Patent Application
20240192229
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 sample or an environmental sample) can be amplified as described herein such that misfolded polypeptides present in the sample can aggregate to form fibrils and/or globular polypeptide aggregates, and the amplified sample can be contacted with a solution containing nanoparticles (e.g., metal nanoparticles such as gold nanoparticles) as described herein to detect the presence or absence of fibrils and/or globular polypeptide aggregates. In another example, a sample (e.g., a biological sample or an environmental sample) can be amplified as described herein such that misfolded polypeptides present in the sample can aggregate to form fibrils and/or globular polypeptide aggregates, and the amplified sample can be contacted with a solution containing one or more organic dyes (e.g., Congo Red) as described herein to detect the presence or absence of fibrils and/or globular polypeptide aggregates. In some cases, methods and materials provided herein can be used to determine if a mammal (e.g., a human) has a proteinopathy based, at least in part, in the presence or absence of misfolded polypeptides in a sample obtained from the mammal.
Prion diseases, collectively identified as transmissible spongiform encephalopathies (TSEs), are caused by an infectious misfolded prion proteins that spread throughout an infected animal, ultimately leading to advanced neurodegeneration and death (Prusiner et al., Proc. Nat. Acad. Sci. USA, 95:13363-13383 (1998); and Williams et al., J. Wildlife Dis., 16:89-98 (1980)). TSEs are known to infect a wide variety of mammals including cattle, sheep, camels, mink, cats, cervids, and humans (Collinge, Ann. Rev. Neurosci., 24:519-550 (2001)). Similarly, protein-misfolding diseases of humans, such as sporadic Alzheimer's Disease (AD), Parkinson's Disease (PD), Pick's disease, Lewy body dementia (LBD), and amyotrophic lateral sclerosis, are thought to originate from the misfolding and pathogenic accumulation of proteins (e.g., amyloid plaques and tau tangles) within the central nervous system (Lin et al., Nature, 443:787-795 (2006)).
This document provides methods and materials for detecting the presence or absence of misfolded polypeptides in a sample. For example, a sample (e.g., a biological sample or an environmental sample) can be amplified such that misfolded polypeptides present in the sample can aggregate to form fibrils and/or globular polypeptide aggregates, and the amplified sample can be contacted with a solution containing nanoparticles (e.g., metal nanoparticles such as gold nanoparticles) to detect the presence or absence of fibrils and/or globular polypeptide aggregates. In another example, a sample (e.g., a biological sample or an environmental sample) can be amplified such that misfolded polypeptides present in the sample can aggregate to form fibrils and/or globular polypeptide aggregates, and the amplified sample can be contacted with a solution containing one or more organic dyes (e.g., Congo Red) to detect the presence or absence of fibrils and/or globular polypeptide aggregates. In some cases, methods and materials provided herein can be used to determine if a mammal (e.g., a human) has a proteinopathy based, at least in part, in the presence or absence of misfolded polypeptides in a sample obtained from the mammal. For example, a sample obtained from a mammal can be amplified such that misfolded polypeptides present in the sample can aggregate to form fibrils, the amplified sample can be contacted with a solution containing nanoparticles (e.g., metal nanoparticles such as gold nanoparticles) to detect the presence or absence of fibrils, and the mammal can be classified as having a proteinopathy if the presence of fibrils is detected. For example, a sample obtained from a mammal can be amplified such that misfolded polypeptides present in the sample can aggregate to form globular polypeptide aggregates, the amplified sample can be contacted with a solution containing nanoparticles (e.g., metal nanoparticles such as gold nanoparticles) to detect the presence or absence of globular polypeptide aggregates, and the mammal can be classified as having a proteinopathy if the presence of globular polypeptide aggregates is detected. In another example, a sample obtained from a mammal can be amplified such that misfolded polypeptides present in the sample can aggregate to form fibrils, the amplified sample can be contacted with a solution containing one or more organic dyes (e.g., Congo Red) to detect the presence or absence of fibrils, and the mammal can be classified as having a proteinopathy if the presence of fibrils is detected. For example, a sample obtained from a mammal can be amplified such that misfolded polypeptides present in the sample can aggregate to form globular polypeptide aggregates, the amplified sample can be contacted with a solution containing one or more organic dyes (e.g., Congo Red) to detect the presence or absence of globular polypeptide aggregates, and the mammal can be classified as having a proteinopathy if the presence of globular polypeptide aggregates is detected.
