Nanoparticle-enhanced method and material for detecting misfolded polypeptides

Abstract

This document relates to methods and materials for detecting the presence or absence of misfolded polypeptides in a sample. For example, a sample (e.g., a biological or environmental sample) can be exposed to nanoparticles (e.g., nanoparticles having a size of 2 μm or less (e.g., 1 μm or less), e.g., silica nanoparticles (siNPs) having a size of 2 μm or less (e.g., siNPs having a size of 1 μm or less)) during a seed amplification assay to accelerate the aggregation of misfolded polypeptides present in the sample into fibrils and / or polypeptide aggregates (e.g., spherical polypeptide aggregates). In some cases, the methods and materials provided herein can be used to determine, at least in part, whether a mammal (e.g., human) has a proteinosis based on the presence or absence of misfolded polypeptides in a sample obtained from that mammal.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the interests of U.S. Patent Application No. 63 / 460,179, filed April 18, 2023. The disclosures of the prior application are deemed to be part of the disclosures of this application and are incorporated therein by reference.

[0002] This document relates to methods and materials for detecting the presence or absence of misfolded polypeptides in a sample. For example, a sample (e.g., a biological or environmental sample) can be exposed to nanoparticles (e.g., nanoparticles having a size of 2 μm or less, e.g., silica nanoparticles (siNPs) having a size of 2 μm or less) during a seed amplification assay to accelerate the aggregation of misfolded polypeptides present in the sample into fibrils and / or polypeptide aggregates (e.g., spherical polypeptide aggregates). For example, a sample (e.g., a biological or environmental sample) can be exposed to nanoparticles (e.g., nanoparticles having a size of 1 μm or less, e.g., siNPs having a size of 1 μm or less) during a seed amplification assay to accelerate the aggregation of misfolded polypeptides present in the sample into fibrils and / or polypeptide aggregates (e.g., spherical polypeptide aggregates). In some cases, the methods and materials provided herein can be used to determine, at least in part, whether a mammal (e.g., a human) has a proteinosis based on the presence or absence of misfolded polypeptides in a sample obtained from that mammal. [Background technology]

[0003] Pathological amyloid formed by misfolded polypeptides is associated with Alzheimer's disease (misfolded Aβ and tau), Parkinson's disease (misfolded α-synuclein), and prion diseases (misfolded cellular prion protein, PrP). CIt is found in many neurodegenerative diseases in both humans and animals, including (Prusiner et al., Proc. Natl. Acad. Sci. USA, 95 (23), 13363-13383 (1998), Soto et al., Arch. Neurol., 65 (2), 184-189 (2008), Marsh et al., J. Virol., 79 (21), 13794-13796 (2005), and Kim et al., Sci. Rep., 6:19548 (2016)). Amyloid formation, observed across pathogenic protein isoforms, occurs via the misfolding of functional polypeptides into insoluble and degradation-resistant amyloid fibrils. A notable feature across a wide range of neurodegenerative diseases is that the generation and deposition of the associated misfolded polypeptides may begin years before the onset of clinical symptoms.

[0004] Real-time quaking-induced conversion (RT-QuIC) has emerged as one of the most sensitive and promising assays for the early diagnosis of various misfold polypeptide-related neurodegenerative diseases (Iranzo et al., Lancet Neurol., 20 (3), 203-212 (2021), Dong et al., Pathogens, 10(3):305 (2021), Coysh et al., Front. Aging Neurosci., 14, 872629 (2022), Telling et al., Proc. Natl. Acad. Sci. USA, 116 (46), 22894-22896 (2019), and Haley et al., Pathogens, 6(3):35 (2017)). RT-QuIC has been successfully applied to detect several misfolded polypeptides associated with various neurodegenerative diseases (e.g., TDP-43, tau, alpha-synuclein, infectious prions, etc.), but the complexity of the biological samples still presents challenges for its clinical diagnostic application. For example, the above assay often takes up to 48 hours to complete the standard test.In addition, natural inhibitors present in biological samples can interfere with reaction kinetics, potentially leading to false negative results (Davenport et al., J. Clin. Microbiol., 56(9):e00947-18 (2018), Coysh et al., Front. Aging Neurosci., 14, 872629 (2022), Li et al., Sci. Rep., 11(1):16759 (2021), and Orru et al., "Real-Time Quaking-Induced Conversion (QuIC) Assays for the Detection and Diagnosis of Human Prion Diseases." pages 621-635 in Prions and Diseases, Zou et al., (Eds); Springer International Publishing: Cham, 2023). [Overview of the project]

[0005] This document provides methods and materials for detecting the presence or absence of misfolded polypeptides, polypeptide fibrils, and / or polypeptide aggregates (e.g., spherical polypeptide aggregates) in a sample. Misfolded polypeptides can accumulate and form aggregates (e.g., aggregates of two or more misfolded polypeptides), such as fibrils (e.g., amyloid fibrils), polypeptide aggregates (e.g., spherical polypeptide aggregates), and / or oligomers. For example, a sample (e.g., a biological or environmental sample) can be exposed to nanoparticles (e.g., nanoparticles having a size of 2 μm or less, e.g., siNPs having a size of 2 μm or less) during a seed amplification assay to accelerate the aggregation of misfolded polypeptides present in the sample into fibrils and / or polypeptide aggregates. For example, a sample (e.g., a biological or environmental sample) can be exposed to nanoparticles (e.g., nanoparticles having a size of 1 μm or less, e.g., siNPs having a size of 1 μm or less) during a seed amplification assay to accelerate the aggregation of misfolded polypeptides present in the sample into fibrils and / or polypeptide aggregates. Seed amplification assays accelerated by the presence of nanoparticles are sometimes called nanoparticle-enhanced seed amplification assays, such as nanoparticle-enhanced RT-QuIC (Nano-QuIC).

[0006] In some cases, a sample (e.g., a biological or environmental sample) can be exposed to nanoparticles (e.g., nanoparticles having a size of 2 μm or less (e.g., 1 μm or less), e.g., siNPs having a size of 2 μm or less (e.g., 1 μm or less)) during a seed amplification assay. For example, a seed amplification assay may include (a) contacting a sample (e.g., a sample suspected of containing misfolded polypeptides) with a polypeptide substrate (e.g., a recombinant PrP substrate, e.g., recombinant hamster PrP (rPrP)) to form a mixture, and (b) stirring the mixture for a certain period of time. In some cases, the methods and materials provided herein can be used to determine, at least in part, whether a mammal (e.g., a human) has a proteinosis based on the presence or absence of misfolded polypeptides in a sample obtained from that mammal. For example, a sample obtained from a mammal can be exposed to nanoparticles (e.g., nanoparticles with a size of 2 μm or less (e.g., 1 μm or less), e.g., siNPs with a size of 2 μm or less (e.g., 1 μm or less)) during a seed amplification assay to accelerate the aggregation of misfolded polypeptides present in the sample into fibrils and / or polypeptide aggregates, allowing for the detection of the presence or absence of fibrils and / or polypeptide aggregates. If fibrils and / or polypeptide aggregates are detected, the mammal can be classified as having a proteinosis.

[0007] As demonstrated herein, adding nanoparticles (e.g., siNPs) having a size of 2 μm or less (e.g., 1 μm or less) to a seed amplification assay (e.g., an RT-QuIC reaction) can accelerate amplification (e.g., aggregation of misfolded polypeptides). For example, adding nanoparticles (e.g., siNPs) having a size of 2 μm or less (e.g., 1 μm or less) to an RT-QuIC reaction can improve the amyloid formation rate during the RT-QuIC reaction. In some cases, adding nanoparticles (e.g., siNPs) having a size of 2 μm or less (e.g., 1 μm or less) to an RT-QuIC reaction can also improve the sensitivity of the RT-QuIC reaction.

[0008] The ability to detect the presence of misfolded polypeptides in a sample as described herein (for example, by exposing the sample to nanoparticles (e.g., siNPs) having a size of 2 μm or less (e.g., 1 μm or less) during a seed amplification assay) provides an accelerated method for rapidly and effectively detecting the presence of misfolded polypeptides. For example, the method described herein provides a protein-based diagnostic method that is relatively easy to use, requires inexpensive reagents and equipment, is highly sensitive and specific to the target protein, can be performed in a variety of settings (e.g., in-situ, in a small laboratory, etc.), and can be performed within about 4 hours (e.g., within about 4 hours, within about 3 hours, within about 2 hours, or less). In some cases, the method and materials described herein can be used to rapidly and easily identify mammals having proteinopathy.

[0009] Overall, one aspect of this document features a method for detecting the presence or absence of misfolded polypeptides in a sample, wherein the method includes, or may essentially consist of, the steps of: (a) exposing a sample to nanoparticles having a size of 2 μm or less (e.g., 1 μm or less) during a seed amplification assay, wherein misfolded polypeptides, if present in the sample, form fibrils; (b) detecting the fibrils formed in step (a); (c) identifying the sample as having the presence of the misfolded polypeptides if the fibrils are detected; and (d) identifying the sample as not having the misfolded polypeptides if the fibrils are not detected. The sample may be a biological sample. Biological samples may be obtained from living mammals. Living mammals may include humans, monkeys, camels, horses, minks, cats, dogs, cattle, sheep, mice, rats, hamsters, mammary deer, axis deer, elk, fallow deer, American marsh deer, mule deer, jayak, moose, pampas deer, red deer, reindeer, roe deer, samba deer, Japanese deer, white-tailed deer, antelope, or goat. Biological samples may include lymphatic tissue, muscle tissue, tonsil tissue, skin tissue, brain tissue, brainstem tissue, blood, cerebrospinal fluid, urine, feces, saliva, mucus, liver tissue, heart tissue, intestinal tissue, semen, spleen tissue, or eye tissue. Biological samples may be obtained from dead mammals. Biological samples may include beef, mutton, lamb, or venison. Samples may include environmental samples. Environmental samples may include soil, water, dust, or plants. Environmental samples can be obtained using cotton swabs or filters. Environmental samples can be obtained from locations selected from the group consisting of natural habitats, waterways, farms, food processing facilities, water treatment facilities, and medical facilities. Environmental samples can be obtained from the food processing facilities, which process food for mammalian consumption. Environmental samples can be obtained from the medical facilities. The method may include a step of isolating polypeptides from the samples prior to step (a).The seed amplification assay may include shaking the sample, sonicating the sample, exposing the sample to sound waves, or irradiating the sample with light. The misfolded polypeptide may be a prion protein (PrP) polypeptide, a tau polypeptide, an amyloid-beta polypeptide, an α-synuclein polypeptide, a TDP-43 polypeptide, an islet amyloid polypeptide (IAPP), a superoxide dismutase 1 (SOD1) polypeptide, a huntingtin polypeptide, a fusion sarcoma (FUS) polypeptide, or a translocation liposarcoma (TLS) polypeptide. The misfolded polypeptide may be associated with proteinopathy. Protein disorders can include chronic wasting disease (CWD), Creutzfeldt-Jakob disease, infectious mink encephalopathy, scrapie, feline spongiform encephalopathy, ungulate spongiform encephalopathy, bovine spongiform encephalopathy, camel spongiform encephalopathy, intermediate pituitary dysfunction (PPID), Alzheimer's disease (AD), Parkinson's disease (PD), Pick's disease, Lewy body dementia (LBD), amyotrophic lateral sclerosis (ALS), multiple system atrophy, progressive supranuclear palsy, corticobasal degeneration, or chronic traumatic encephalopathy. The seed amplification assay can be completed in less than approximately 500 hours. The seed amplification assay can be completed in less than approximately 96 hours. The nanoparticles can be silica nanoparticles, gold nanoparticles, silver nanoparticles, platinum nanoparticles, iron nanoparticles, copper nanoparticles, dielectric nanoparticles with a metal shell, titanium dioxide nanoparticles, tin dioxide nanoparticles, cerium oxide nanoparticles, zirconium dioxide nanoparticles, iron oxide nanoparticles, cadmium telluride quantum dots, or lead sulfide quantum dots.

