom-in of (2A) in the baseline region showing the incubation phase of the assay before large a-synuclein aggregates were detected. The gray sensorgram represents a negative control sample.

[0018] Fig. 3. Sensorgrams of 6 positive samples from PD patients in the label-free a- synuclein aggregate detection assay. DESCRIPTION OF EXEMPLARY EMBODIMENTS

[0019] Unless defined otherwise herein, 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. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.

[0020] All patents and publications, including all sequences disclosed within such patents and publications, referred to herein are expressly incorporated by reference.

[0021] The headings provided herein are not limitations of the various aspects or embodiments of the invention. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.

[0022] As noted above, this disclosure provides a label-free a-synuclein aggregate detection assay that comprises: (a) binding a-synuclein to a biolayer interferometry (BLI) microprobe; (b) contacting the BLI microprobe with a biological sample after (a); (c) contacting the BLI microprobe with a-synuclein after (b); and (d) measuring an optical signal from the BLI microprobe, wherein the optical signal indicates whether a-synuclein aggregates have formed on the microprobe in (c).

[0023] Methods

[0024] Biolayer interferometry (BLI) is an optical biosensing technology that relies on the phase shift-wavelength correlation created between interference patterns off of two unique surfaces on the tip of a biosensor (see, e.g., Muller-Esparza, Frontiers in Molecular Biosciences 2020 7: 98) BLI can be used to quantify binding strength, measure protein interactions, and identify properties of reaction kinetics, such as rate constants and reaction rates (see, e.g., Rich, Analytical Biochemistry 2007 361: 1-6). In the current method, BLI is used to detect and / or analyze a-synuclein aggregation. BLI measures kinetics and biomolecular interactions on a basis of wave interference. In this method, the probe is dipped into a series of solution and molecules into the solutions associate with the probe to produce a layer the surface of the biosensor. This creates two separate surfaces: the substrate itself and the substrate interacting with the molecule immobilized on the biosensor tip. This creates a thin-fi I m interference, in which the created layer acts as a thin film bound by these two surfaces. Light, e.g., from a tungsten lamp, can be shone onto the biosensor tip and reflected off both surfaces, creating two unique reflection patterns with different intensities. The wavelength shift (AX) between these two reflection patterns creates an interference pattern from which all desired results can be obtained. Because the wavelength shift is direct measure of the change in thickness of the biological layer and the biological layer thickness will change in response to molecules associating to and dissociating from the biosensor, the interference pattern will allow for real-time monitoring of molecular interactions on the biosensor surface.

[0025] In some embodiments, this microprobe may be made by binding a-synuclein to a BLI microprobe that is coated in a capture agent. The BLI microprobe (also referred to simply as a 'BLI probe' or 'probe' herein) may be composed of any suitable solid substrate such as, e.g., glass, quartz glass, or plastic (e.g., polylactic acid, polyethylene, polypropylene, and polytetrafluouroethylene plastics). In some embodiments, the capture agent may be a polypeptide that is capable of binding to an a-synuclein protein directly, or an a-synuclein protein that has been modified to add a heterologous moiety (e.g., chemically conjugated a small molecule, or a heterologous fusion polypeptide) that is capable of being bound by the capture agent. In certain embodiments, the capture agent may be a protein (e.g., an antibody or an avidin) that has been immobilized onto the probe. Different immobilization chemistries may be used to covalently link the capture agents to the surface of the probe. By way of example, bifunctional reagents containing a siloxane group for chemical conjugation to quartz (i.e., SiCh) and a hydroxyl, amine, carboxyl or other suitable reaction groups may be used for the attachment of biological molecules such as proteins (e.g., antibodies, antigens, avidins).

[0026] In some embodiments the capture agent may comprise an antibody that binds specifically to a-synuclein. Anti a-synuclein antibodies are known and readily commercially available (e.g., MA5-12272 from Invitrogen, and sc-12767 from Santa Cruz Biotech). In some cases, the capture agent may comprise a polypeptide that binds specifically to a heterologous moiety that has been added to the a-synuclein protein. For example, the capture agent may be a polypeptide (e.g., an antibody) specific for an affinity tag known in the art (e.g., FLAG, HA, Myc, or GST affinity tag) in the case that the a-synuclein is fused to a heterologous affinity tag. Affinity tags and corresponding antibodies that bind specifically to the affinity tags are known in the art and readily commercially available. In some embodiments, the capture agent may be an avidin (e.g., an avidin, streptavidin, or neuravidin) which can the enable capture of a-synuclein that has been conjugated to a biotin moiety, the conjugation performed using known methods in the art.

