WO2026024610A1 - Dosage de détection d'agrégat d'alpha-synucléine sans étiquette - Google Patents

Dosage de détection d'agrégat d'alpha-synucléine sans étiquette

Info

Publication number
WO2026024610A1
WO2026024610A1 PCT/US2025/038446 US2025038446W WO2026024610A1 WO 2026024610 A1 WO2026024610 A1 WO 2026024610A1 US 2025038446 W US2025038446 W US 2025038446W WO 2026024610 A1 WO2026024610 A1 WO 2026024610A1
Authority
WO
WIPO (PCT)
Prior art keywords
synuclein
bli
microprobe
prior
probe
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/038446
Other languages
English (en)
Inventor
Ruben Yiqi Luo
Thomas MONTINE
Kathleen Poston
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Leland Stanford Junior University
Original Assignee
Leland Stanford Junior University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Leland Stanford Junior University filed Critical Leland Stanford Junior University
Publication of WO2026024610A1 publication Critical patent/WO2026024610A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G01N2800/2828—Prion diseases
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2835—Movement disorders, e.g. Parkinson, Huntington, Tourette

Definitions

  • 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.
  • Alpha-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. 1 a-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).
  • PD Parkinson's disease
  • DLB dementia with Lewy bodies
  • MSA multiple system atrophy
  • 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.
  • Mutations 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.
  • 3 In 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.
  • PMCA 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. 8-10 This 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.
  • 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.
  • ThT could to some extent promote or inhibit the a- synuclein aggregation.
  • the presence of ThT may have impact on the aggregation process to obscure the effects of drug candidates.
  • an a-synuclein aggregate assay without using any additive is in demand.
  • 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.
  • BLI biolayer interferometry
  • 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.
  • 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.
  • 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.
  • the a-synuclein aggregates can be isolated and manipulated for further measurement.
  • the kinetics of a-synuclein aggregate formation can be observable.
  • Fig. 1 Experiment steps of the label-free a-synuclein aggregate detection assay.
  • 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.
  • 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).
  • BLI biolayer interferometry
  • Biolayer interferometry 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).
  • BLI is used to detect and/or analyze a-synuclein aggregation. BLI measures kinetics and biomolecular interactions on a basis of wave interference.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • bifunctional reagents containing a siloxane group for chemical conjugation to quartz i.e., SiCh
  • 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).
  • 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).
  • the capture agent may comprise a polypeptide that binds specifically to a heterologous moiety that has been added to the a-synuclein protein.
  • 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 tag known in the art
  • Affinity tags and corresponding antibodies that bind specifically to the affinity tags are known in the art and readily commercially available.
  • 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.
  • 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.
  • BLI biolayer interferometry
  • a-synuclein may be directly captured onto the BLI probe via the different immobilization chemistries as discussed herein.
  • 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).
  • 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:
  • 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:
  • 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:
  • the a-synuclein that may be captured on a subject BLI probe comprises a heterologous moiety.
  • the heterologous moiety is a heterologous polypeptides (e.g., a heterologous polypeptide fused to the a-synuclein protein).
  • the heterologous polypeptide is an affinity.
  • suitable affinity tags can include, without limitation, FLAG, GST, HA, and Myc tags.
  • the heterologous moiety may be a small molecule that has been conjugated to the a-synuclein protein.
  • the heterologous moiety may be a biotin molecule, which can be conjugated to a-synuclein using known methods in the art.
  • step (a) of binding a-synuclein to a subject biolayer interferometry (BLI) microprobe 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).
  • a-synuclein e.g., a solution of a subject a-synuclein as discussed herein.
  • concentration of a-synuclein in the solution may vary depending on the particular embodiment of the methods.
  • the a-synuclein solution may range in concentration from 1 pg/ml to 100 pg/ml of a-synuclein.
  • 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.
  • 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.
  • 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.
  • the concentration of a-synuclein may be less than 1 pg/ml.
  • the concentration of a-synuclein may be more than 100 pg/ml.
  • the temperature of the a-synuclein solution may range from 4°C to 40°C.
  • 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.
  • 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.
  • the BLI probe may be dipped in the a-synuclein solution for 1 to 30 or more minutes.
  • 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.
  • 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.
  • 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.
  • 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.
  • the optical signal may be measured at least before step (c) and during or after (c), thereby allowing aggregation formation to be observed.
  • the optical signal may be wave interference, measured over time.
  • step (d) may produces a BLI sensorgram.
  • the determination of whether a-synuclein aggregates have formed is done by analysis of the BLI sensorgram.
  • 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.
  • CSF cerebrospinal fluid
  • a different type of biological fluid sample may be used, such as blood, plasma, serum, saliva, tears, or urine.
  • 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.
  • PD Parkinson's disease
  • DLB dementia with Lewy bodies
  • MSA multiple system atrophy
  • 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.
  • 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.
  • the BLI probe may be contacted with the biological sample for an amount of time less than an hour or more than 90 hrs.