[0010] In another embodiment, this document features a method for detecting the presence or absence of misfolded polypeptides in a sample, wherein the method includes, or may essentially consist of, the steps of: (a) exposing the sample to nanoparticles having a size of 2 μm or less (e.g., 1 μm or less) during a seed amplification assay, wherein the misfolded polypeptides, if present in the sample, form spherical polypeptide aggregates; (b) detecting the spherical polypeptide aggregates if formed in step (a); (c) identifying the sample as having the presence of the misfolded polypeptides if the spherical polypeptide aggregates are detected; and (d) identifying the sample as not having the misfolded polypeptides if the spherical polypeptide aggregates are not detected. The sample may be a biological sample. Biological samples may be obtained from living mammals. Living mammals may include humans, monkeys, camels, horses, minks, cats, dogs, cattle, sheep, mice, rats, hamsters, mammary deer, axis deer, elk, fallow deer, American marsh deer, mule deer, jayak, moose, pampas deer, red deer, reindeer, roe deer, samba deer, Japanese deer, white-tailed deer, antelope, or goat. Biological samples may include lymphatic tissue, muscle tissue, tonsil tissue, skin tissue, brain tissue, brainstem tissue, blood, cerebrospinal fluid, urine, feces, saliva, mucus, liver tissue, heart tissue, intestinal tissue, semen, spleen tissue, or eye tissue. Biological samples may be obtained from dead mammals. Biological samples may include beef, mutton, lamb, or venison. Samples may include environmental samples. Environmental samples may include soil, water, dust, or plants. Environmental samples can be obtained using cotton swabs or filters. Environmental samples can be obtained from locations selected from the group consisting of natural habitats, waterways, farms, food processing facilities, water treatment facilities, and medical facilities. Environmental samples can be obtained from the food processing facilities, which process food for mammalian consumption. Environmental samples can be obtained from the medical facilities. The method may include a step of isolating polypeptides from the samples prior to step (a).The seed amplification assay may include shaking the sample, sonicating the sample, exposing the sample to sound waves, or irradiating the sample with light. The misfolded polypeptide may be PrP polypeptide, tau polypeptide, amyloid-beta polypeptide, α-synuclein polypeptide, TDP-43 polypeptide, IAPP, SOD1 polypeptide, huntingtin polypeptide, FUS polypeptide, or TLS polypeptide. The misfolded polypeptide may be associated with proteinopathy. Proteinopathy may be CWD, Creutzfeldt-Jakob disease, infectious mink encephalopathy, scrapie, feline spongiform encephalopathy, ungulate spongiform encephalopathy, bovine spongiform encephalopathy, camel spongiform encephalopathy, PPID, AD, PD, Pick's disease, LBD, ALS, multiple system atrophy, progressive supranuclear palsy, corticobasal degeneration, or chronic traumatic encephalopathy. The seed amplification assay can be completed in less than approximately 500 hours. The seed amplification assay can be completed in less than approximately 96 hours. The nanoparticles can be silica nanoparticles, gold nanoparticles, silver nanoparticles, platinum nanoparticles, iron nanoparticles, copper nanoparticles, dielectric nanoparticles with a metal shell, titanium dioxide nanoparticles, tin dioxide nanoparticles, cerium oxide nanoparticles, zirconium dioxide nanoparticles, iron oxide nanoparticles, cadmium telluride quantum dots, or lead sulfide quantum dots.

[0011] In another embodiment, this document features a composition comprising nanoparticles having a size of 2 μm or less (e.g., 1 μm or less) for use in accelerating a seed amplification assay. The seed amplification assay may include shaking the sample, sonicating the sample, exposing the sample to sound waves, or irradiating the sample with light. The nanoparticles may be silica nanoparticles, gold nanoparticles, silver nanoparticles, platinum nanoparticles, iron nanoparticles, copper nanoparticles, dielectric nanoparticles with a metal shell, titanium dioxide nanoparticles, tin dioxide nanoparticles, cerium oxide nanoparticles, zirconium dioxide nanoparticles, iron oxide nanoparticles, cadmium telluride quantum dots, or lead sulfide quantum dots. The seed amplification assay can be completed in less than approximately 500 hours. The seed amplification assay can be completed in less than approximately 96 hours.

[0012] In another aspect, this document features a method for detecting the presence or absence of misfolded polypeptides in a sample, wherein the method includes, or may essentially consist of, the steps of: (a) exposing the sample to a nanoporous material having pore sizes from 2 nm to about 200 nm (e.g., from 2 nm to about 100 nm) during a seed amplification assay, wherein the misfolded polypeptides, if present in the sample, form fibrils; (b) detecting the fibrils formed in step (a); (c) identifying the sample as having the presence of the misfolded polypeptides, if the fibrils are detected; and (d) identifying the sample as not having the misfolded polypeptides, if the fibrils are not detected. The sample may be a biological sample. Biological samples may be obtained from living mammals. Living mammals may include humans, monkeys, camels, horses, minks, cats, dogs, cattle, sheep, mice, rats, hamsters, mammary deer, axis deer, elk, fallow deer, American marsh deer, mule deer, jayak, moose, pampas deer, red deer, reindeer, roe deer, samba deer, Japanese deer, white-tailed deer, antelope, or goat. Biological samples may include lymphatic tissue, muscle tissue, tonsil tissue, skin tissue, brain tissue, brainstem tissue, blood, cerebrospinal fluid, urine, feces, saliva, mucus, liver tissue, heart tissue, intestinal tissue, semen, spleen tissue, or eye tissue. Biological samples may be obtained from dead mammals. Biological samples may include beef, mutton, lamb, or venison. Samples may include environmental samples. Environmental samples may include soil, water, dust, or plants. Environmental samples can be obtained using cotton swabs or filters. Environmental samples can be obtained from locations selected from the group consisting of natural habitats, waterways, farms, food processing facilities, water treatment facilities, and medical facilities. Environmental samples can be obtained from the food processing facility, where the food processing facility processes food for consumption by mammals. The method of claim 56, wherein the environmental sample can be obtained from the medical facility. The method may include a step of isolating polypeptides from the sample prior to step (a).The seed amplification assay may include shaking the sample, sonicating the sample, exposing the sample to sound waves, or irradiating the sample with light. The misfolded polypeptide may be PrP polypeptide, tau polypeptide, amyloid-beta polypeptide, α-synuclein polypeptide, TDP-43 polypeptide, IAPP, SOD1 polypeptide, huntingtin polypeptide, FUS polypeptide, or TLS polypeptide. The misfolded polypeptide may be associated with proteinopathy. Proteinopathy may be CWD, Creutzfeldt-Jakob disease, infectious mink encephalopathy, scrapie, feline spongiform encephalopathy, ungulate spongiform encephalopathy, bovine spongiform encephalopathy, camel spongiform encephalopathy, PPID, AD, PD, Pick's disease, LBD, ALS, multiple system atrophy, progressive supranuclear palsy, corticobasal degeneration, or chronic traumatic encephalopathy. The seed amplification assay can be completed in less than approximately 500 hours. The seed amplification assay can be completed in less than approximately 96 hours. The nanoporous material can be mesoporous silica, mesoporous polytetrafluoroethylene, zeolite particles, or metal-organic frameworks (MOFs).

[0013] In another embodiment, this document features a method for detecting the presence or absence of misfolded polypeptides in a sample, wherein the method includes, or may essentially consist of, the steps of: (a) exposing the sample to a nanoporous material having pore sizes from 2 nm to about 200 nm (e.g., from 2 nm to about 100 nm) during a seed amplification assay, wherein the misfolded polypeptides, if present in the sample, form spherical polypeptide aggregates; (b) detecting the spherical polypeptide aggregates if formed in step (a); (c) identifying the sample as having the presence of the misfolded polypeptides if the spherical polypeptide aggregates are detected; and (d) identifying the sample as not having the misfolded polypeptides if the spherical polypeptide aggregates are not detected. The sample may be a biological sample. Biological samples may be obtained from living mammals. Living mammals may include humans, monkeys, camels, horses, minks, cats, dogs, cattle, sheep, mice, rats, hamsters, mammary deer, axis deer, elk, fallow deer, American marsh deer, mule deer, jayak, moose, pampas deer, red deer, reindeer, roe deer, samba deer, Japanese deer, white-tailed deer, antelope, or goat. Biological samples may include lymphatic tissue, muscle tissue, tonsil tissue, skin tissue, brain tissue, brainstem tissue, blood, cerebrospinal fluid, urine, feces, saliva, mucus, liver tissue, heart tissue, intestinal tissue, semen, spleen tissue, or eye tissue. Biological samples may be obtained from dead mammals. Biological samples may include beef, mutton, lamb, or venison. Samples may include environmental samples. Environmental samples may include soil, water, dust, or plants. Environmental samples can be obtained using cotton swabs or filters. Environmental samples can be obtained from locations selected from the group consisting of natural habitats, waterways, farms, food processing facilities, water treatment facilities, and medical facilities. Environmental samples can be obtained from the food processing facilities, which process food for mammalian consumption. Environmental samples can be obtained from the medical facilities. The method may include a step of isolating polypeptides from the samples prior to step (a).The seed amplification assay may include shaking the sample, sonicating the sample, exposing the sample to sound waves, or irradiating the sample with light. The misfolded polypeptide may be PrP polypeptide, tau polypeptide, amyloid-beta polypeptide, α-synuclein polypeptide, TDP-43 polypeptide, IAPP, SOD1 polypeptide, huntingtin polypeptide, FUS polypeptide, or TLS polypeptide. The misfolded polypeptide may be associated with proteinopathy. Proteinopathy may be CWD, Creutzfeldt-Jakob disease, infectious mink encephalopathy, scrapie, feline spongiform encephalopathy, ungulate spongiform encephalopathy, bovine spongiform encephalopathy, camel spongiform encephalopathy, PPID, AD, PD, Pick's disease, LBD, ALS, multiple system atrophy, progressive supranuclear palsy, corticobasal degeneration, or chronic traumatic encephalopathy. The seed amplification assay can be completed in less than approximately 500 hours. The seed amplification assay can be completed in less than approximately 96 hours. The nanoporous material can be mesoporous silica, mesoporous polytetrafluoroethylene, zeolite particles, or MOF.

[0014] In another embodiment, this document features a composition comprising a nanoporous material having a pore size from 2 nm to about 200 nm (e.g., from 2 nm to about 100 nm) for use in accelerating a seed amplification assay. The seed amplification assay may include shaking the sample, sonicating the sample, exposing the sample to sound waves, or irradiating the sample with light. The nanoporous material may be mesoporous silica, mesoporous polytetrafluoroethylene, zeolite particles, or MOFs. The seed amplification assay can be completed in less than about 500 hours. The seed amplification assay can be completed in less than about 96 hours.

[0015] In another embodiment, this document features a method for detecting the presence or absence of misfolded polypeptides in a sample, the method comprising, (a) exposing the sample to nanoparticles having a size of 2 μm or less (e.g., 1 μm or less) in a seed amplification assay comprising exposing the sample to sound waves, wherein the misfolded polypeptides form fibrils if present in the sample; (b) detecting the fibrils if formed in step (a); (c) identifying the sample as having the presence of the misfolded polypeptides if the fibrils are detected; and (d) identifying the sample as not having the misfolded polypeptides if the fibrils are not detected, or which may essentially consist of these steps.

[0016] In another embodiment, this document features a method for detecting the presence or absence of misfolded polypeptides in a sample, wherein the method includes, or may essentially consist of, the steps of: (a) exposing the sample to nanoparticles having a size of 2 μm or less (e.g., 1 μm or less) in a seed amplification assay comprising exposing the sample to sound waves, wherein the misfolded polypeptides, if present in the sample, form spherical polypeptide aggregates; (b) detecting the spherical polypeptide aggregates if formed in step (a); (c) identifying the sample as having the presence of the misfolded polypeptides if the spherical polypeptide aggregates are detected; and (d) identifying the sample as not having the misfolded polypeptides if the spherical polypeptide aggregates are not detected.

[0017] In another embodiment, this document features a method for detecting the presence or absence of misfolded polypeptides in a sample, the method comprising, (a) exposing the sample to metal nanoparticles having a size of 2 μm or less (e.g., 1 μm or less) during a seed amplification assay including light irradiation of the sample, wherein the misfolded polypeptides form fibrils if present in the sample; (b) detecting the fibrils formed in step (a); (c) identifying the sample as having the presence of the misfolded polypeptides if the fibrils are detected; and (d) identifying the sample as not having the misfolded polypeptides if the fibrils are not detected, or which may essentially consist of these steps.

[0018] In another embodiment, this document features a method for detecting the presence or absence of misfolded polypeptides in a sample, wherein the method includes, or may essentially consist of, the steps of: (a) exposing the sample to metal nanoparticles having a size of 2 μm or less (e.g., 1 μm or less) during a seed amplification assay including light irradiation of the sample, wherein the misfolded polypeptides, if present in the sample, form spherical polypeptide aggregates; (b) detecting the spherical polypeptide aggregates if formed in step (a); (c) identifying the sample as having the presence of the misfolded polypeptides if the spherical polypeptide aggregates are detected; and (d) identifying the sample as not having the misfolded polypeptides if the spherical polypeptide aggregates are not detected.

[0019] Unless otherwise indicated, 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 methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0020] Details of one or more embodiments of the invention are described in the accompanying drawings and the following description of the modes for carrying out the invention. Other features, objects, and advantages of the invention will become apparent from the description of the modes for carrying out the invention and the drawings and from the claims.