[0027] In some embodiments, the a-synuclein may be bound to a biolayer interferometry (BLI) microprobe via a biotin / avidin moiety interaction, e.g., by binding biotinylated a- synuclein to a streptavidin-coated BLI microprobe, where the term avidin moiety is intended to cover avidin, streptavidin, neutravidin and other similar proteins that bind to biotin.

[0028] Alternative methods for tethering proteins to surfaces are well known, e.g., amine coupling and sulfhydryl coupling, and may be employed herein. In some embodiments, the a-synuclein may be directly captured onto the BLI probe via the different immobilization chemistries as discussed herein.

[0029] In any embodiment, the a-synuclein protein may be any known a-synuclein splice isoform and / or variant a-synuclein protein of interest (e.g., any known human a-synuclein splice isoform and / or variant a-synuclein protein).

[0030] In certain embodiments, the amino acid sequence of the a-synuclein that may be captured on a subject BLI probe comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following a-synuclein isoform amino acid sequence, or a fragment thereof:

[0031] MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHGVATVAEKTKEQ VTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSE EGYQDYEPEA (SEQ ID NO:1).

[0032] In certain embodiments, the amino acid sequence of the a-synuclein that may be captured on a subject BLI probe comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following a-synuclein isoform amino acid sequence, or a fragment thereof:

[0033] MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHGVATVAEKTKEQ VTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKEGYQDYEPEA (SEQ ID NO:2).

[0034] In certain embodiments, the amino acid sequence of the a-synuclein that may be captured on a subject BLI probe comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following a-synuclein isoform amino acid sequence, or a fragment thereof:

[0035] MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVVAEKTKEQVTNVGGAVVTGVTA VAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEA (SEQ ID NO:3).

[0036] In some embodiments, the a-synuclein that may be captured on a subject BLI probe comprises a heterologous moiety. In some cases, the heterologous moiety is a heterologous polypeptides (e.g., a heterologous polypeptide fused to the a-synuclein protein). In some embodiments, the heterologous polypeptide is an affinity. For example, suitable affinity tags can include, without limitation, FLAG, GST, HA, and Myc tags. In some embodiments, the heterologous moiety may be a small molecule that has been conjugated to the a-synuclein protein. For example, the heterologous moiety may be a biotin molecule, which can be conjugated to a-synuclein using known methods in the art.

[0037] In any embodiment, step (a) of binding a-synuclein to a subject biolayer interferometry (BLI) microprobe (e.g., a BLI probe coated in a capture agent) may be performed by dipping the BLI probe into an aqueous solution of a-synuclein (e.g., a solution of a subject a-synuclein as discussed herein). The concentration of a-synuclein in the solution may vary depending on the particular embodiment of the methods. In some embodiments, the a-synuclein solution may range in concentration from 1 pg / ml to 100 pg / ml of a-synuclein. For example, the a-synuclein solution may range from 1 to 10 pg / ml, from 2 to 9 pg / ml, from 3 to 8 pg / ml, from 4 to 7 pg / ml, or from 5 to 6 pg / ml of a-synuclein. As another example, the a-synuclein solution may range from 10 to 100 pg / ml, from 20 to 90 pg / ml, from from 30 to 80 pg / ml, from 40 to 70 pg / ml, or from 50 to 60 pg / ml of a-synuclein. As yet another example, the a-synuclein solution may range from 5 to 15 pg / ml of a-synuclein, e.g., from 6 to 14 pg / ml, from 7 to 13 pg / ml, from 8 to 12 pg / ml, or from 9 to 11 pg / ml of a-synuclein. In some cases, the concentration of a-synuclein may be less than 1 pg / ml. In some cases, the concentration of a-synuclein may be more than 100 pg / ml. In some embodiments, the temperature of the a-synuclein solution may range from 4°C to 40°C. For example, the temperature of the a-synuclein solution may range from 4°C to 15°C, from 6°C to 13°C, or from 8°C to 11°C. As another example, the temperature of the a-synuclein solution may range from 15°C to 45°C, e.g., from 16°C to 44°C, from 18°C to 42°C, from 20°C to 40°C, from 22°C to 38°C, from 24°C to 36°C, from 26°C to 34°C, from 28°C to 32°C, or from 29°C to 31°C. In some embodiments, the BLI probe may be dipped in the a-synuclein solution for 1 to 30 or more minutes. For example, the BLI probe may be dipped in the a-synuclein solution for 1 to 30 minutes, for 5 to 25 minutes, for 10 to 20 minutes, or for 15 minutes. In certain embodiments, the subject a-synuclein solution may have any combination of a-synuclein concentration and temperature described above and the BLI probe may be contacted to the a-synuclein for any of the lengths of time described above. For example, in some embodiments, a subject BLI probe may be dipped in an a-synuclein solution that may range in concentration from 5 to 15 pg / ml of a-synuclein and have a temperature in the range from 15°C to 45°C for a length of time ranging from 1 to 30 minutes. In addition, in any of the above embodiments the a-synuclein solution may be mechanically agitated (e.g., by means of a shaker or stir bar and stir plate) during the dipping of the BLI probe in the a-synuclein solution. In any embodiment, the optical signal may be measured at least before step (c) and during or after (c), thereby allowing aggregation formation to be observed. In some embodiments, the optical signal may be wave interference, measured over time. In this method, step (d) may produces a BLI sensorgram. In these embodiments, the determination of whether a-synuclein aggregates have formed is done by analysis of the BLI sensorgram.