As demonstrated herein, amplifying a sample suspected of containing misfolded polypeptides, and contacting the amplified sample with a solution of gold nanoparticles or Congo Red can be used to detect the presence or absence of the misfolded polypeptides. For example, samples containing prions from deer having chronic wasting disease (CWD) were amplified such that the prions within the samples formed fibrils, the amplified samples were contacted with a solution of gold nanoparticles, and the presence of the amplified prions was visually detected by the color of the solution and was detected by the peak light absorbance of the solution. In another example, samples containing misfolded α-synuclein polypeptides were amplified such that the misfolded α-synuclein polypeptides within the samples formed fibrils, the amplified samples were contacted with a solution of gold nanoparticles or Congo Red, and the presence of the amplified α-synuclein polypeptides was visually detected by the color of the solution and was detected by the peak light absorbance of the solution.
Having the ability to detect the presence of misfolded polypeptides in a sample as described herein (e.g., by amplifying the sample and contacting the amplified sample with a solution containing metal nanoparticles or one or more organic dyes such as Congo Red) provides a unique method to quickly and effectively detect the presence of misfolded polypeptides. For example, the methods described herein provide a protein-based diagnostic method that is relatively easy to use, requires inexpensive reagents and equipment, is highly sensitive and specific to the targeted protein, and can be performed in diverse settings (e.g., in field settings, small laboratories, etc.). In some cases, the methods described herein can be used to quickly and easily identify a mammal as having a proteinopathy.
In general, one aspect of this document features methods for detecting the presence or absence of misfolded polypeptides in a sample. The methods can include, or consist essentially of, (a) amplifying a sample under conditions where the misfolded polypeptides, when present, form fibrils; (b) contacting the sample with a solution containing metal nanoparticles; (c) detecting the fibrils in the solution containing the metal nanoparticles; (d) identifying the sample as having the presence of the misfolded polypeptides if the fibrils are detected; and (e) identifying the sample as lacking the misfolded polypeptides if the fibrils are not detected. The sample can be a biological sample. The biological sample can be obtained from a living mammal. The living mammal can be a human, a monkey, a camel, a horse, a mink, a cat, a cow, a sheep, a mouse, a rat, a hamster, a brocket, a chital, an elk, a fallow deer, a marsh deer, a mule deer, a muntjac, a moose, a pampas deer, a red deer, a reindeer, a roe deer, a sambar deer, a sika, a white-tailed deer, an antelope, or a goat. The biological sample can be lymph tissue, muscle tissue, tonsil tissue, skin tissue, brain tissue, brain-stem tissue, blood, cerebrospinal fluid, urine, feces, saliva, mucus, liver tissue, heart tissue, intestinal tissue, spleen tissue, or eye tissue. The biological can be obtained from a mammal post-mortem. The biological sample can be beef or venison. The sample can be an environmental sample. The environmental sample can be soil, water, dust, or a plant. The environmental sample can be obtained using a swab or a filter. The environmental sample can be obtained from a location selected from group consisting of a natural habitat, a waterway, a farm, a food processing facility, a water-treatment facility, and a hospital. When the environmental sample is obtained from a food processing facility, the food processing facility can process food intended for mammalian consumption. The environmental sample can be obtained from a hospital. The method can include, prior to the amplifying step, isolating polypeptides from the sample. The amplifying step can include shaking the sample or sonicating the sample. The metal nanoparticles can be gold nanoparticles. The detecting step can include visually detecting a color shift, where the color shift is indicative of the absence of the misfolded polypeptide. In some cases, the detecting step can include detecting light absorbance, where an absorbance of from about 510 nm to about 525 nm (e.g., about 521 nm) is indicative of the presence of the misfolded polypeptide, and where an absorbance of from about 530 nm to about 600 nm is indicative of the absence of the misfolded polypeptide. In some cases, the detecting step can include detecting light absorbance, where an absorbance of from about 510 nm to about 521 nm (e.g., about 517 nm) is indicative of the presence of the misfolded polypeptide, and where an absorbance of from about 525 nm to about 600 nm is indicative of the absence of the misfolded polypeptide. The misfolded polypeptide can be a prion protein (PrP) polypeptide, a tau polypeptide, an amyloid ß polypeptide, an α-synuclein polypeptide, or a TDP-43 polypeptide. The misfolded polypeptide can be associated with a proteinopathy. The proteinopathy can be chronic wasting disease (CWD), Cruzefeldt-Jakob Disease, transmissible mink encephalopathy, feline spongiform encephalopathy, ungulate spongiform encephalopathy, bovine-spongiform encephalapothy, camilid spongiform encephalopathy, pituitary pars intermedia dysfunction (PPID), Alzheimer's Disease (AD), Parkinson's Disease (PD), Pick's disease, Lewy body dementia (LBD), amyotrophic lateral sclerosis (ALS), multiple systems atrophies, progressive supranuclear palsies, corticobasal degenerations, or a chronic traumatic encephalopathy.