Brief Description of the Drawings

[0021] [Figure 1A-B] Figure 1A) is a schematic diagram of fibril growth showing the nucleation, growth, and stationary phases. The sigmoid curve represents the detection of misfolded protein (MP), and the flat line represents the absence of MP detection. Figure 1B) is a graph of the results from Nano-QuIC and conventional RT-QuIC. Nano-QuIC conditions: 48°C, 2.5 mg / mL 50 nm silica NPs. [Figure 1C] Figure 1C) is a schematic diagram of the Nano-QuIC seed amplification mechanism. In Nano-QuIC, silica NPs promote primary nucleation at a higher rate than the bulk solution method, e.g., RT-QuIC. [Figure 2A-B] Figure 2A) is a graph of the relative amyloid formation rate (RAF) for different diameters and concentrations of silica NP solutions seeded with CWD-positive or -negative tissue homogenates and amplified at 42°C. Figure 2B) is a graph of the relative RAF of solutions seeded with CWD-positive or -negative tissue with different concentrations of 50 nm silica NPs amplified at different temperatures. [Figure 2C-D]Figure 2C) shows the nucleation phase lag time versus the concentration of 50 nm silica NP at 48°C. Figure 2D) shows the fibril growth time constant (τ) versus the concentration of 50 nm silica NP at 48°C. Unless otherwise noted, error bars indicate the standard deviation. [Figure 3] This figure shows the relative RAF (ratio of RAF of the reaction with 15nm AuNP to the reaction without 15nm AuNP). The assay was run at 42°C. Error bars indicate the standard deviation. [Figure 4] Figure 4A) This figure shows the raw RAF of a 10-fold dilution series of CWD-positive tissue amplified at 42°C with RT-QuIC versus Nano-QuIC (50nm siNP, 2.5 mg / mL). Arrows highlight the absence of signal from RT-QuIC (false negative), which is likely due to the inhibitor. Figure 4B) This figure shows the kinetic curve of dilutions of 10-1 CWD-positive tissue amplified at 42°C with RT-QuIC versus Nano-QuIC (50nm siNP, 2.5 mg / mL). Unless otherwise noted, error bars indicate standard deviation. [Figure 5] Figure 5A) shows the relative RAF of Nano-QuIC versus 20nm silica NP at various temperatures and concentrations. Figure 5B) shows the relative RAF of Nano-QuIC versus 100nm silica NP at various temperatures and concentrations. [Figure 6] This graph shows the kinetic curves of positive and negative CWD samples measured using Nano-QuIC (50 nm, 2.5 mg / ml, 48°C). [Figure 7A]This is an illustrative figure demonstrating that the addition of nanoparticles (NPs) with a diameter of less than 1 micron accelerates the detection of misfolded polypeptides by the seed amplification assay (SAA). (Figure 7A) This is a diagram illustrating the SAA. SAA is a highly sensitive assay used to detect misfolded protein seeds by amplifying protein misfolds. SAA involves multiple cycles of amplifying protein misfolds. In each cycle, if misfolded protein seeds are present, the assay provides a protein substrate to support the growth of misfolded protein fibrils (amyloid) initiated by the seeds. Mechanical forces (e.g., shaking or sonication) are then applied to fragment the misfolded protein fibrils, resulting in the creation of more seeds for the next cycle of fibril amplification. [Figure 7B-C] Figure 7B) The addition of NP reduced the number of SAA cycles required to detect misfolded polypeptides. ThT, thioflavin T, is the phosphor used to detect misfolded polypeptides; NP, is a nanoparticle less than 1 micron in diameter; threshold is the fluorescence level used to determine whether misfolded polypeptides are detected. Figure 7C) The addition of NP reduced the amyloid formation rate required to detect misfolded polypeptides. Amyloid formation rate = 1 / time to threshold, a term used to describe how quickly amyloid forms in SAA. [Figure 8]Figure 8A) shows Nano-QuIC applied to human samples. It compares Nano-QuIC with conventional RT-QuIC for detecting artificially misfolded α-synuclein (a Parkinson's disease-related protein) in human plasma at final spike concentrations ranging from 9 μg / mL to 90 pg / mL. Nano-QuIC addresses inhibitors, enabling a 100-fold improvement in sensitivity. Figure 8B shows Nano-QuIC data from two human plasma samples, one from patients diagnosed with Parkinson's disease (solid line) versus one from healthy controls (dotted line). Nano-QuIC detected more seeding activity in the Parkinson's disease samples than in the healthy controls. [Modes for carrying out the invention]

[0022] This document provides methods and materials for detecting the presence or absence of misfolded polypeptides, polypeptide fibrils, and / or polypeptide aggregates (e.g., spherical polypeptide aggregates) in a sample. In some cases, a sample (e.g., a biological or environmental sample) can be exposed to nanoparticles (e.g., nanoparticles having a size of 2 μm or less, e.g., siNPs having a size of 2 μm or less) during a seed amplification assay to accelerate the aggregation of misfolded polypeptides present in the sample into fibrils and / or polypeptide aggregates, thereby enabling the detection of the presence or absence of fibrils and / or polypeptide aggregates. For example, a sample suspected of containing misfolded polypeptides can be exposed to nanoparticles (e.g., nanoparticles having a size of 2 μm or less, e.g., siNPs having a size of 2 μm or less) during a seed amplification assay so that, if misfolded polypeptides are present, they can aggregate to form fibrils (e.g., aggregates of two or more misfolded polypeptides), thereby enabling the detection of the presence or absence of fibrils. In some cases, a sample (e.g., a biological or environmental sample) can be exposed to nanoparticles (e.g., nanoparticles with a size of 1 μm or less, e.g., siNPs with a size of 1 μm or less) during a seed amplification assay to accelerate the aggregation of misfolded polypeptides present in the sample into fibrils and / or polypeptide aggregates, thereby enabling the detection of the presence or absence of fibrils and / or polypeptide aggregates. For example, a sample suspected of containing misfolded polypeptides can be exposed to nanoparticles (e.g., nanoparticles with a size of 1 μm or less, e.g., siNPs with a size of 1 μm or less) during a seed amplification assay so that, if misfolded polypeptides are present, they can aggregate to form fibrils (e.g., aggregates of two or more misfolded polypeptides), thereby enabling the detection of the presence or absence of fibrils.

[0023] A sample (e.g., a biological sample or an environmental sample) can be exposed to nanoparticles (e.g., nanoparticles having a size of 2 μm or less (e.g., 1 μm or less), e.g., siNPs having a size of 2 μm or less (e.g., 1 μm or less)) using any type of seed amplification assay. In some cases, the seed amplification assay may include (a) contacting the sample (e.g., a sample suspected of containing misfolded polypeptides) with a polypeptide substrate (e.g., a recombinant PrP substrate, e.g., recombinant hamster PrP (rPrP)) to form a mixture, and (b) stirring the mixture for a certain period of time.

[0024] A seed amplification assay may involve contacting a sample (e.g., a sample suspected of containing misfolded polypeptides) with any suitable polypeptide substrate. In some cases, the polypeptide substrate may include one or more recombinant polypeptides. Examples of polypeptide substrates that can be used in a seed amplification assay include, but are not limited to, recombinant PrP polypeptides (e.g., rPrP polypeptides) and α-synuclein polypeptides. In some cases, the polypeptide substrate may be in the form of a tissue homogenate (e.g., a tissue homogenate containing one or more polypeptide substrates).

[0025] Examples of seed amplification assays, but not limited to, include oscillation-induced conversion assays (e.g., RT-QuIC assay, MN-QuIC assay, and photonic-QuIC (Photo-QuIC) assay) and protein-misfolding cyclic amplification (PMCA) assays. In some cases, a sample (e.g., a biological or environmental sample) may be exposed to nanoparticles (e.g., nanoparticles with a size of 2 μm or less (e.g., 1 μm or less), e.g., siNPs with a size of 2 μm or less (e.g., 1 μm or less)) in seed amplification assays described in other literature (see, for example, WO 2002 / 221683, Haley et al., Pathogens, 6:35 (2017), Dong et al., Pathogens, 10:305 (2021), and Schwabenlander et al., J. Wildl. Dis., 58(1):50-62 (2021)). In some cases, a sample (e.g., a biological sample or an environmental sample) can be exposed to nanoparticles (e.g., nanoparticles having a size of 2 μm or less (e.g., 1 μm or less), e.g., siNPs having a size of 2 μm or less (e.g., 1 μm or less)) in a seed amplification assay performed with a microfluidic device.

[0026] In some cases, the presence or absence of misfolded polypeptides can be detected in less than 500 hours (for example, less than 450 hours, less than 400 hours, less than 350 hours, less than 300 hours, less than 250 hours, less than 200 hours, less than 150 hours, less than 100 hours, or less than 50 hours) using the methods and materials described herein (e.g., methods for detecting the presence or absence of misfolded polypeptides). For example, using the methods and materials described herein for detecting the presence or absence of misfolded polypeptides, the presence or absence of misfolded polypeptides can be detected from about 2 hours to about 500 hours (for example, from about 2 hours to about 400 hours, from about 2 hours to about 300 hours, from about 2 hours to about 200 hours, from about 2 hours to about 100 hours, from about 2 hours to about 50 hours, from about 50 hours to about 500 hours, from about 100 hours to about 500 hours, from about 150 hours to about 500 hours, from about 200 hours to about 500 hours, from about 250 hours to about 500 hours, It can be detected between approximately 300 hours and 500 hours, between approximately 350 hours and 500 hours, between approximately 400 hours and 500 hours, between approximately 450 hours and 500 hours, between approximately 50 hours and 450 hours, between approximately 100 hours and 400 hours, between approximately 150 hours and 350 hours, between approximately 200 hours and 300 hours, between approximately 50 hours and 150 hours, between approximately 100 hours and 200 hours, between approximately 150 hours and 250 hours, between approximately 250 hours and 350 hours, between approximately 300 hours and 400 hours, or between approximately 350 hours and 450 hours.

[0027] In some cases, the presence or absence of misfolded polypeptides can be detected in less than 96 hours (e.g., less than 48 hours, less than 14 hours, less than 10 hours, less than 8 hours, or less than about 5 hours) using the methods and materials described herein (e.g., methods for detecting the presence or absence of misfolded polypeptides). For example, the presence or absence of misfolded polypeptides can be detected from about 2 hours to about 96 hours (e.g., from about 2 hours to about 48 hours, from about 2 hours to about 14 hours, from about 2 hours to about 8 hours, from about 2 hours to about 6 hours, from about 2 hours to about 5 hours, from about 2 hours to about 4 hours, from about 4 hours to about 96 hours, from about 6 hours to about 96 hours, from about 12 hours to about 96 hours, from about 3 hours to about 48 hours, from about 5 hours to about 14 hours, from about 3 hours to about 12 hours, from about 4 hours to about 10 hours, from about 5 hours to about 8 hours, from about 3 hours to about 5 hours, from about 4 hours to about 6 hours, from about 5 hours to about 7 hours, from about 6 hours to about 8 hours, from about 7 hours to about 9 hours, from about 8 hours to about 10 hours, or from about 9 hours to about 11 hours) using the methods and materials for detecting the presence or absence of misfolded polypeptides described herein.

[0028] In some cases, the methods and materials described herein (e.g., methods for detecting the presence or absence of misfolded polypeptides) can be used to detect the presence or absence of any misfolded polypeptide, polypeptide fibrils, and / or polypeptide aggregates (e.g., spherical polypeptide aggregates). For example, the methods and materials described herein can be used to detect the presence or absence of amyloid plaques. For example, the methods and materials described herein can be used to detect the presence or absence of tau tangles.

[0029] In some cases, the methods and materials described herein (e.g., methods for detecting the presence or absence of misfolded polypeptides) can be used to determine, at least in part, whether a mammal (e.g., human) has a proteinosis based on the presence or absence of misfolded polypeptides in a sample obtained from that mammal. For example, a sample obtained from a mammal can be exposed to nanoparticles (e.g., nanoparticles having a size of 2 μm or less (e.g., 1 μm or less), e.g., siNPs having a size of 2 μm or less (e.g., 1 μm or less)) during a seed amplification assay to accelerate the aggregation of misfolded polypeptides present in the sample into fibrils and / or polypeptide aggregates, the presence or absence of fibrils and / or polypeptide aggregates can be detected, and the mammal can be classified as having a proteinosis if the presence of fibrils and / or polypeptide aggregates is detected. For example, a sample obtained from a mammal can be exposed to nanoparticles (e.g., nanoparticles with a size of 2 μm or less (e.g., 1 μm or less), e.g., siNPs with a size of 2 μm or less (e.g., 1 μm or less)) during a seed amplification assay to accelerate the aggregation of misfolded polypeptides present in the sample into fibrils and / or polypeptide aggregates. The presence or absence of fibrils and / or polypeptide aggregates can be detected, and if the absence of fibrils and polypeptide aggregates is detected, the mammal can be classified as not having a proteinosis.

[0030] The sample can be exposed to nanoparticles of any suitable size (e.g., any suitable longest dimension, e.g., diameter). In some cases, the nanoparticles can have a diameter of approximately 2 μm or less. For example, the nanoparticles can range in diameter from approximately 2 nm to approximately 2 μm (e.g., approximately 2 nm to approximately 1.8 μm, approximately 2 nm to approximately 1.5 μm, approximately 2 nm to approximately 1.3 μm, approximately 2 nm to approximately 100 nm, approximately 2 nm to approximately 75 nm, approximately 2 nm to approximately 50 nm, approximately 2 nm to approximately 25 nm, approximately 25 nm to approximately 2 μm, approximately 50 nm to approximately 2 μm, approximately 75 nm to approximately 2 μm, approximately 100 nm to approximately 2 μm, approximately 250 nm to approximately 2 μm, approximately 500 nm or They can have diameters of approximately 2 μm, from approximately 750 nm to approximately 2 μm, from approximately 1 μm to approximately 2 μm, from approximately 1.3 μm to approximately 2 μm, from approximately 1.5 μm to approximately 2 μm, from approximately 18 μm to approximately 2 μm, from approximately 100 nm to approximately 1.75 μm, from approximately 250 nm to approximately 1.5 μm, from approximately 500 nm to approximately 1.25 μm, from approximately 750 nm to approximately 1 μm, from approximately 250 nm to approximately 750 nm, from approximately 500 nm to approximately 1 μm, or from approximately 750 nm to approximately 1.5 μm. In some cases, the nanoparticles can have a diameter of approximately 1 μm or less. For example, nanoparticles range in diameter from approximately 2 nm to approximately 1 μm (for example, approximately 2 nm to approximately 800 nm, approximately 2 nm to approximately 600 nm, approximately 2 nm to approximately 500 nm, approximately 2 nm to approximately 400 nm, approximately 2 nm to approximately 300 nm, approximately 2 nm to approximately 200 nm, approximately 2 nm to approximately 100 nm, approximately 2 nm to approximately 50 nm, approximately 50 nm to approximately 1 μm, approximately 100 nm to approximately 1 μm, approximately 200 nm to approximately 1 μm, approximately 300 nm to approximately 1 μm, approximately 500 nm to approximately 1 μm, approximately 700 nm They can have a range from nm to approximately 1 μm, from approximately 900 nm to approximately 1 μm, from approximately 50 nm to approximately 750 nm, from approximately 100 nm to approximately 500 nm, from approximately 200 nm to approximately 400 nm, from approximately 50 nm to approximately 100 nm, from approximately 100 nm to approximately 300 nm, from approximately 200 nm to approximately 400 nm, from approximately 300 nm to approximately 500 nm, from approximately 400 nm to approximately 600 nm, from approximately 500 nm to approximately 700 nm, with a diameter of approximately 600 nm to approximately 800 nm, or from approximately 700 nm to approximately 900 nm.Nanoparticles may have any shape (e.g., spherical, rod-shaped, nanowire-shaped, shell-shaped, cubic, elliptical, and star-shaped).