[0038] Following step (a), the BLI microprobe can be contacted with a biological sample. The biological sample used in the method may be cerebrospinal fluid (CSF), which may be obtained from a suitable subject, e.g., a human or other mammal. However, in some embodiments a different type of biological fluid sample may be used, such as blood, plasma, serum, saliva, tears, or urine. In these embodiments, the sample may be obtained from an asymptomatic subject undergoing disease screening, or a subject that has or is suspected of having a neurodegenerative disease, e.g., Parkinson's disease ( PD), dementia with Lewy bodies (DLB), or multiple system atrophy (MSA), to be used for diagnosis and for assessing progression of disease. In some embodiments, the subject from which the biological sample has been obtained may have been treated (e.g., treated with a test compound) and, as such, the method may be used for assessing response to treatment. In any embodiment, the BLI probe may be contacted with the biological sample for an amount of time in the range of 1 to 90 hrs, e.g., in the range of 6 to 84 hrs, 12 to 78 hrs, 18 to 72 hrs, 24 to 72 hrs, 30 to 66 hrs, 36 to 60 hrs, 42 to 54 hrs, 44 to 52 hrs, or 46 to 50 hrs. In some cases, the BLI probe may be contacted with the biological sample for an amount of time less than an hour or more than 90 hrs. In some embodiments, the biological sample may be adjusted to a desired temperature. In some cases, the temperature of the biological sample may be in the range from 4°C to 60°C. For example, in some cases the temperature of the biological sample may be in the range from 4°C to 20°C, e.g., from 8°C to 16°C, or from 10°C to 14°C. In some cases, the temperature of the biological sample may be in the range from 20°C to 60°C, e.g., from 22°C to 58°C, from 24°C to 56°C, from 26°C to 54°C, from 28°C to 52°C, from 30°C to 50°C, from 32°C to 48°C, from 34°C to 46°C, from 36°C to 44°C, from 38 to 42°C, or from 39°C to 41°C. In certain embodiments, the BLI probe may be contacted with a subject biological sample having any of the temperatures and for any of the lengths of time described above. For example, in some embodiments, a subject BLI probe may be contacted with a biological sample having a temperature in the range from 20°C to 60°C for a length of time ranging from 1 to 90 hrs. In addition, in any of the above embodiments the biological sample may be mechanically agitated (e.g., by means of a shaker or stir bar and stir plate) during the contacting of the BLI probe with the biological sample.

[0039] Following step (b), the BLI microprobe can be contacted with a-synuclein. In any embodiment, the contacting of the BLI microprobe to a-synuclein may be performed by contacting the BLI microprobe to an aqueous solution of a-synuclein. In any embodiment, the a-synuclein protein may be any known a-synuclein splice isoform and / or variant a-synuclein protein of interest (e.g., any known human a-synuclein splice isoform and / or variant a-synuclein protein).

[0040] In certain embodiments, the amino acid sequence of the a-synuclein in the solution of step (c) comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following a-synuclein isoform amino acid sequence, or a fragment thereof:

[0041] MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHGVATVAEKTKEQ VTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSE EGYQDYEPEA (SEQ ID NO:1).

[0042] In certain embodiments, the amino acid sequence of the a-synuclein in the solution of step (c) comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following a-synuclein isoform amino acid sequence, or a fragment thereof: MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHGVATVAEKTKEQ VTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKEGYQDYEPEA (SEQ ID N0:2).

[0043] In certain embodiments, the amino acid sequence of the a-synuclein in the solution of step (c) comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following a-synuclein isoform amino acid sequence, or a fragment thereof:

[0044] MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVVAEKTKEQVTNVGGAVVTGVTA VAQKTVEGAGSIAAATGFVKKDQLGKN EEGAPQEGILEDMPVDPDNEAYEM PSEEGYQDYEPEA (SEQ ID NO:3).