  • the biological sample may be adjusted to a desired temperature.
  • the temperature of the biological sample may be in the range from 4°C to 60°C.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the BLI microprobe can be contacted with a-synuclein.
  • the contacting of the BLI microprobe to a-synuclein may be performed by contacting the BLI microprobe to an aqueous solution of a-synuclein.
  • 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).
  • 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:
  • 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).
  • 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:
  • the concentration of a-synuclein in the solution of step (c) may vary depending on the particular embodiment of the methods.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the optical signal may be measured at least before step (c) and during or after (c), thereby allowing aggregation formation to be observed.
  • 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).
  • 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.
  • step (d) may produce a BLI sensorgram.
  • the determination of whether a- synuclein aggregates have formed is done by analysis of the BLI sensorgram.
  • 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).
  • 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.
  • 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.
  • the method may further comprise diagnosing a neurodegenerative disease based on the results.
  • the method may further comprise diagnosing Parkinson's disease (PD), dementia with Lewy bodies (DLB), or multiple system atrophy (MSA) based on the results.
  • 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.
  • the method may comprise monitoring a neurodegenerative disease based on the results.
  • the method may comprise monitoring Parkinson's disease (PD), dementia with Lewy bodies (DLB), or multiple system atrophy (MSA) based on the results.
  • 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.
  • a BLI detection system that comprises a biolayer interferometry (BLI) microprobe that is coated in a-synuclein, e.g., biotinylated a-synuclein.
  • BLI biolayer interferometry
  • (BLI) microprobe may further comprise a-synuclein aggregates.
  • the system may further comprise a light source (e.g., a tungsten light) and a detector.
  • BLI microprobes also sometimes referred to as thin-film interferometry (TFI) microprobes
  • TFI thin-film interferometry
  • 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.
  • a capture agent e.g., an antibody or an avidin protein
  • 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).
  • 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.
  • the BLI probe may be coated with avidins that are directly immobilized on the surface of the BLI probe.
  • 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.
  • the BLI probe may further comprise a-synuclein aggregates on the surface of the BLI probe.
  • a-synuclein aggregates may be formed on the surface of the microprobe according to the methods described herein.
  • a subject BLI detection system further comprises a light source (e.g., a tungsten lamp) and a detector.
  • a light source e.g., a tungsten lamp
  • 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.
  • kits comprising (a) biolayer interferometry (BLI) microprobe; and (b) a-synuclein modified with a binding agent, e.g., biotinylated a-synuclein.
  • BLI biolayer interferometry
  • the BLI microprobe may be coated in the binding partner, e.g., streptavidin.
  • the modified a-synuclein may bound to its binding partner or in a separate container.
  • 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.
  • 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).
  • the BLI microprobe may be coated in a capture agent.
  • 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.
  • 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.
  • 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).
  • 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).
  • the capture agent may comprise a polypeptide that binds specifically to a heterologous moiety that has been added to the a-synuclein protein.
  • 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 tag known in the art (e.g., FLAG, HA, Myc, or GST affinity tag)
  • Affinity tags and corresponding antibodies that bind specifically to the affinity tags are known in the art and readily commercially available.
  • 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.
  • an avidin e.g., an avidin, streptavidin, or neuravidin
  • the a-synuclein comprises a heterologous moiety.
  • the heterologous moiety is a heterologous polypeptides (e.g., a heterologous polypeptide fused to the a-synuclein protein).
  • the heterologous polypeptide is an affinity.
  • suitable affinity tags can include, without limitation, FLAG, GST, HA, and Myc tags.
  • the heterologous moiety may be a small molecule that has been conjugated to the a-synuclein protein.
  • 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.
  • 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).
  • a subject kit may further comprise one or more containers for storing the BLI probe, the capure agent, and/or the a-synuclein.
  • 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.
  • 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.
  • 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.
  • 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.
  • the container is configured to hold a volume ranging from 0.1 ml to 200 ml.
  • 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.
  • the container may be composed of glass, such as, but not limited to, silicate glass, borosilicate glass, sodium borosilicate glass (e.g., PYREXTM), fused quartz glass, fused silica glass, and the like.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a barrier e.g., a water-tight and/or air-tight seal
  • 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).
  • the label-free a-synuclein aggregate detection assay is carried out in three steps:
  • 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.
  • 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).
  • 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.
  • the testing throughput of the label-free a-synuclein aggregate detection assay has been demonstrated.
  • BLI analyzer models with the capability to run multiple BLI microprobes simultaneously, such as 8-channel and 32-channel models.
  • 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.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Biotechnology (AREA)
  • General Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Pathology (AREA)
  • Microbiology (AREA)
  • Neurology (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Neurosurgery (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