[0031] A sample can be exposed to any suitable type of nanoparticle. Nanoparticles having a size of 2 μm or less (e.g., 1 μm or less) are sometimes called quantum dots (QDs). In some cases, the nanoparticles can be metallic nanoparticles. Examples of nanoparticles that can be used to expose a sample (e.g., a biological or environmental sample) include, but are not limited to, gold nanoparticles (AuNPs), silver nanoparticles, platinum nanoparticles, iron nanoparticles, copper nanoparticles, dielectric nanoparticles with a metal shell, SiNPs, titanium dioxide nanoparticles, tin dioxide nanoparticles, cerium oxide nanoparticles, zirconium dioxide nanoparticles, iron oxide nanoparticles, cadmium telluride QDs, and lead sulfide QDs.

[0032] In some cases, the methods and materials provided herein may include, in place of or in addition to nanoparticles, one or more nanoporous materials (e.g., one or more materials containing nanometer-scale pores). For example, a sample (e.g., a biological or environmental sample) may be exposed to one or more nanoporous materials in a seed amplification assay (e.g., to accelerate the aggregation of misfolded polypeptides present in the sample into fibrils and / or polypeptide aggregates). The nanoporous materials may contain pores having any suitable size (e.g., pores having any suitable longest dimension, e.g., diameter). In some cases, nanoporous materials range from 2 nm to approximately 200 nm (for example, 2 nm to approximately 180 nm, 2 nm to approximately 150 nm, 2 nm to approximately 130 nm, 2 nm to approximately 100 nm, 2 nm to approximately 75 nm, 2 nm to approximately 50 nm, 2 nm to approximately 25 nm, 25 nm to approximately 200 nm, 50 nm to approximately 200 nm, 75 nm to approximately 200 nm, 100 nm to approximately 200 nm, 1 It can contain pores having sizes (e.g., longest dimension, e.g., diameter) of 25 nm to about 200 nm, 150 nm to about 200 nm, 175 nm to about 200 nm, 25 nm to about 175 nm, 50 nm to about 150 nm, 75 nm to about 125 nm, 25 nm to about 75 nm, 50 nm to about 100 nm, or 100 nm to about 150 nm, or 125 nm to about 175 nm. In some cases, nanoporous materials can contain pores having sizes (e.g., longest dimension, e.g., diameter) ranging from 2 nm to about 100 nm (e.g., 2 nm to about 75 nm, 2 nm to about 50 nm, 2 nm to about 25 nm, 25 nm to about 100 nm, 50 nm to about 100 nm, 75 nm to about 100 nm, 25 nm to about 75 nm, 30 nm to about 50 nm, 40 nm to about 60 nm, 50 nm to about 70 nm, or 60 nm to about 80 nm).In some cases, nanoporous materials can have any suitable size (e.g., any suitable longest dimension, e.g., diameter), provided that the nanoporous material contains pores on a nanometer scale. In some cases, the nanoporous material can be a porous oxide (e.g., a mesoporous oxide). Examples of nanoporous materials that can be exposed to samples (e.g., biological or environmental samples) include, but are not limited to, porous silica (e.g., mesoporous silica), porous polytetrafluoroethylene (e.g., mesoporous polytetrafluoroethylene, e.g., mesoporous TEFLON®), zeolite particles, and metal-organic frameworks (MOFs). In some cases, the nanoporous material can be in the form of nanoporous beads. For example, the nanoporous material may contain one or more mesoporous silica beads. For example, the nanoporous material may contain one or more mesoporous polytetrafluoroethylene beads (e.g., one or more TEFLON® beads).

[0033] A sample (e.g., a sample suspected of containing misfolded polypeptides) can be stirred in the presence of nanoparticles (e.g., nanoparticles having a size of 2 μm or less (e.g., 1 μm or less), e.g., siNPs having a size of 2 μm or less (e.g., 1 μm or less)) using any suitable method. For example, the sample can be stirred by exposing it to one or more mechanical forces. In some cases, a sample exposed to nanoparticles (e.g., nanoparticles having a size of 2 μm or less (e.g., 1 μm or less), e.g., siNPs having a size of 2 μm or less (e.g., 1 μm or less)) can be stirred by shaking. The sample can be shaken for any suitable time. For example, the sample can be shaken for a period of time ranging from approximately 2 hours to approximately 96 hours (e.g., approximately 2 hours to approximately 48 hours, approximately 2 hours to approximately 24 hours, approximately 2 hours to approximately 12 hours, approximately 2 hours to approximately 8 hours, approximately 8 hours to approximately 96 hours, approximately 12 hours to approximately 96 hours, approximately 24 hours to approximately 96 hours, approximately 48 hours to approximately 96 hours, approximately 12 hours to approximately 48 hours, approximately 12 hours to approximately 24 hours, or approximately 24 hours to approximately 48 hours). The sample can be shaken at any appropriate speed. For example, the sample can be run from approximately 100 RPM to approximately 1200 RPM (for example, from approximately 100 RPM to approximately 1000 RPM, from approximately 100 RPM to approximately 700 RPM, from approximately 100 RPM to approximately 500 RPM, from approximately 300 RPM to approximately 1200 RPM, from approximately 500 RPM to approximately 1200 RPM, from approximately 800 RPM to approximately 1200 RPM, from approximately 1000 RPM to approximately 1200 RPM, from approximately 300 RPM to The sample can be shaken at speeds up to approximately 1000 RPM, from approximately 500 RPM to approximately 800 RPM, from approximately 300 RPM to approximately 500 RPM, from approximately 40 RPM to approximately 600 RPM, from approximately 500 RPM to approximately 700 RPM, from approximately 600 RPM to approximately 800 RPM, from approximately 700 RPM to approximately 900 RPM, from approximately 800 RPM to approximately 1000 RPM, or from approximately 900 RPM to approximately 1100 RPM. The sample can be shaken at any suitable temperature.For example, the sample can be shaken from approximately 20°C to approximately 60°C (e.g., from approximately 20°C to approximately 50°C, from approximately 20°C to approximately 40°C, from approximately 20°C to approximately 30°C, from approximately 30°C to approximately 60°C, from approximately 40°C to approximately 60°C, from approximately 50°C to approximately 60°C, or from approximately 30°C to approximately 50°C).

[0034] In some cases, a sample exposed to nanoparticles (e.g., nanoparticles having a size of 2 μm or less (e.g., 1 μm or less), e.g., siNPs having a size of 2 μm or less (e.g., 1 μm or less)) can be stirred by sonication. The sample can be sonicated for any appropriate required time. For example, the sample can be sonicated for about 5 seconds to about 30 seconds (e.g., about 5 seconds to about 25 seconds, about 5 seconds to about 20 seconds, about 5 seconds to about 15 seconds, about 5 seconds to about 10 seconds, about 10 seconds to about 30 seconds, about 15 seconds to about 30 seconds, about 20 seconds to about 30 seconds, about 25 seconds to about 30 seconds, about 10 seconds to about 25 seconds, about 15 seconds to about 20 seconds, about 10 seconds to about 15 seconds, or about 20 seconds to about 25 seconds). The sonication step can be performed any number of times. For example, a sample can be ultrasonically treated approximately 2 to 10 times (e.g., approximately 2 to 8 times, approximately 2 to 6 times, approximately 2 to 4 times, approximately 4 to 10 times, approximately 6 to 10 times, approximately 8 to 10 times, approximately 4 to 8 times, approximately 4 to 6 times, or approximately 6 to 8 times). When ultrasonically treating a sample more than twice, the ultrasonic treatment stage may include a rest period between ultrasonic treatment cycles. The pause period can be approximately 10 to 40 minutes (for example, approximately 10 to 35 minutes, 10 to 30 minutes, 10 to 25 minutes, 10 to 20 minutes, 10 to 15 minutes, 15 to 40 minutes, 20 to 40 minutes, 25 to 40 minutes, 30 to 40 minutes, 35 to 40 minutes, 15 to 35 minutes, 20 to 30 minutes, 15 to 25 minutes, or 25 to 35 minutes). The sample can be ultrasonically treated at any appropriate amplitude. For example, a sample can be ultrasonically treated at a power level ranging from approximately 50 watts to approximately 500 watts (e.g., approximately 50 watts to approximately 300 watts, approximately 50 watts to approximately 100 watts, approximately 100 watts to approximately 500 watts, approximately 300 watts to approximately 500 watts, approximately 100 watts to approximately 300 watts, approximately 100 watts to approximately 200 watts, approximately 200 watts to approximately 300 watts, or approximately 300 watts to approximately 400 watts).The sample can be ultrasonically treated at any suitable temperature. For example, the sample can be ultrasonically treated from approximately 20°C to approximately 65°C (e.g., from approximately 20°C to approximately 50°C, from approximately 20°C to approximately 40°C, from approximately 30°C to approximately 65°C, from approximately 40°C to approximately 65°C, from approximately 50°C to approximately 65°C, or from approximately 30°C to approximately 50°C).

[0035] In some cases, a sample exposed to nanoparticles (e.g., nanoparticles having a size of 2 μm or less (e.g., 1 μm or less), e.g., siNPs having a size of 2 μm or less (e.g., 1 μm or less)) can be stirred using sound waves. The sample can be exposed to sound waves for any suitable required time. For example, the sample can be exposed to sound waves for about 1 hour to about 24 hours (e.g., about 1 hour to about 24 hours, about 1 hour to about 18 hours, about 1 hour to about 12 hours, about 1 hour to about 8 hours, about 1 hour to about 4 hours, about 8 hours to about 24 hours, about 12 hours to about 24 hours, about 18 hours to about 24 hours, about 4 hours to about 18 hours, about 8 hours to about 12 hours, about 4 hours to about 12 hours, or about 8 hours to about 18 hours). The sample can be exposed to sound waves having any suitable frequency. For example, a sample can be exposed to sound waves having frequencies from approximately 100 Hz to approximately 100,000 Hz (e.g., approximately 100 Hz to approximately 50,000 Hz, approximately 100 Hz to approximately 10,000 Hz, approximately 100 Hz to approximately 1,000 Hz, approximately 100 Hz to approximately 500 Hz, approximately 500 Hz to approximately 100,000 Hz, approximately 1,000 Hz to approximately 100,000 Hz, approximately 10,000 Hz to approximately 100,000 Hz, approximately 50,000 Hz to approximately 100,000 Hz, or approximately 1,000 Hz to approximately 50,000 Hz).

[0036] In some cases, a sample exposed to nanoparticles (e.g., nanoparticles having a size of 2 μm or less (e.g., 1 μm or less), e.g., siNPs having a size of 2 μm or less (e.g., 1 μm or less)) can be stirred using light irradiation. The sample can be exposed to light irradiation for any appropriate required time. For example, a sample can be exposed to light irradiation for a period of approximately 1 microsecond to approximately 1 hour (e.g., approximately 1 nanosecond to approximately 1 hour, approximately 1 picosecond to approximately 1 hour, approximately 1 millisecond to approximately 1 hour, approximately 1 second to approximately 1 hour, approximately 1 minute to approximately 1 hour, approximately 1 microsecond to approximately 1 minute, approximately 1 microsecond to approximately 1 second, approximately 1 microsecond to approximately 1 millisecond, approximately 1 microsecond to approximately 1 picosecond, approximately 1 microsecond to approximately 1 nanosecond, approximately 1 nanosecond to approximately 1 minute, approximately 1 picosecond to approximately 1 second, approximately 1 nanosecond to approximately 1 picosecond, approximately 1 picosecond to approximately 1 millisecond, approximately 1 millisecond to approximately 1 second, or approximately 1 second to approximately 1 minute). The light irradiation step can be performed any number of times. If the sample is exposed to light irradiation more than once, the light irradiation step may include a rest period between light irradiation cycles. The light irradiation step can be performed any number of times. For example, the sample can be exposed to light irradiation at any appropriate intensity. For example, the sample can be exposed to light irradiation at approximately 10 5 W / m 2 From about 10 10 W / m 2 The sample can be exposed to light irradiation up to a certain point. Light irradiation can be provided by any suitable light source. Examples of light sources that can be used to provide light irradiation include, but are not limited to, laser sources, light-emitting diodes (LEDs), lamps, and arc lamps. In some cases, light irradiation may involve applying heat (e.g., using an external heater). In some cases, light irradiation can be used to stir the sample (e.g., a sample suspected of containing misfolded polypeptides) in the presence of metal nanoparticles. Seed amplification assays that use light-based amplification (e.g., stirring using light irradiation) are sometimes called photonic seed amplification assays, or Photo-QuIC assays.