[0045] The concentration of a-synuclein in the solution of step (c) may vary depending on the particular embodiment of the methods. In some embodiments, the a-synuclein solution may range in concentration from 0.1 mg / ml to 2 mg / ml of a-synuclein, e.g., the a-synuclein solution may range in concentration from 0.2 mg / ml to 1.9 mg / ml, from 0.3 mg / ml to 1.7 mg / ml, from 0.4 mg / ml to 1.6 mg / ml, from 0.5 mg / ml to 1.5 mg / ml, 0.6 mg / ml to 1.4 mg / ml, from 0.7 mg / ml to 1.3 mg / ml, from 0.8 mg / ml to 1.2 mg / ml, or from 0.9 mg / ml to 1.1 mg / m l. In some cases, the concentration of a-synuclein may be less than 0.1 mg / ml. In some cases, the concentration of a-synuclein may be more than 2 mg / ml. In some embodiments, the temperature of the a-synuclein solution may range from 15°C to 45°C, e.g., from 16°C to 44°C, from 18°C to 42°C, from 20°C to 40°C, from 22°C to 38°C, from 24°C to 36°C, from 26°C to 34°C, from 28°C to 32°C, or from 29°C to 31°C. In some embodiments, the BLI microprobe may be contacted with the a-synuclein solution of step (c) for an amount of time in the range from 20 to 200 hrs, e.g. from 30 to 200 hrs, from 40 to 190 hrs, from 50 to 180 hrs, from 60 to 170 hrs, from 70 to 160 hrs, from 80 to 150 hrs, from 90 to 140 hrs, from 100 to 130 hrs, or from 110 to 120 hrs. In certain embodiments, the subject a-synuclein solution of step (c) may have any combination of a-synuclein concentration and temperature described above and the BLI probe may be contacted to the a-synuclein for any of the lengths of time described above. For example, in some embodiments, the subject BLI probe may be contacted to the an a-synuclein solution of step (c) that may range in concentration from 0.1 mg / ml to 2 mg / ml of a-synuclein and have a temperature in the range from 15°C to 45°C for a length of time ranging from 20 to 200 hrs. In addition, in any of the above embodiments the a-synuclein solution of step (c) may be mechanically agitated (e.g., by means of a shaker or stir bar and stir plate) during the dipping of the BLI probe in the a-synuclein solution.

[0046] As discussed above, in any embodiment, the optical signal may be measured at least before step (c) and during or after (c), thereby allowing aggregation formation to be observed. For example, the optical signal may be measured at least before step (c) and during or after (c) to indicate whether a-synuclein aggregates have formed on the microprobe during step (c). In some embodiments, the optical signal may be wave interference, measured over time. In some cases, the wave interference may be measured over an amount of time in the range from 20 to 200 hrs, e.g. from 30 to 200 hrs, from 40 to 190 hrs, from 50 to 180 hrs, from 60 to 170 hrs, from 70 to 160 hrs, from 80 to 150 hrs, from 90 to 140 hrs, from 100 to 130 hrs, or from 110 to 120 hrs. In this method, step (d) may produce a BLI sensorgram. In these embodiments, the determination of whether a- synuclein aggregates have formed is done by analysis of the BLI sensorgram.

[0047] In any embodiment, the method may comprise analyzing the kinetics of the formation of any a-synuclein aggregates or removing any a-synuclein aggregates from the probe after step (d). In some cases, the removing any a-synuclein aggregates from the probe after step (d) may comprise contacting the probe to a fluid that does not comprise a-synuclein, thereby allowing any a-synuclein aggregates to dissociate from the probe. In some cases, the removing any a-synuclein aggregates from the probe after step (d) may comprise washing the probe to remove any a-synuclein aggregates from the probe. In any embodiment, the method may further comprise diagnosing a neurodegenerative disease based on the results. In certain embodiments, the method may further comprise diagnosing Parkinson's disease (PD), dementia with Lewy bodies (DLB), or multiple system atrophy (MSA) based on the results. By way of example, the method may further comprise diagnosing Parkinson's disease (PD), dementia with Lewy bodies (DLB), or multiple system atrophy (MSA) in a subject from which the biological sample was obtained.

[0048] In any embodiment, the method may comprise monitoring a neurodegenerative disease based on the results. In certain embodiments, the method may comprise monitoring Parkinson's disease (PD), dementia with Lewy bodies (DLB), or multiple system atrophy (MSA) based on the results. By way of example, the method may comprise monitoring Parkinson's disease (PD), dementia with Lewy bodies (DLB), or multiple system atrophy (MSA) in a subject from which the biological sample was obtained. Systems

[0049] Also provided is a BLI detection system that comprises a biolayer interferometry (BLI) microprobe that is coated in a-synuclein, e.g., biotinylated a-synuclein. In these embodiments, (BLI) microprobe may further comprise a-synuclein aggregates. The system may further comprise a light source (e.g., a tungsten light) and a detector.