Est proposé un procédé comprenant : la liaison d'α-synucléine à une microsonde d'interférométrie de biocouche (BLI), la mise en contact de la microsonde BLI avec un échantillon biologique, la mise en contact de la microsonde BLI avec l'α-synucléine, et la mesure d'un signal optique provenant de la microsonde BLI, le signal optique indiquant si des agrégats d'α-synucléine se sont formés sur la microsonde.
PCT/US2025/038446 2024-07-23 2025-07-21 Dosage de détection d'agrégat d'alpha-synucléine sans étiquette Pending WO2026024610A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202463674640P 2024-07-23 2024-07-23
US63/674,640 2024-07-23

Publications (1)

Publication Number Publication Date
WO2026024610A1 true WO2026024610A1 (fr) 2026-01-29

Family

ID=98566380

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2025/038446 Pending WO2026024610A1 (fr) 2024-07-23 2025-07-21 Dosage de détection d'agrégat d'alpha-synucléine sans étiquette

Country Status (1)

Country Link
WO (1) WO2026024610A1 (fr)

Similar Documents

Publication Publication Date Title
Kulenkampff et al. Quantifying misfolded protein oligomers as drug targets and biomarkers in Alzheimer and Parkinson diseases
Concepcion et al. Label-free detection of biomolecular interactions using BioLayer interferometry for kinetic characterization
Dutra et al. An SPR immunosensor for human cardiac troponin T using specific binding avidin to biotin at carboxymethyldextran-modified gold chip
CN103460049B (zh) 扩增和检测朊病毒的方法
Heckler et al. Phosphorylated neurofilament heavy chain: a potential diagnostic biomarker in amyotrophic lateral sclerosis
Soltermann et al. Label-free methods for optical in vitro characterization of protein–protein interactions
US20060003372A1 (en) Integration of direct binding label-free biosensors with mass spectrometry for functional and structural characterization of molecules
JP7425138B2 (ja) デジタルアッセイのために横からの照射を使用した光学イメージングシステム
JP2011514517A (ja) 環状シトルリン化ペプチドに対する抗体のアッセイ方法
EP3295174B1 (fr) Procédé de réutilisation d'une sonde d'essai et de réactifs dans un immunodosage
Gupta et al. Epitope imprinting of iron binding protein of Neisseria meningitidis bacteria through multiple monomers imprinting approach
Horne et al. Detection of protein oligomers with nanopores
KR20080114685A (ko) 분자 상호작용을 특징화하는 방법
Zhang et al. Detection of Myoglobin with an Open‐Cavity‐Based Label‐Free Photonic Crystal Biosensor
WO2026024610A1 (fr) Dosage de détection d'agrégat d'alpha-synucléine sans étiquette
CN117881791A (zh) 用于进行测定的物品和方法
Haab et al. Using antibody arrays to measure protein abundance and glycosylation: considerations for optimal performance
US20240125805A1 (en) Methods for polypeptide processing and analysis
CA2523745C (fr) Epreuves pour prion a haut rendement
Blomkvist Development and optimization of Gyrolab immunoassay targeting biomarker neurofilament light chain: Implementation of BOLD signal amplification on the Gyrolab platform
Schlichtiger et al. Biosensor approaches for the detection of autoantibodies in human serum
US20260118273A1 (en) Instruments and methods for performing optical assays
JP7658969B2 (ja) 凝集体の決定方法
JP2025186587A (ja) 検体の前処理方法
Herries et al. Brain biomarkers: follow-up of RNA expression discovery approach: CSF assays for neurogranin, SNAP-25, and VILIP-1

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 25844966

Country of ref document: EP

Kind code of ref document: A1