[0037] The presence or absence of fibrils (e.g., fibrils formed from misfolded polypeptides) can be detected using any suitable method. In some cases, the presence or absence of fibrils can be detected using fluorescence. For example, a sample (e.g., a biological or environmental sample) can be exposed to nanoparticles (e.g., nanoparticles having a size of 2 μm or less (e.g., 1 μm or less), e.g., siNPs having a size of 2 μm or less (e.g., 1 μm or less)) and one or more phosphors during a seed amplification assay, and the presence of fibrils can be indicated by the presence of a fluorescent signal. Examples of such phosphors include, but are not limited to, thioflavin T (ThT), Congo red, stilbene, curcumin, thiophene, alkatrines, chrysamine G, PIB, BF-227, and borondipyrrometene (e.g., BODIPY®). In some cases, a sample containing nanoparticles (e.g., nanoparticles having a size of 2 μm or less (e.g., 1 μm or less), e.g., siNPs having a size of 2 μm or less (e.g., 1 μm or less)) and fibrils exposed to one or more phosphors (e.g., a biological sample or an environmental sample) may emit a fluorescence signal from about 400 nm to about 800 nm (e.g., from about 400 nm to about 700 nm, from about 400 nm to about 600 nm, from about 400 nm to about 500 nm, from about 500 nm to about 800 nm, from about 600 nm to about 800 nm, from about 700 nm to about 800 nm, or from about 500 nm to about 700 nm) during a seed amplification assay. In some cases, fibril-less samples (e.g., biological or environmental samples) exposed to nanoparticles (e.g., nanoparticles with a size of 2 μm or less (e.g., 1 μm or less), e.g., siNPs with a size of 2 μm or less (e.g., 1 μm or less)) and one or more phosphors during seed amplification assays may emit little to no fluorescence signal (e.g., from about 400 nm to about 800 nm).

[0038] In some cases, the methods provided herein are not antibody-based methods. For example, the methods provided herein can be carried out without antibody-based techniques.

[0039] In some cases, the methods provided herein can be carried out without any stimulation. For example, the methods provided herein can be carried out without electrochemical stimulation.

[0040] In some cases, the methods provided herein can be carried out without any sensors. For example, the methods provided herein can be carried out without any colorimetric sensors. For example, the methods provided herein can be carried out without any electrochemical sensors.

[0041] The presence or absence of any misfolded polypeptide can be detected using the methods described herein (e.g., methods for detecting the presence or absence of misfolded polypeptides). In some cases, misfolded polypeptides may be associated with disease. Examples of polypeptides that may be misfolded and can be detected as described herein include, but are not limited to, prion protein (PrP) polypeptides, tau polypeptides, amyloid-beta polypeptides, alpha-synuclein polypeptides, TDP-43 polypeptides, islet amyloid polypeptides (IAPP), superoxide dismutase 1 (SOD1) polypeptides, huntingtin polypeptides, fusion sarcoma (FUS) polypeptides, and translocation liposarcoma (TLS) polypeptides.

[0042] The presence or absence of misfolded polypeptides associated with any proteinopathy can be detected using the methods described herein (for example, methods for detecting the presence or absence of misfolded polypeptides). As used herein, proteinopathy is any disease associated with misfolded polypeptides, and possibly with the aggregation of one or more misfolded polypeptides. In some cases, proteinopathy may be infectious spongiform encephalopathy (TES). In some cases, proteinopathy may be protein misfold disease (PMD). In some cases, proteinopathy may be tauopathy. In some cases, proteinopathy may be alpha-synucleinopathy. Examples of proteinopathy-related polypeptides that may be misfolded and in which misfolded polypeptides may be detected as described herein include, but are not limited to, chronic wasting disease (CWD), Creutzfeldt-Jakob disease, infectious mink encephalopathy, scrapie, feline spongiform encephalopathy, ungulate spongiform encephalopathy, bovine spongiform encephalopathy, camel spongiform encephalopathy, Alzheimer's disease (AD), Parkinson's disease (PD), Pick's disease, Lewy body dementia (LBD), amyotrophic lateral sclerosis (ALS), multiple system atrophy, progressive supranuclear palsy, corticobasal degeneration, chronic traumatic encephalopathy, and type II diabetes mellitus.

[0043] The methods described herein (e.g., methods for detecting the presence or absence of misfolded polypeptides) can be used to detect the presence or absence of misfolded polypeptides in any suitable sample. In some cases, the sample may be a biological sample (e.g., a sample obtained from a mammal). In some cases, the sample may be an environmental sample. The sample may be a fresh sample or a fixed sample (e.g., a formaldehyde-fixed sample or a formalin-fixed sample). In some cases, the sample may be a pre-treated sample. For example, a pre-treated sample may be homogenized. For example, a pre-treated sample may be diluted (e.g., diluted with a buffer, e.g., phosphate-buffered saline (PBS)).

[0044] In some cases, the methods provided herein may include a step of isolating one or more biomolecules (e.g., polypeptides) from a sample (e.g., a biological or environmental sample). For example, polypeptides can be isolated from a sample and enriched or concentrated prior to amplification as described herein. In some cases, one or more biomolecules (e.g., polypeptides) isolated from a sample (e.g., a biological or environmental sample) may be exposed to nanoparticles (e.g., nanoparticles having a size of 2 μm or less (e.g., 1 μm or less), e.g., siNPs having a size of 2 μm or less (e.g., 1 μm or less)) during a seed amplification assay as described herein.

[0045] In some cases, the methods provided herein do not involve the step of isolating one or more biomolecules (e.g., polypeptides) from a sample (e.g., a biological or environmental sample). For example, a sample may contain one or more inhibitors (e.g., one or more inhibitors that may be naturally present in the sample). Examples of inhibitors that may be present in the samples described herein (e.g., biological or environmental samples) include, but are not limited to, mucin polypeptides, proteases, lipids (e.g., polar lipids), and hemoglobin. For example, the methods described herein (e.g., methods for detecting the presence or absence of misfolded polypeptides) can be used to detect the presence or absence of misfolded polypeptides in a sample containing one or more inhibitors (e.g., one or more inhibitors that may be naturally present in the sample). In some cases, the step of exposing a sample containing one or more inhibitors (e.g., one or more inhibitors that may be naturally present in the sample) to nanoparticles (e.g., nanoparticles having a size of 2 μm or less (e.g., 1 μm or less), e.g., siNPs having a size of 2 μm or less (e.g., 1 μm or less)) during a seed amplification assay may be effective in addressing the inhibitors present in the sample (e.g., in improving the sensitivity of the seed amplification assay).

[0046] In some cases, a sample (e.g., a biological or environmental sample) may contain less than approximately 20 pg (μg / mL) of misfolded polypeptides per mL of sample.

[0047] When detecting the presence or absence of misfolded polypeptides, polypeptide fibrils, and / or polypeptide aggregates (e.g., spherical polypeptide aggregates) in a biological sample using the methods described herein (e.g., methods for detecting the presence or absence of misfolded polypeptides), the biological sample can be obtained from any suitable mammal. In some cases, the sample can be obtained from a living mammal. In some cases, the sample can be obtained from a postmortem mammalian sample. For example, the postmortem sample may be mammalian tissue or by-products intended for consumption by another mammal (e.g., human), such as beef, mutton, lamb, or venison. In some cases, the mammal may be a member of the Cervidae family (e.g., a member of the family Cervidae). Examples of mammals in which a sample can be obtained and the presence or absence of misfolded polypeptides can be evaluated in the manner described herein (for example, by exposing the sample to nanoparticles (e.g., siNPs) having a size of 2 μm or less (e.g., 1 μm or less) during a seed amplification assay) include, but are not limited to, humans, non-human primates (e.g., monkeys), camels, minks, cats, dogs, cattle, sheep, mice, rats, hamsters, mammary deer, axis deer, elk, fallow deer, marsh deer, mule deer, jayak, moose, pampas deer, red deer, reindeer, roe deer, sambar deer, sika deer, white-tailed deer, antelope, and goats.

[0048] When detecting the presence or absence of misfolded polypeptides, polypeptide fibrils and / or polypeptide aggregates (e.g., spherical polypeptide aggregates) in a biological sample using the methods described herein (e.g., methods for detecting the presence or absence of misfolded polypeptides), the biological sample can be any type of biological sample. In some cases, the biological sample can be a secretion (e.g., somatic secretion). Examples of biological samples that can be evaluated for the presence or absence of misfolded polypeptides as described herein (e.g., by exposing the sample to nanoparticles (e.g., siNPs) having a size of 2 μm or less (e.g., 1 μm or less) during a seed amplification assay include, but are not limited to, lymphoid tissue, muscle tissue, tonsil tissue, skin tissue, brain tissue, brainstem tissue, blood (e.g., whole blood, serum and plasma), cerebrospinal fluid, urine, feces, saliva, mucus, liver tissue, heart tissue, intestinal tissue, semen, spleen tissue and eye tissue.

[0049] When detecting the presence or absence of misfolded polypeptides in an environmental sample using the methods described herein (e.g., methods for detecting the presence or absence of misfolded polypeptides), the environmental sample can be obtained from any suitable source. Examples of sources from which a sample can be obtained and which can be evaluated for the presence or absence of misfolded polypeptides as described herein (e.g., by exposing the sample to nanoparticles (e.g., siNPs) having a size of 2 μm or less (e.g., 1 μm or less) during a seed amplification assay include, but are not limited to, soil, water, dust, and plants.

[0050] When detecting the presence or absence of misfolded polypeptides in an environmental sample using the methods described herein (e.g., methods for detecting the presence or absence of misfolded polypeptides), the environmental sample can be obtained by any suitable method. Examples of methods that can be used to obtain an environmental sample that can be evaluated for the presence or absence of misfolded polypeptides as described herein (e.g., by exposing the sample to nanoparticles (e.g., siNPs) having a size of 2 μm or less (e.g., 1 μm or less) during a seed amplification assay) include, but are not limited to, cotton swabs and filters (e.g., air filtration system filters).

[0051] When detecting the presence or absence of misfolded polypeptides in an environmental sample using the methods described herein (e.g., methods for detecting the presence or absence of misfolded polypeptides), the environmental sample can be obtained from any environmental setting. Examples of environmental settings from which such an environmental sample can be obtained, which can be evaluated for the presence or absence of misfolded polypeptides as described herein (e.g., by exposing the sample to nanoparticles (e.g., siNPs) having a size of 2 μm or less (e.g., 1 μm or less) during a seed amplification assay), include, but are not limited to, natural habitats, waterways, farms, food processing facilities (e.g., meat processing facilities), medical facilities, animal feed processing facilities, water treatment facilities, and medical facilities (e.g., clinics, emergency rooms, emergency clinics, and hospitals, e.g., human hospitals and veterinary hospitals). When the environmental sample is obtained from a food processing facility, the food processing facility may be processing food for mammalian (e.g., human) consumption. For example, the environmental sample can be obtained from a food processing facility that processes agricultural products for mammalian consumption (e.g., alfalfa, corn, beets, soybeans, oats, pasture grass, potatoes, straw, and related by-products). When environmental samples are obtained from a medical facility, they can be obtained from any surface (e.g., a stainless steel surface) that frequently comes into contact with patients and / or bodily fluids (e.g., blood, urine, and feces). For example, environmental samples obtained from a medical facility may be obtained from surgical instruments, examination surfaces, and countertops.

[0052] In some cases, the presence or absence of misfolded polypeptides in a sample can be confirmed using one or more techniques conventionally used to detect the presence or absence of misfolded polypeptides in a sample. For example, enzyme-linked immunosorbent assay (ELISA), immunohistochemistry (IHC), and / or RT-QuIC testing can be used to confirm the presence or absence of misfolded polypeptides in a sample.

[0053] This document also provides methods and materials for treating mammals (e.g., humans) identified as having proteinopathy as described herein (e.g., at least in part on the presence of fibrils formed from misfolded polypeptides). For example, a mammal identified as having proteinopathy, at least in part on the presence of fibrils formed from misfolded polypeptides in a sample obtained from the mammal, can be subjected to one or more (e.g., one, two, three, four, five or more) therapies that can be used to treat one or more symptoms of proteinopathy. Examples of therapies that can be used to treat one or more symptoms of proteinopathy include, but are not limited to, physiotherapy, occupational therapy, speech therapy, electrical stimulation, and any combination thereof.

[0054] In some cases, non-human mammals can be selected for treatment using the methods and materials provided herein. For example, non-humans identified as having a proteinosis can be selected for isolation based at least partially on the presence of fibrils formed from misfolded polypeptides in a sample obtained from a mammal. In some cases, non-humans identified as having a proteinosis can be isolated from other mammals based at least partially on the presence of fibrils formed from misfolded polypeptides in a sample obtained from a mammal. For example, non-humans identified as having a proteinosis can be selected for euthanasia based at least partially on the presence of fibrils formed from misfolded polypeptides in a sample obtained from a mammal. In some cases, non-humans identified as having a proteinosis can be euthanized based at least partially on the presence of fibrils formed from misfolded polypeptides in a sample obtained from a mammal.

[0055] In some cases, facilities can be selected for treatment using the methods and materials provided herein. For example, a facility identified as having the presence of one or more misfolded polypeptides in a sample obtained from the facility (e.g., an environmental sample) can be selected for one or more treatments (e.g., one, two, three, four, five, or more) that can be used to disinfect the facility. In some cases, a facility identified as having the presence of one or more misfolded polypeptides in a sample obtained from the facility (e.g., an environmental sample) can be disinfected.