[0050] BLI microprobes, also sometimes referred to as thin-film interferometry (TFI) microprobes, systems containing the same and methods for their use are described in, e.g., US20110305599A1, Luo et al (Clin Chim Acta 2020 502:128-132), Luo et al (Clinical Infectious Diseases 2021 73: e3095-e3097) and Luo et al (Clin Chem 202066:1319-1328), which are incorporated by reference herein for disclosure of the probes, systems containing the same and methods. Different immobilization chemistries may be used to covalently link the surface of the probe to a capture agent (e.g., an antibody or an avidin protein) or to a-synuclein directly. By way of example, bifunctional reagents containing a siloxane group for chemical conjugation to quartz (i.e., SiOz) and a hydroxyl, amine, carboxyl or other suitable reaction groups may be used for the attachment of if biological molecules such as proteins (e.g., antibodies, avidins). As another example, a variety of chemical conjugation methods are available with polymers (e.g., plastics) which utilize available chemically-active surface groups, such as amine, hydroxyl, and carboxyl groups. In some cases, the BLI probe may be coated with avidins that are directly immobilized on the surface of the BLI probe. In these embodiments, the BLI probe may be additionally coated with biotinylated a-synuclein that is bound to the avidin that is directly immobilized onto the surface of the probe. In any of these embodiments, the BLI probe may further comprise a-synuclein aggregates on the surface of the BLI probe. In certain embodiments, a-synuclein aggregates may be formed on the surface of the microprobe according to the methods described herein.

[0051] In some embodiments, a subject BLI detection system further comprises a light source (e.g., a tungsten lamp) and a detector. In certain embodiments, the light source and detector are part of a spectrophotometer. The spectrophotometer may be operably connected to the BLI probe to detect changes in the interference pattern of light reflected from the tip of the biosensor. Spectrophotometers and their use in BLI / TFI systems are described, e.g., in US20110305599A1, Luo et al (Clin Chim Acta 2020502:128-132), Luo et al (Clinical Infectious Diseases 202173: e3095-e3097) and Luo et al (Clin Chem 2020 66:1319-1328), which are incorporated by reference herein for disclosure of the spectrophotometers, systems containing the same, and methods.

[0052] Kits

[0053] Also provided is a kit comprising (a) biolayer interferometry (BLI) microprobe; and (b) a-synuclein modified with a binding agent, e.g., biotinylated a-synuclein. In some embodiments, the BLI microprobe may be coated in the binding partner, e.g., streptavidin. In these embodiments, the modified a-synuclein may bound to its binding partner or in a separate container.

[0054] BLI probes generally contain a quartz glass rod having a diameter of less than 1mm having specialized optical layers and specialized surface chemistry built at the distal end (the sensing end) of the probe. BLI / TFI probes, systems containing the same and methods for their use are described in, e.g., US20110305599A1, Luo et al (Clin Chim Acta 2020 502:128-132), Luo et al (Clinical Infectious Diseases 2021 73: e3095-e3097) and Luo et al (Clin Chem 2020 66:1319-1328), which are incorporated by reference herein for disclosure of the probes, systems containing the same and methods.

[0055] The BLI microprobe (also referred to simply as a 'BLI probe' or 'probe' herein) may be composed of any suitable solid substrate such as, e.g., glass, quartz glass, or plastic (e.g., polylactic acid, polyethylene, polypropylene, and polytetrafluouroethylene plastics). In certain embodiments, the BLI microprobe may be coated in a capture agent. In some embodiments, the capture agent may be a polypeptide that is capable of binding to an a-synuclein protein directly, or an a-synuclein protein that has been modified to add a heterologous moiety (e.g., chemically conjugated a small molecule, or a heterologous fusion polypeptide) that is capable of being bound by the capture agent. In certain embodiments, the capture agent may be a protein (e.g., an antibody or an avidin) that has been immobilized onto the probe. Different immobilization chemistries may be used to covalently link the capture agents to the surface of the probe. By way of example, bifunctional reagents containing a siloxane group for chemical conjugation to quartz (i.e., SiO?) and a hydroxyl, amine, carboxyl or other suitable reaction groups may be used for the attachment of biological molecules such as proteins (e.g., antibodies, antigens, avidins).

[0056] In some embodiments the capture agent may comprise an antibody that binds specifically to a-synuclein. Anti a-synuclein antibodies are known and readily commercially available (e.g., MA5-12272 from Invitrogen, and sc-12767 from Santa Cruz Biotech). In some cases, the capture agent may comprise a polypeptide that binds specifically to a heterologous moiety that has been added to the a-synuclein protein. For example, the capture agent may be a polypeptide (e.g., an antibody) specific for an affinity tag known in the art (e.g., FLAG, HA, Myc, or GST affinity tag) in the case that the a-synuclein is fused to a heterologous affinity tag. Affinity tags and corresponding antibodies that bind specifically to the affinity tags are known in the art and readily commercially available.

[0057] In some embodiments, the capture agent may be an avidin (e.g., an avidin, streptavidin, or neuravidin) which can the enable capture of a-synuclein that has been conjugated to a biotin moiety, the conjugation performed using known methods in the art.

[0058] In some embodiments, the a-synuclein comprises a heterologous moiety. In some cases, the heterologous moiety is a heterologous polypeptides (e.g., a heterologous polypeptide fused to the a-synuclein protein). In some embodiments, the heterologous polypeptide is an affinity. For example, suitable affinity tags can include, without limitation, FLAG, GST, HA, and Myc tags. In some embodiments, the heterologous moiety may be a small molecule that has been conjugated to the a-synuclein protein. For example, the heterologous moiety may be a biotin molecule, which can be conjugated to a-synuclein using known methods in the art. In these cases, the a-synuclein is a biotinylated a-synuclein protein.

[0059] In any embodiment, the a-synuclein protein may be any known a-synuclein splice isoform and / or variant a-synuclein protein of interest (e.g., any known human a-synuclein splice isoform and / or variant a-synuclein protein).

[0060] In certain embodiments, the amino acid sequence of the a-synuclein (e.g., the biotinylated a-synuclein) included in the kit comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following a-synuclein isoform amino acid sequence, or a fragment thereof:

[0061] MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHGVATVAEKTKEQ VTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSE EGYQDYEPEA (SEQ ID NO:1).

[0062] In certain embodiments, the amino acid sequence of the a-synuclein (e.g., the biotinylated a-synuclein) included in the kit comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following a-synuclein isoform amino acid sequence, or a fragment thereof:

[0063] MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHGVATVAEKTKEQ VTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKEGYQDYEPEA (SEQ ID NO:2).

[0064] In certain embodiments, the amino acid sequence of the a-synuclein (e.g., the biotinylated a-synuclein) included in the kit comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following a-synuclein isoform amino acid sequence, or a fragment thereof:

[0065] MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVVAEKTKEQVTNVGGAVVTGVTA VAQKTVEGAGSIAAATGFVKKDQLGKN EEGAPQEGILEDMPVDPDNEAYEM PSEEGYQDYEPEA (SEQ ID NO:3).

[0066] A subject kit may further comprise one or more containers for storing the BLI probe, the capure agent, and / or the a-synuclein. In some cases, the size of the one more containers may depend on the volume of the capture agent and / or the a-synuclein (in lyophilized or liquid form) to be held in the one or more containers. In certain embodiments, a container may be configured to hold an amount of a subject capture agent or the a-synuclein, ranging from 0.1 mg to 1000 mg, such as from 0.1 mg to 900 mg, such as from 0.1 mg to 800 mg, such as from 0.1 mg to 700 mg, such as from 0.1 mg to 600 mg, such as from 0.1 mg to 500 mg, such as from 0.1 mg to 400 mg, or 0.1 mg to 300 mg, or 0.1 mg to 200 mg, or 0.1 mg to 100 mg, 0.1 mg to 90 mg, or 0.1 mg to 80 mg, or 0.1 mg to 70 mg, or 0.1 mg to 60 mg, or 0.1 mg to 50 mg, or 0.1 mg to 40 mg, or 0.1 mg to 30 mg, or 0.1 mg to 25 mg, or 0.1 mg to 20 mg, or 0.1 mg to 15 mg, or 0.1 mg to 10 mg, or 0.1 mg to 5 mg, or 0.1 mg to 1 mg, or 0.1 mg to 0.5 mg. In some cases, the container is configured to hold an amount of a capture agent or a-synuclein (in lyophilized or liquid form), ranging from 0.1 g to 10 g, or 0.1 g to 5 g, or 0.1 g to 1 g, or 0.1 g to 0.5 g. In some instances, the container may be configured to hold a volume (e.g., a volume of a liquid) ranging from 0.1 ml to 1000 ml, such as from 0.1 ml to 900 ml, or 0.1 ml to 800 ml, or 0.1 ml to 700 ml, or 0.1 ml to 600 ml, or 0.1 ml to 500 ml, or 0.1 ml to 400 ml, or 0.1 ml to 300 ml, or 0.1 ml to 200 ml, or 0.1 ml to 100 ml, or 0.1 ml to 50 ml, or 0.1 ml to 25 ml, or 0.1 ml to 10 ml, or 0.1 ml to 5 ml, or 0.1 ml to 1 ml, or 0.1 ml to 0.5 ml. In some cases, the container is configured to hold a volume ranging from 0.1 ml to 200 ml.