[0056] The present invention will be further described in the following embodiments, but these embodiments are not intended to limit the scope of the present invention as described in the claims.

[0057] [Examples] [Example 1] Nanoparticle-enhanced RT-QuIC diagnostic assay This embodiment describes the discovery that nanoparticles can improve the sensitivity of RT-QuIC. For example, when silica nanoparticles were added to an RT-QuIC experiment (defined as Nano-QuIC) for CWD diagnosis, the performance was significantly improved, resulting in a halving of the time to disease detection, mitigating the effects of inhibitory factors, and thus improving sensitivity by, for example, 10 times.

[0058] The impact of silica nanoparticles (siNPs) as reagents in the RT-QuIC reaction on the detection of CWD prions in the lymphoid tissue of wild white-tailed deer was investigated. All experiments were performed using recombinant hamster prP (rHaPrP) as the RT-QuIC reaction substrate. The performance of RT-QuIC was observed to be significantly improved in the presence of siNPs, depending on their size. Optimal RT-QuIC conditions, represented by the highest rate of amyloid formation (RAF, Figure 1B) and the lowest false-positive rate, were identified by investigating various combinations of NP diameter and concentration and reaction temperature. Addition of 50 nm siNPs was found to optimize RT-QuIC performance by significantly improving both RAF and ThT fluorescence (Figure 1B).

[0059] Using sigmoid-like curves, kinetic fluorescence data were fitted to extract key parameters representing the nucleation lag time and growth phase time constant. This information revealed that the majority of the rate improvement was likely due to the higher nucleation rate of MPs on the siNP surface compared to the nucleation rate of the bulk solution (Figure 1C). In addition, the accelerating effect of NPs was not limited to siNPs alone, but could also be induced by adding gold nanoparticles (AuNPs) to the RT-QuIC reaction. Finally, to test whether RT-QuIC using siNPs could overcome the effects of inhibitors, serial dilutions of CWD-positive lymphoid tissue showed that NP-enhanced RT-QuIC (Nano-QuIC) could easily detect the presence of CWD prions at concentrations that had not been achieved previously.

[0060] result Effects of silica NP (siNP) diameter and concentration To investigate the effect of nanoparticles on the performance of RT-QuIC, siNPs ranging from 20 nm to 100 nm were added as reagents to RT-QuIC reactions seeded with CWD-positive or CWD-negative tissue homogenates (parotid lymph nodes). The siNP concentrations were 0.1 mg / mL, 0.5 mg / mL, and 2.5 mg / mL (referred to as 1 / 25×, 1 / 5×, and 1×, respectively). The reaction was run at 42°C for 48 hours. All siNP diameters examined here affected the RAF when compared to the RAF of the reaction without siNPs (Figure 2A). The ratio of the RAFs between the reactions with and without siNPs gives a parameter known as the relative RAF. For all siNP diameters, the relative RAF increased as the siNP concentration increased (Figure 2A). This resulted in an average time to CWD detection of 50 nm siNPs at 42°C of 6.3 hours (95% confidence interval [CI]: ±0.96 hours), which is 4.9 hours faster than the conventional RT-QuIC reaction of the same sample (average time to detection of 11.2 hours (95% CI: ±1.00 hours)). Importantly, the PrP investigated in this specification... CWD The optimal siNP reaction parameter for diagnostic evaluation of positive tissue (50 nm siNP at 42°C) did not result in false positives.

[0061] The effect of temperature The temperature of a specific RT-QuIC experiment directly affects the sensitivity and specificity of the diagnosis. Generally, higher temperatures result in a higher RAF for a given true-positive diagnostic sample, however, such conditions increase the risk of spontaneous misfolding of substrate proteins. Lower temperatures yield lower RAF values, potentially leading to prolonged diagnostic time and false-negative results, but the false-positive rate decreases. To identify the optimal conditions for maximizing the sensitivity and specificity of Nano-QuIC, experiments were conducted at 37°C, 42°C, and 48°C for siNPs ranging from 20 nm to 100 nm at concentrations in the range of 0.1 to 2.5 mg / mL. Here again, relative RAF was determined by comparing the reactions with and without siNPs. In the case of the reaction at 37°C, all diameters of the siNPs resulted in a higher relative RAF for CWD-positive samples compared to the same samples tested with conventional RT-QuIC (Figures 2B, 5A, and 5B). However, compared to siNP experiments performed at higher temperatures, CWD-positive reactions performed at 37°C exhibited a slower RAF. In the Nano-QuIC experiments performed at 48°C, all diameters of siNPs also resulted in a higher relative RAF (Figures 2B, 5A, and 5B). In addition, experiments performed at 48°C had a shorter time to detection (higher raw RAF) compared to reactions at lower temperatures. Nano-QuIC performed using 50 nm siNPs at 2.5 mg / mL and a temperature of 48°C yielded the highest relative RAF and the fastest time to detection, but this was only a 2.5× improvement compared to the conventional RT-QuIC of 10.1 hours (95% CI: ±1.44 hours), at 4.1 hours (95% CI: ±0.45 hours) (Figure 2B). No repeated false positives were observed across our CWD-negative samples (see Discussion). Using these parameters, Nano-QuIC was performed on a blinded set of 10 CWD-positive and 10 CWD-negative repospharyngeal lymph node samples from wild white-tailed deer, and all tissues were classified with 100% sensitivity and specificity (Table 1).

[0062] [Table 1]

[0063] Aggregation rate and mechanism The formation of proteinaceous fibrils is a process by which misfolded polypeptides interact with each other to form large linear structures. Modeling of fibril formation in RT-QuIC is obtained using three phases: a nucleation (lag) phase, a growth (elongation) phase, and a stationary phase (Figure 1A). During the nucleation phase, native polypeptides misfold into nuclear units. Since ThT fluoresces when bound to fibrils but not when bound to individual monomers, the nucleation phase is characterized by zero or low fluorescence values. During the elongation phase, the nucleus functions as a template to efficiently misfold other native polypeptides, resulting in the eventual production of linear fibrils. In the presence of mechanical vibration or sonication, these fibrils can break, thus creating additional nuclei for native polypeptides to misfold and producing more fibrils. The elongation phase was characterized by the exponential growth of fibrils, as recorded by the exponential growth of ThT fluorescence (Figure 1A and Figure 2B). In the final stationary phase, ThT fluorescence stops increasing exponentially and stabilizes. This model explains both the propagation of infectious prions observed in transmissible spongiform encephalopathies (e.g., CWD or Creutzfeldt-Jakob disease) and the misfolding and spread of polypeptides associated with many neurodegenerative diseases (e.g., alpha-synuclein in Parkinson's disease, TDP-43 in ALS, and tau in Alzheimer's disease, etc.).

[0064] The three phases of protein amplification using RT-QuIC were modeled using a sigmoid-like curve.

[0065]

Number

[0066] To characterize the kinetics of how siNPs affect the RT-QuIC reaction (50 nm siNPs at 48°C), we developed a fitting using Equation 1 that gives a semi-quantitative value of the lag time and the time constant (τ) of fibril growth for concentrations of 1 / 25 × ~ 1 × (see supporting methods). We observed that the lag time decreased as the siNP concentration increased (Figure 2C), which suggests that siNPs may play a crucial role in the formation of the initial nuclei of fibrilization.

[0067] At pH 7.4, rHaPrP, used as the RT-QuIC substrate, had a net positive charge, while siNPs had a net negative charge, resulting in an attractive force. This attractive force likely promoted the absorption of rHaPrP onto the silica NP surface, thus increasing the local effective concentration of the RT-QuIC substrate. In addition, infectious prions can be absorbed onto glass and silica surfaces. The absorbed polypeptides were not fixed to a single location on a given siNP, but rather diffused across the surface and could interact with each other. Because the local concentration of polypeptides on the siNP surface was higher than in the bulk reaction space, the RT-QuIC substrate had more opportunities to interact with misfolded protein seeds, which directly affected the reaction kinetics by facilitating a more efficient nucleation phase. Furthermore, polypeptides absorbed onto the surface often undergo conformational changes. These conformational changes made rHaPrP more susceptible to misfolding in the presence of prion seeds, which could therefore increase the nucleation rate. The three characteristic phases of protein amplification (discussed above) were completely absent in the negative samples.

[0068] The fibril growth phase was compared between different RT-QuIC reaction conditions using the fibril growth time constant (τ). For 50 nm siNPs at 48°C, it was observed that the growth phase was characterized by a longer τ as the nanoparticle concentration increased (Figure 2D). In addition, the maximum ThT fluorescence of the NP-containing solution was greater than that of the NP-free solution (Figure 2B). This observation can be explained if, in the presence of mechanical shaking, siNPs cleaved the fibrils more efficiently into smaller units. More fibril nuclei can recruit more haPrP substrates, and this therefore contributes to the exponential growth phase. Such a mechanism can effectively explain the unique sigmoid curve observed in Nano-QuIC.

[0069] gold nanoparticles To demonstrate that the accelerating effect of NPs on RT-QuIC performance can be applied to other NP types, further experiments were conducted using AuNPs. AuNPs with 15 nm citrate caps were added as reagents to the RT-QuIC reaction to obtain final AuNP concentrations of 20.75 μg / mL, 62.5 μg / mL, or 187.5 μg / mL. CWD-positive or CWD-negative tissue homogenates were added to the solution, and the RT-QuIC reaction was run at 42°C for 48 hours. RAF was higher for all solutions containing AuNPs compared to RAF for reactions without AuNPs (Figure 3). The fastest mean time to detection was found to be 8.2 hours (95% CI: ±1.21 hours), compared to the mean detection time for solutions without AuNPs: 12.5 hours (95% CI: ±1.26 hours).

[0070] Addressing RT-QuIC inhibitors To compare the sensitivity of Nano-QuIC and RT-QuIC, a tissue dilution series experiment was conducted on 50 nm siNPs at 42°C. A 10-fold dilution of CWD-positive tissue seed was used. -1 from 10 -9The following was prepared. Next, subsamples of these dilutions (i.e., seeds) were added to the Nano-QuIC reaction and the conventional RT-QuIC reaction. Nano-QuIC and RT-QuIC produced 10 -9 and 10 -8 Seeding activity was detected in reactions involving diluted seeds, demonstrating the diagnostic sensitivity of these assays. However, with low-dilution seeds, Nano-QuIC performed significantly better than conventional RT-QuIC. PrP CWD Positive tissue 10 -1 At this dilution, conventional RT-QuIC did not exhibit seeding activity, while Nano-QuIC showed high RAF value, as reflected in PrP CWD The presence of was clearly detected (Figures 4A and 4B). Furthermore, PrP CWD Positive tissue 10 -2 At this dilution, Nano-QuIC provided twice the RAF compared to conventional RT-QuIC (Figure 4A).

[0071] Biological samples, particularly those derived from clinical settings, are extremely complex, which makes diagnosis difficult. Similar to inhibitors that negatively impact PCR performance, RT-QuIC is susceptible to inhibitors that interfere with detection and / or protein substrate misfolding. RT-QuIC inhibitors may include mucin family polypeptides and polar lipids, but a diverse range of other inhibitors may exist. Traditionally, RT-QuIC inhibitors have been addressed by diluting tissue samples and, in harmony with the reaction-limiting compound, until the desired diagnostic sensitivity functions for true-positive samples. However, the amount of MP present in a given biological sample is directly affected by heterogeneity of MPs both within and between individuals, as well as the stage of neurodegenerative disease (i.e., early vs. late stages). Therefore, practicing dilution of diagnostic samples to address inhibitors may contribute to false negatives (i.e., the diagnostic sample is true-positive but has very low levels of MP that are beyond the detection limit and subsequently diluted). These data suggest that Nano-QuIC has the potential to address false negatives related to sample dilution and / or inhibitors, as the siNP improvement assay exhibited 100% sensitivity in repeats, something that was not possible with conventional RT-QuIC (Figure 3). While not bound by theory, it is thought that the protein substrate (rHaPrP) can bind to the siNP surface, and therefore this increases the local rHaPrP concentration, enabling more efficient interaction with MPs and subsequent misfolding, thereby addressing inhibitors and ultimately improving diagnostic sensitivity.

[0072] In summary, these results demonstrate that incorporating siNP as a reagent into the RT-QuIC reaction can improve diagnostic performance.

[0073] method Tissue preparation CWD-positive and CWD-negative parotid lymph nodes from white-tailed deer (WTD) were selected as tissue samples for this study. The parotid lymph nodes were classified using the Bio-Rad TeSeE Short Assay Protocol (SAP) combo kit (BioRad Laboratories Inc., Hercules, CA, USA). The tissues were homogenized in PBS (10% w:v) in 2 mL tubes containing 1.5 mm zirconium beads using a BeadBug homogenizer (Benchmark Scientific, Sayreville, New Jersey, USA) at maximum speed for 90 seconds. The samples were divided into fixed volumes and frozen. These samples were referred to as 10% homogenates. In this study, the same homogenate was used for all experiments except for the blinded sample set. All CWD-positive and negative samples were selected based on independent ELISA, IHC, and / or RT-QuIC results and subsampled as described by Schwabenlander et al. (Schwabenlander et al., J. Wildl. Dis., 58(1):50-62 (2021)).