[0067] Suitable containers for the BLI probe, the capure agent, and / or the a-synuclein include, for example, boxes, bottles, vials, syringes, and test tubes. Containers can be formed from a variety of suitable materials, including glass or plastic. For example, the container may be composed of glass, such as, but not limited to, silicate glass, borosilicate glass, sodium borosilicate glass (e.g., PYREX™), fused quartz glass, fused silica glass, and the like. Other examples of suitable materials for the containers include plastics, such as, but not limited to, polypropylene, polymethylpentene, polytetrafluoroethylene (PTFE), perfluoroethers (PFE), fluorinated ethylene propylene (FEP), perfluoroalkoxy alkanes (PFA), polyethylene terephthalate (PET), polyethylene (PE), polyetheretherketone (PEEK), and the like.

[0068] In some embodiments, the container may be sealed. That is, the container may include a seal that substantially prevents the contents of the container from exiting the container. The seal of the container may also substantially prevent other substances from entering the container. For example, the seal may be a water-tight seal that substantially prevents liquids from entering or exiting the container, or may be an air-tight seal that substantially prevents gases from entering or exiting the container. In some instances, the seal is a removable or breakable seal, such that the contents of the container may be exposed to the surrounding environment when so desired, e.g., if it is desired to remove a portion of the contents of the container. In some instances, the seal is made of a resilient material to provide a barrier (e.g., a water-tight and / or air-tight seal) for retaining a sample in the container. Particular types of seals include, but are not limited to, films, such as polymer films, caps, etc., depending on the type of container. Suitable materials for the seal include, for example, rubber or polymer seals, such as, but not limited to, silicone rubber, natural rubber, styrene butadiene rubber, ethylene-propylene copolymers, polychloroprene, polyacrylate, polybutadiene, polyurethane, styrene butadiene, and the like, and combinations thereof. A container may have a sterile access port (for example, the container may be a vial having a stopper pierceable by a hypodermic injection needle).

[0069] EXAMPLES

[0070] To further illustrate some embodiments of the present invention, the following specific examples are given with the understanding that they are being offered to illustrate examples of the present invention and should not be construed in any way as limiting its scope.

[0071] Methods

[0072] In this example, the label-free a-synuclein aggregate detection assay is carried out in three steps:

[0073] (1) A BLI microprobe pre-coated with streptavidin is dipped into a 10 pg / ml biotinylated a-synuclein solution for 15 min at 30°C and 1000 rpm shaking speed to load the capture agent.

[0074] (2) The BLI microprobe is dipped into a CSF sample for 48 hr at 40°C and 400 rpm shaking speed to capture small a-synuclein aggregates (seeds).

[0075] (3) The BLI microprobe is dipped into an a-synuclein solution for 120 hr at 30°C and 1000 rpm shaking speed to form large a-synuclein aggregates.

[0076] A time trace of label-free optical sensing responses (BLI sensorgram) was recorded during the experiment process.

[0077] These steps are illustrated in Figure 1

[0078] Preliminary Results

[0079] In preliminary tests, the label-free a-synuclein aggregate detection assay successfully identified positive samples from PD patients, as shown in Figure 2.

[0080] The testing throughput of the label-free a-synuclein aggregate detection assay has been demonstrated. There are different BLI analyzer models with the capability to run multiple BLI microprobes simultaneously, such as 8-channel and 32-channel models. Depending on the BLI analyzer in use, it is possible to analyze multiple samples to increase the testing throughput. As shown in Figure 3, six positive samples from PD patients and two negative samples could be analyzed at the same time. Higher throughput may be possible by using others BLI analyzer models.

[0081] References

[0082] 1. Goedert M. Alpha-synuclein and neurodegenerative diseases. Nat Rev Neurosci. 2001;2(7):492-501. doi: 10.1038 / 35081564 Spil lantini MG, Schmidt ML, Lee VMY, Trojanowski JQ, Jakes R, Goedert M. a-Synuclein in Lewy bodies. Nature. 1997;388(6645):839-840. doi:10.1038 / 42166 Li W, Lesuisse C, Xu Y, Troncoso JC, Price DL, Lee MK. Stabilization of a-Synuclein Protein with Aging and Familial Parkinson's Disease-Linked A53T Mutation. J Neurosci. 2004;24(33):7400-7409. doi: 10.1523 / J NEU ROSCI.1370-04.2004 Lashuel HA, Overk CR, Oueslati A, Masliah E. The many faces of a-synuclein: from structure and toxicity to therapeutic target. Nat Rev Neurosci. 2013;14(l):38-48. doi:10.1038 / nrn3406 Gao L, Tang H, Nie K, et al. Cerebrospinal fluid alpha-synuclein as a biomarker for Parkinson's disease diagnosis: a systematic review and meta-analysis. Int J Neurosci. 2015;125(9):645-654. doi:10.3109 / 00207454.2014.961454 Majbour NK, Vaikath NN, Van Dijk KD, et al. Oligomeric and phosphorylated alpha- synuclein as potential CSF biomarkers for Parkinson's disease. Mol Neurodegener. 2016;ll(l):7. doi:10.1186 / sl3024-016-0072-9 Bellomo G, De Luca CMG, Paoletti FP, Gaetani L, Moda F, Parnetti L. a-Synuclein Seed Amplification Assays for Diagnosing Synucleinopathies: The Way Forward. Neurology. 2022;99(5) : 195-205. doi:10.1212 / WNL.0000000000200878 Fairfoul G, McGuire LI, Pal S, et al. Alpha-synuclein RT -Qu IC in the CSF of patients with alpha-synucleinopathies. Ann Clin Transl Neurol. 2016;3(10):812-818. doi:10.1002 / acn3.338 Shahnawaz M, Tokuda T, Waragai M, et al. Development of a Biochemical Diagnosis of Parkinson Disease by Detection of a-Synuclein Misfolded Aggregates in Cerebrospinal Fluid. JAMA Neurol. 2017;74(2):163. doi:10.1001 / jamaneurol.2016.4547 Groveman BR, Orru CD, Hughson AG, et al. Rapid and ultra-sensitive quantitation of disease-associated a-synuclein seeds in brain and cerebrospinal fluid by aSyn RT-QulC. Acta Neuropathol Commun. 2018;6(l):7. doi:10.1186 / s40478-018-0508-2 Luo YR, Chakraborty I, Lazar-Molnar E, Wu AHB, Lynch KL. Development of Label-Free Immunoassays as Novel Solutions for the Measurement of Monoclonal Antibody Drugs and Antidrug Antibodies. Clin Chem. 2020;66(10):1319-1328. doi:10.1093 / clinchem / hvaal79 Luo YR, Yun C, Chakraborty I, Wu AHB, Lynch KL. A SARS-CoV-2 Label-Free Surrogate Virus Neutralization Test and a Longitudinal Study of Antibody Characteristics in COVID-

[0083] 19 Patients. Tang YW, ed. J Clin Microbiol. 2021;59(7):e00193-21. doi:10.1128 / JCM.00193-21

[0084] Although the foregoing embodiments have been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the above teachings that certain changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.

Claims

CLAIMSWhat is claimed is:

1. A method comprising:(a) binding a-synuclein to a biolayer interferometry (BLI) microprobe;(b) contacting the BLI microprobe with a biological sample after (a);(c) contacting the BLI microprobe with a-synuclein after (b); and(d) measuring an optical signal from the BLI microprobe, wherein the optical signal indicates whether a-synuclein aggregates have formed on the microprobe in (c).

2. The method of claim 1, wherein (a) is done by binding a-synuclein to a BLI microprobe that is coated in a capture agent.

3. The method of claim 1 or 2, wherein the optical signal is wave interference, measured over time.

4. The method of any prior claim, wherein the optical signal is at least measured:(i) before (c) and(ii) during or after (c).

5. The method of any prior claim, wherein the sample is cerebrospinal fluid (CSF).

6. The method of any prior claim, wherein (d) produces a BLI sensorgram, and wherein the determination of whether a-synuclein aggregates have formed is done by analysis of the BLI sensorgram.

7. The method of any prior claim, wherein the sample is obtained from a subject that has or is suspected of having a neurodegenerative disease.

8. The method of claim 7, wherein the neurodegenerative disease is Parkinson's disease (PD), dementia with Lewy bodies (DLB), or multiple system atrophy (MSA).

9. The method of any prior claim, wherein the subject from which the biological sample has been obtained has been treated with a test compound.

10. The method of any prior claim, further comprising analyzing the kinetics of the formation of any a-synuclein aggregates.

11. The method of any prior claim, further comprising removing any a-synuclein aggregates from the probe after step (d).

12. The method of any prior claim, further comprising diagnosing a neurodegenerative disease based on the results of step (d).

13. The method of any prior claim, further comprising monitoring a neurodegenerative disease based on the results of step (d).

14. A biolayer interferometry (BLI) system comprising a microprobe that is coated in a- synuclein.

15. The BLI microprobe of claim 14, wherein the a-synuclein is biotinylated a-synuclein.

16. The BLI microprobe of claim 14 or 15, further comprises a-synuclein aggregates on the surface of the BLI microprobe.

17. A kit comprising:(a) biolayer interferometry (BLI) microprobe; and(b) biotinylated a-synuclein.

18. The kit of claim 17, wherein the BLI microprobe is coated in an avidin moiety.

19. The kit of claim 17 or 18, wherein the biotinylated a-synuclein is bound to streptavidin.

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