[0074] Preparation of recombinant substrates. Recombinant PrP (rPrP) was generated and purified as described in other literature (Schwabenlander et al., J. Wildl. Dis., 58(1):50-62 (2021)). The substrate was derived from a cleaved form (amino acids 90-231) of the Syrian hamster PRNP gene cloned into pD431-SR (ATUM, Newark, CA, USA) and expressed in Rosetta(DE3) Escherichia coli (Sigma-Aldrich, St. Louis, MO, USA). Recombinant hamster PrP was used in this study due to its apparent universal tendency to misfold in the presence of infectious prions across a wide range of species.

[0075] QuIC for organizations. For Nano-QuIC analysis, the master mix was prepared according to the following specifications: 1×PBS, 1 mM ethylenediaminetetraacetic acid (EDTA), 170 mM NaCl, 10 μM thioflavin T (ThT), 0.1 mg / mL recombinant hamster PrP substrate (rPrP), and siNP (nanoComposix a Fortis Life Sciences Company, San Diego, CA, USA) at final well concentrations of 2.5 mg / mL, 0.5 mg / mL, 0.1 mg / mL, or without NP. For the AuNP reaction, AuNP (Nanopartz, Loveland, CO, USA) with a 15 nm citrate cap was added to the master mix solution at final well concentrations of 20.8 μg / mL, 62.5 μg / mL, and 187.5 μg / mL. For the RT-QuIC reaction, the master mix was the same except for the absence of NP. For temperature and concentration experiments, 10% tissue homogenate (prepared as described above) was further diluted 100-fold in dilution buffer (0.1% sodium dodecyl sulfate (SDS), 1×PBS, 1×N-2 supplement (number of μL of N2 per 100 μL SDS / PBS) (Thermofisher Scientific, Waltham, MA, USA)). For dilution experiments (Figures 4A and 4B), 10-fold serial dilutions were prepared from the 10% homogenate (for example, 10 μL of 10% homogenate dissolved in 90 μL of dilution buffer was 10-fold). -1 (Dilution, etc.). 2 μL of diluent was added to each well containing 98 μL of master mix. For temperature and concentration experiments, four cycles were used for each condition. For dilution experiments, eight cycles were used. Nano-QuIC plates were amplified for 48 hours using a FLUOstar® Omega plate reader (BMG Labtech, Cary, North Carolina, USA, various temperatures, 700 rpm, double orbital, 57 seconds shaking, 83 seconds rest). Fluorescence readings were taken at 45-minute intervals. Following the method of Rowden et al. (Pathogens, 12(2):309 (2023)), the reciprocal of the number of seconds required for the curve to exceed a threshold of twice the background fluorescence signal in cycle 4 of the reaction (s) was used. -1) was decided upon as RAF.

[0076] Blinded sample set Similar to the parotid lymph node samples described above, 10 CWD-negative and 10 CWD-positive samples were prepared from the retropharyngeal lymph nodes. Samples were blinded and run at 48°C using both RT-QuIC and Nano-QuIC. The siNP used was 2.5 mg / mL at 50 nm. Using the maxpoint method (Rowden et al., Pathogens, 12(2):309 (2023)), samples were classified as positive if they were positive in two or more of four repeats.

[0077] Evaluation of kinetic data The ThT kinetic curve was modeled as having nucleation, growth, and stationary phases. The maximum change in fluorescence between time points was found. To better fit the target region (nucleation and growth phases), data from the start of the nucleation phase to 10 points after the maximum change in fluorescence was used. This captured the start regions of the growth and stationary phases. The fitting for Eq 1 was created using Origin version 9.9.5. Average R 2 The values ​​and their corresponding standard deviations can be seen in Table 2.

[0078] [Table 2]

[0079] [Example 2] Nanoparticle-enhanced RT-QuIC diagnostic assay method: For the artificial spike in the human plasma experiment (Figure 8A), EDTA plasma samples were obtained. The master mix was prepared to the following specifications: 1× phosphate buffer (9.2 mM NaH2PO4, 2.8 mM Na2HPO4, 2.7 mM KCl, and 137 mM NaCl), 1 mM ethylenediaminetetraacetic acid (EDTA), 170 mM NaCl (in addition to the NaCl in the phosphate buffer), 10 μM thioflavin T (ThT), and recombinant human alpha-synuclein 0.09 mg / mL. The pH was approximately 6.3. For the Nano-QuIC reaction, 50 nm silica nanoparticles were used at a concentration of 2.5 mg / mL. 10 μL of spontaneously misfolded α-synuclein (0.09 mg / mL) was sonicated (200W, 15 seconds on, 15 seconds off, 3 times) and spiked into 90 μL of human plasma, but this solution was 10 0 This is called the diluent (Figure 8A). Next, this solution is added to human plasma to reach a final spike concentration of 90 pg / mL (10 -5 The solution was then sequentially diluted 10-fold until it reached the final dilution (called the final dilution). Each spike dilution was then diluted 100-fold with SDS / PBS (0.1% sodium dodecyl sulfate (SDS), 1×PBS). 2 μL of this final dilution was then added to 98 μL of the master mix in a 96-well plate. The plate was placed on a plate reader (BMG Labtech, Cary, North Carolina, USA). The mixture was incubated at 700 rpm, double orbital, 42°C for at least 48 hours (shaking for 60 seconds, then resting for 60 seconds).

[0080] For the human plasma sample test (Figure 8B), plasma samples were obtained from subjects diagnosed with and undiagnosed with Parkinson's disease. 200 µl of human plasma was centrifuged at 21,000 g for 40 minutes. 185 µl of supernatant was removed, and the remaining liquid / pellet was resuspended in 20 µl of 0.1% SDS in 1 × PBS. 2 µl of this mixture was added to a master mix containing 98 µl of silica nanoparticles (see above) on a 96-well plate, and 1 µl of 0.1% SDS was added. The samples were placed on a BMG plate reader at 700 rpm, 42C, with 60-second on / off intervals for 260 hours.

[0081] In summary, these results demonstrate that Nano-QuIC can overcome the barriers of inhibitory factors commonly associated with blood / plasma samples in conventional RT-QuIC assays. Whole blood samples are extremely complex and difficult to diagnose. For example, in CSF samples, blood contamination is considered to significantly hinder QuIC diagnosis. Even in the presence of robust inhibitory factors present in human plasma, Nano-QuIC performed better than RT-QuIC, detecting misfolded α-synuclein at a level of 900 pg / mL, which is 1 / 100th lower than RT-QuIC, without repeated false positives (Figure 8A).

[0082] In addition, pooled human plasma from patients diagnosed with Parkinson's disease versus those not diagnosed was examined. Parkinson's samples were observed to show higher seeding activity (Figure 8B). It took approximately 160 hours to observe the seeding activity of this reaction, which was longer than that required for the spike plasma experiment in Figure 8A; however, the activity was still present. These data support the use of Nano-QuIC for human disease diagnosis. The longer seeding time may be due to extremely low levels of misfolded α-synuclein in the plasma (likely close to pg), coupled with high concentrations of inhibitors.

[0083] Other Embodiments While the present invention has been described along with embodiments for carrying it out, it should be understood that the foregoing description is intended to illustrate, and not limit, the scope of the present invention as defined by the appended claims. Other embodiments, advantages, and modifications are within the scope of the following claims.

Claims

1. A method for detecting the presence or absence of misfolded polypeptides in a sample, (a) A step of exposing the sample to nanoparticles having a size of 1 μm or less during a seed amplification assay, wherein if misfolded polypeptides are present in the sample, fibrils are formed. (b) A step of detecting the fibril if it has been formed in step (a), (c) If the fibril is detected, the step of identifying the sample as having the presence of the misfolded polypeptide, and (d) If no fibrils are detected, the step of identifying the sample as not having misfolded polypeptides. Methods that include...

2. The method according to claim 1, wherein the sample is a biological sample.

3. The method according to claim 2, wherein the biological sample is obtained from a living mammal.

4. The method according to claim 3, wherein the living mammal is selected from the group consisting of humans, monkeys, camels, horses, minks, cats, dogs, cows, sheep, mice, rats, hamsters, mammal deer, axis deer, elk, fallow deer, American marsh deer, mule deer, munjak, moose, pampas deer, red deer, reindeer, roe deer, samba deer, sika deer, white-tailed deer, antelope, and goats.

5. The method according to claim 3 or claim 4, wherein the biological sample is selected from the group consisting of lymphoid tissue, muscle tissue, tonsil tissue, skin tissue, brain tissue, brainstem tissue, blood, cerebrospinal fluid, urine, feces, saliva, mucus, liver tissue, heart tissue, intestinal tissue, semen, spleen tissue, and eye tissue.

6. The method according to claim 2, wherein the biological sample is obtained from a postmortem mammal.

7. The method according to claim 6, wherein the biological sample is beef, lamb, mutton, or venison.

8. The method according to claim 1, wherein the sample is an environmental sample.

9. The method according to claim 8, wherein the environmental sample is selected from soil, water, dust, and plants.

10. The method according to claim 8 or 9, wherein the environmental sample is obtained using a cotton swab or a filter.

11. The method according to any one of claims 8 to 10, wherein the environmental sample is obtained from a location selected from the group consisting of natural habitats, waterways, farms, food processing facilities, water treatment facilities, and medical facilities.

12. The method according to claim 11, wherein the environmental sample is obtained from the food processing facility, and the food processing facility processes food for consumption by mammals.

13. The method according to claim 11, wherein the environmental sample is obtained from the medical facility.

14. The method according to any one of claims 1 to 13, comprising the step of isolating a polypeptide from the sample prior to step (a).

15. The method according to any one of claims 1 to 14, wherein the seed amplification assay includes shaking the sample, ultrasonically treating the sample, exposing the sample to sound waves, or irradiating the sample with light.

16. The method according to any one of claims 1 to 15, wherein the misfolded polypeptide is selected from the group consisting of prion protein (PrP) polypeptide, tau polypeptide, amyloid-beta polypeptide, α-synuclein polypeptide, TDP-43 polypeptide, islet amyloid polypeptide (IAPP), superoxide dismutase 1 (SOD1) polypeptide, huntingtin polypeptide, fusion sarcoma (FUS) polypeptide, and translocation liposarcoma (TLS) polypeptide.

17. The method according to any one of claims 1 to 16, wherein the misfolded polypeptide is associated with proteinosis.

18. The method according to claim 17, wherein the protein disorder is selected from the group consisting of chronic wasting disease (CWD), Creutzfeldt-Jakob disease, infectious mink encephalopathy, scrapie, feline spongiform encephalopathy, ungulate spongiform encephalopathy, bovine spongiform encephalopathy, camel spongiform encephalopathy, intermediate pituitary dysfunction (PPID), Alzheimer's disease (AD), Parkinson's disease (PD), Pick's disease, Lewy body dementia (LBD), amyotrophic lateral sclerosis (ALS), multiple system atrophy, progressive supranuclear palsy, corticobasal degeneration, and chronic traumatic encephalopathy.

19. The method according to any one of claims 1 to 18, wherein the seed amplification assay is completed in less than approximately 96 hours.

20. The method according to any one of claims 1 to 19, wherein the nanoparticles are selected from the group consisting of silica nanoparticles, gold nanoparticles, silver nanoparticles, platinum nanoparticles, iron nanoparticles, copper nanoparticles, dielectric nanoparticles having a metal shell, titanium dioxide nanoparticles, tin dioxide nanoparticles, cerium oxide nanoparticles, zirconium dioxide nanoparticles, iron oxide nanoparticles, cadmium telluride quantum dots, and lead sulfide quantum dots.

21. A method for detecting the presence or absence of misfolded polypeptides in a sample, (a) A step of exposing the sample to nanoparticles having a size of 1 μm or less during a seed amplification assay, wherein if misfolded polypeptides are present in the sample, spherical polypeptide aggregates are formed. (b) A step of detecting the spherical polypeptide aggregates if they are formed in step (a), (c) If the spherical polypeptide aggregates are detected, the step of identifying the sample as having the presence of the misfolded polypeptides, and (d) If no spherical polypeptide aggregates are detected, the step of identifying the sample as not containing misfolded polypeptides. Methods that include...

22. The method according to claim 21, wherein the sample is a biological sample.

23. The method according to claim 22, wherein the biological sample is obtained from a living mammal.

24. The method according to claim 23, wherein the living mammal is selected from the group consisting of humans, monkeys, camels, horses, minks, cats, dogs, cows, sheep, mice, rats, hamsters, mammary deer, axis deer, elk, fallow deer, American marsh deer, mule deer, jayak, moose, pampas deer, red deer, reindeer, roe deer, samba deer, sika deer, white-tailed deer, antelope, and goats.

25. The method according to claim 23 or claim 24, wherein the biological sample is selected from the group consisting of lymphoid tissue, muscle tissue, tonsil tissue, skin tissue, brain tissue, brainstem tissue, blood, cerebrospinal fluid, urine, feces, saliva, mucus, liver tissue, heart tissue, intestinal tissue, semen, spleen tissue, and eye tissue.

26. The method according to claim 22, wherein the biological sample is obtained from a postmortem mammal.

27. The method according to claim 26, wherein the biological sample is beef, lamb, mutton, or venison.

28. The method according to claim 21, wherein the sample is an environmental sample.

29. The method according to claim 28, wherein the environmental sample is selected from soil, water, dust, and plants.

30. The method according to claim 28 or claim 29, wherein the environmental sample is obtained using a cotton swab or a filter.

31. The method according to any one of claims 28 to 30, wherein the environmental sample is obtained from a location selected from the group consisting of natural habitats, waterways, farms, food processing facilities, water treatment facilities, and medical facilities.

32. The method according to claim 31, wherein the environmental sample is obtained from the food processing facility, and the food processing facility processes food for consumption by mammals.

33. The method according to claim 31, wherein the environmental sample is obtained from the medical facility.

34. The method according to any one of claims 21 to 33, comprising the step of isolating a polypeptide from the sample prior to step (a).

35. The method according to any one of claims 21 to 33, wherein the seed amplification assay includes shaking the sample, ultrasonically treating the sample, exposing the sample to sound waves, or irradiating the sample with light.

36. The method according to any one of claims 21 to 35, wherein the misfolded polypeptide is selected from the group consisting of PrP polypeptide, tau polypeptide, amyloid-beta polypeptide, α-synuclein polypeptide, TDP-43 polypeptide, IAPP, SOD1 polypeptide, huntingtin polypeptide, FUS polypeptide, and TLS polypeptide.

37. The method according to any one of claims 21 to 36, wherein the misfolded polypeptide is associated with proteinosis.

38. The method according to claim 37, wherein the protein disorder is selected from the group consisting of CWD, Creutzfeldt-Jakob disease, infectious mink encephalopathy, scrapie, feline spongiform encephalopathy, ungulate spongiform encephalopathy, bovine spongiform encephalopathy, camel spongiform encephalopathy, PPID, AD, PD, Pick's disease, LBD, ALS, multiple system atrophy, progressive supranuclear palsy, corticobasal degeneration, and chronic traumatic encephalopathy.

39. The method according to any one of claims 21 to 38, wherein the seed amplification assay is completed in less than approximately 96 hours.

40. The method according to any one of claims 21 to 39, wherein the nanoparticles are selected from the group consisting of silica nanoparticles, gold nanoparticles, silver nanoparticles, platinum nanoparticles, iron nanoparticles, copper nanoparticles, dielectric nanoparticles having a metal shell, titanium dioxide nanoparticles, tin dioxide nanoparticles, cerium oxide nanoparticles, zirconium dioxide nanoparticles, iron oxide nanoparticles, cadmium telluride quantum dots, and lead sulfide quantum dots.

41. A composition comprising nanoparticles having a size of 1 μm or less for use in accelerating seed amplification assays.

42. The composition according to claim 41, wherein the seed amplification assay includes shaking the sample, ultrasonically treating the sample, exposing the sample to sound waves, or irradiating the sample with light.

43. The composition according to claim 41 or claim 42, wherein the nanoparticles are selected from the group consisting of silica nanoparticles, gold nanoparticles, silver nanoparticles, platinum nanoparticles, iron nanoparticles, copper nanoparticles, dielectric nanoparticles having a metal shell, titanium dioxide nanoparticles, tin dioxide nanoparticles, cerium oxide nanoparticles, zirconium dioxide nanoparticles, iron oxide nanoparticles, cadmium telluride quantum dots, and lead sulfide quantum dots.

44. The composition according to any one of claims 41 to 43, wherein the seed amplification assay is completed in less than approximately 96 hours.

45. A method for detecting the presence or absence of misfolded polypeptides in a sample, (a) A step of exposing the sample to a nanoporous material having a pore size from 2 nm to about 200 nm during a seed amplification assay, wherein if misfolded polypeptides are present in the sample, fibrils are formed. (b) A step of detecting the fibril if it has been formed in step (a), (c) If the fibril is detected, the step of identifying the sample as having the presence of the misfolded polypeptide, and (d) If no fibrils are detected, the step of identifying the sample as not having misfolded polypeptides. Methods that include...

46. The method according to claim 45, wherein the sample is a biological sample.

47. The method according to claim 46, wherein the biological sample is obtained from a living mammal.

48. The method according to claim 47, wherein the living mammal is selected from the group consisting of humans, monkeys, camels, horses, minks, cats, dogs, cows, sheep, mice, rats, hamsters, mammal deer, axis deer, elk, fallow deer, American marsh deer, mule deer, jayak, moose, pampas deer, red deer, reindeer, roe deer, sambar deer, sika deer, white-tailed deer, antelope, and goats.

49. The method according to claim 47 or claim 48, wherein the biological sample is selected from the group consisting of lymphoid tissue, muscle tissue, tonsil tissue, skin tissue, brain tissue, brainstem tissue, blood, cerebrospinal fluid, urine, feces, saliva, mucus, liver tissue, heart tissue, intestinal tissue, semen, spleen tissue, and eye tissue.

50. The method according to claim 46, wherein the biological sample is obtained from a postmortem mammal.

51. The method according to claim 50, wherein the biological sample is beef, mutton, lamb, or venison.

52. The method according to claim 45, wherein the sample is an environmental sample.

53. The method according to claim 52, wherein the environmental sample is selected from soil, water, dust, and plants.

54. The method according to claim 52 or claim 53, wherein the environmental sample is obtained using a cotton swab or a filter.

55. The method according to any one of claims 52 to 54, wherein the environmental sample is obtained from a location selected from the group consisting of natural habitats, waterways, farms, food processing facilities, water treatment facilities, and medical facilities.

56. The method according to claim 55, wherein the environmental sample is obtained from the food processing facility, and the food processing facility processes food for consumption by mammals.

57. The method according to claim 56, wherein the environmental sample is obtained from the medical facility.

58. The method according to any one of claims 45 to 57, comprising the step of isolating a polypeptide from the sample prior to step (a).

59. The method according to any one of claims 45 to 58, wherein the seed amplification assay includes shaking the sample, ultrasonically treating the sample, exposing the sample to sound waves, or irradiating the sample with light.

60. The method according to any one of claims 45 to 59, wherein the misfolded polypeptide is selected from the group consisting of PrP polypeptide, tau polypeptide, amyloid-beta polypeptide, α-synuclein polypeptide, TDP-43 polypeptide, IAPP, SOD1 polypeptide, huntingtin polypeptide, FUS polypeptide, and TLS polypeptide.

61. The method according to any one of claims 45 to 60, wherein the misfolded polypeptide is associated with proteinosis.

62. The method according to claim 61, wherein the protein disorder is selected from the group consisting of CWD, Creutzfeldt-Jakob disease, infectious mink encephalopathy, scrapie, feline spongiform encephalopathy, ungulate spongiform encephalopathy, bovine spongiform encephalopathy, camel spongiform encephalopathy, PPID, AD, PD, Pick's disease, LBD, ALS, multiple system atrophy, progressive supranuclear palsy, corticobasal degeneration, and chronic traumatic encephalopathy.

63. The method according to any one of claims 45 to 62, wherein the seed amplification assay is completed in less than approximately 96 hours.

64. The method according to any one of claims 45 to 63, wherein the nanoporous material is selected from the group consisting of mesoporous silica, mesoporous polytetrafluoroethylene, zeolite particles, and metal-organic frameworks (MOFs).

65. A method for detecting the presence or absence of misfolded polypeptides in a sample, (a) A step of exposing the sample to a nanoporous material having a pore size from 2 nm to about 200 nm during a seed amplification assay, wherein if misfolded polypeptides are present in the sample, spherical polypeptide aggregates are formed. (b) A step of detecting the spherical polypeptide aggregates if they are formed in step (a), (c) If the spherical polypeptide aggregates are detected, the step of identifying the sample as having the presence of the misfolded polypeptides, and (d) If no spherical polypeptide aggregates are detected, the step of identifying the sample as not containing misfolded polypeptides. Methods that include...

66. The method according to claim 65, wherein the sample is a biological sample.

67. The method according to claim 66, wherein the biological sample is obtained from a living mammal.

68. The method according to claim 67, wherein the living mammal is selected from the group consisting of humans, monkeys, camels, horses, minks, cats, dogs, cows, sheep, mice, rats, hamsters, mammal deer, axis deer, elk, fallow deer, American marsh deer, mule deer, jayak, moose, pampas deer, red deer, reindeer, roe deer, sambar deer, sika deer, white-tailed deer, antelope, and goats.

69. The method according to claim 67 or claim 68, wherein the biological sample is selected from the group consisting of lymphoid tissue, muscle tissue, tonsil tissue, skin tissue, brain tissue, brainstem tissue, blood, cerebrospinal fluid, urine, feces, saliva, mucus, liver tissue, heart tissue, intestinal tissue, semen, spleen tissue, and eye tissue.

70. The method according to claim 66, wherein the biological sample is obtained from a postmortem mammal.

71. The method according to claim 70, wherein the biological sample is beef, lamb, mutton, or venison.

72. The method according to claim 65, wherein the sample is an environmental sample.

73. The method according to claim 72, wherein the environmental sample is selected from soil, water, dust, and plants.

74. The method according to claim 72 or claim 73, wherein the environmental sample is obtained using a cotton swab or a filter.

75. The method according to any one of claims 72 to 74, wherein the environmental sample is obtained from a location selected from the group consisting of natural habitats, waterways, farms, food processing facilities, water treatment facilities, and medical facilities.

76. The method according to claim 75, wherein the environmental sample is obtained from the food processing facility, and the food processing facility processes food for consumption by mammals.

77. The method according to claim 75, wherein the environmental sample is obtained from the medical facility.

78. The method according to any one of claims 65 to 77, comprising the step of isolating a polypeptide from the sample prior to step (a).

79. The method according to any one of claims 65 to 77, wherein the seed amplification assay includes shaking the sample, ultrasonically treating the sample, exposing the sample to sound waves, or irradiating the sample with light.

80. The method according to any one of claims 65 to 79, wherein the misfolded polypeptide is selected from the group consisting of PrP polypeptide, tau polypeptide, amyloid-beta polypeptide, α-synuclein polypeptide, TDP-43 polypeptide, IAPP, SOD1 polypeptide, huntingtin polypeptide, FUS polypeptide, and TLS polypeptide.

81. The method according to any one of claims 65 to 80, wherein the misfolded polypeptide is associated with proteinosis.

82. The method according to claim 81, wherein the protein disorder is selected from the group consisting of CWD, Creutzfeldt-Jakob disease, infectious mink encephalopathy, scrapie, feline spongiform encephalopathy, ungulate spongiform encephalopathy, bovine spongiform encephalopathy, camel spongiform encephalopathy, PPID, AD, PD, Pick's disease, LBD, ALS, multiple system atrophy, progressive supranuclear palsy, corticobasal degeneration, and chronic traumatic encephalopathy.

83. The method according to any one of claims 65 to 82, wherein the seed amplification assay is completed in less than approximately 96 hours.

84. The method according to any one of claims 65 to 83, wherein the nanoporous material is selected from the group consisting of mesoporous silica, mesoporous polytetrafluoroethylene, zeolite particles, and MOF.

85. A composition comprising a nanoporous material having pore sizes ranging from 2 nm to approximately 100 nm, for use in accelerating seed amplification assays.

86. The composition according to claim 85, wherein the seed amplification assay includes shaking the sample, ultrasonically treating the sample, exposing the sample to sound waves, or irradiating the sample with light.

87. The composition according to claim 85 or claim 86, wherein the nanoporous material is selected from the group consisting of mesoporous silica, mesoporous polytetrafluoroethylene, zeolite particles, and MOF.

88. The composition according to any one of claims 85 to 87, wherein the seed amplification assay is completed in less than approximately 96 hours.

89. A method for detecting the presence or absence of misfolded polypeptides in a sample, (a) A step of exposing the sample to nanoparticles having a size of 1 μm or less during a seed amplification assay, wherein the seed amplification assay includes exposing the sample to sound waves, wherein misfolded polypeptides are present in the sample and form fibrils. (b) A step of detecting the fibril if it has been formed in step (a), (c) If the fibril is detected, the step of identifying the sample as having the presence of the misfolded polypeptide, and (d) If no fibrils are detected, the step of identifying the sample as not having misfolded polypeptides. Methods that include...

90. A method for detecting the presence or absence of misfolded polypeptides in a sample, (a) A step of exposing the sample to nanoparticles having a size of 1 μm or less during a seed amplification assay, wherein the seed amplification assay includes exposing the sample to sound waves, wherein if misfolded polypeptides are present in the sample, they form spherical polypeptide aggregates. (b) A step of detecting the spherical polypeptide aggregates if they are formed in step (a), (c) If the spherical polypeptide aggregates are detected, the step of identifying the sample as having the presence of the misfolded polypeptides, and (d) If no spherical polypeptide aggregates are detected, the step of identifying the sample as not containing misfolded polypeptides. Methods that include...

91. A method for detecting the presence or absence of misfolded polypeptides in a sample, (a) A step of exposing the sample to metal nanoparticles having a size of 1 μm or less during a seed amplification assay, wherein the seed amplification assay includes exposing the sample to light irradiation, wherein misfolded polypeptides are present in the sample and form fibrils. (b) A step of detecting the fibril if it has been formed in step (a), (c) If the fibril is detected, the step of identifying the sample as having the presence of the misfolded polypeptide, and (d) If no fibrils are detected, the step of identifying the sample as not having misfolded polypeptides. Methods that include...

92. A method for detecting the presence or absence of misfolded polypeptides in a sample, (a) A step of exposing the sample to metal nanoparticles having a size of 1 μm or less during a seed amplification assay, wherein the seed amplification assay includes exposing the sample to light irradiation, wherein if misfolded polypeptides are present in the sample, they form spherical polypeptide aggregates. (b) A step of detecting the spherical polypeptide aggregates if they are formed in step (a), (c) If the spherical polypeptide aggregates are detected, the step of identifying the sample as having the presence of the misfolded polypeptides, and (d) If no spherical polypeptide aggregates are detected, the step of identifying the sample as not containing misfolded polypeptides. Methods that include...

Images