In another aspect, this document features methods for detecting the presence or absence of misfolded polypeptides in a sample. The methods can include, or consist essentially of, (a) amplifying a sample under conditions where the misfolded polypeptides, when present, form globular polypeptide aggregates; (b) contacting the sample with a solution containing metal nanoparticles; (c) detecting the globular polypeptide aggregates in the solution containing the metal nanoparticles; (d) identifying the sample as having the presence of the misfolded polypeptides if the globular polypeptide aggregates are detected; and (e) identifying the sample as lacking the misfolded polypeptides if the globular polypeptide aggregates are not detected. The sample can be a biological sample. The biological sample can be obtained from a living mammal. The living mammal can be a human, a monkey, a camel, a horse, a mink, a cat, a cow, a sheep, a mouse, a rat, a hamster, a brocket, a chital, an elk, a fallow deer, a marsh deer, a mule deer, a muntjac, a moose, a pampas deer, a red deer, a reindeer, a roe deer, a sambar deer, a sika, a white-tailed deer, an antelope, or a goat. The biological sample can be lymph tissue, muscle tissue, tonsil tissue, skin tissue, brain tissue, brain-stem tissue, blood, cerebrospinal fluid, urine, feces, saliva, mucus, liver tissue, heart tissue, intestinal tissue, spleen tissue, or eye tissue. The biological can be obtained from a mammal post-mortem. The biological sample can be beef or venison. The sample can be an environmental sample. The environmental sample can be soil, water, dust, or a plant. The environmental sample can be obtained using a swab or a filter. The environmental sample can be obtained from a location selected from group consisting of a natural habitat, a waterway, a farm, a food processing facility, a water-treatment facility, and a hospital. When the environmental sample is obtained from a food processing facility, the food processing facility can process food intended for mammalian consumption. The environmental sample can be obtained from a hospital. The method can include, prior to the amplifying step, isolating polypeptides from the sample. The amplifying step can include shaking the sample or sonicating the sample. The metal nanoparticles can be gold nanoparticles. The detecting step can include visually detecting a color shift, where the color shift is indicative of the absence of the misfolded polypeptide. In some cases, the detecting step can include detecting light absorbance, where an absorbance of from about 510 nm to about 525 nm (e.g., about 521 nm) is indicative of the presence of the misfolded polypeptide, and where an absorbance of from about 530 nm to about 600 nm is indicative of the absence of the misfolded polypeptide. In some cases, the detecting step can include detecting light absorbance, where an absorbance of from about 510 nm to about 521 nm (e.g., about 517 nm) is indicative of the presence of the misfolded polypeptide, and where an absorbance of from about 525 nm to about 600 nm is indicative of the absence of the misfolded polypeptide. The misfolded polypeptide can be a PrP polypeptide, a tau polypeptide, an amyloid ß polypeptide, an α-synuclein polypeptide, or a TDP-43 polypeptide. The misfolded polypeptide can be associated with a proteinopathy. The proteinopathy can be CWD, Cruzefeldt-Jakob Disease, transmissible mink encephalopathy, feline spongiform encephalopathy, ungulate spongiform encephalopathy, bovine-spongiform encephalapothy, camilid spongiform encephalopathy, PPID, AD, PD, Pick's disease, LBD, ALS, multiple systems atrophies, progressive supranuclear palsies, corticobasal degenerations, or a chronic traumatic encephalopathy.
Unless otherwise defined, 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 pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the 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.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
This document provides methods and materials for detecting the presence or absence of misfolded polypeptides, polypeptide fibrils, and/or polypeptide aggregates (e.g., globular polypeptide aggregates) in a sample. For example, a sample (e.g., a biological sample or an environmental sample) can be amplified such that misfolded polypeptides present in the sample form fibrils and/or globular polypeptide aggregates, and the amplified sample can be contacted with a solution containing nanoparticles (e.g., metal nanoparticles such as gold nanoparticles) to detect the presence or absence of fibrils and/or polypeptide aggregates (e.g., globular polypeptide aggregates). Nanoparticles (e.g., metal nanoparticles such as gold nanoparticles) used in the methods described herein have unique optical properties. For example, when light interacts with the surface electrons of a metal nanoparticle, it causes the surface electrons to oscillate. At certain wavelengths, oscillations can be in phase and can cause plasmonic resonance, and at the wavelength of plasmonic resonance, metal nanoparticles exhibit very large absorption. When metal nanoparticles within a solution aggregate, the absorption of the solutions shifts. Accordingly, solutions containing metal nanoparticles (e.g., gold nanoparticles) can be used to detect the presence or absence of misfolded polypeptides. For example, a sample suspected of containing misfolded polypeptides can be amplified such that misfolded polypeptides, when present, can aggregate to form fibrils (e.g., aggregates of two or more misfolded polypeptides), and the amplified sample can be contacted with a solution containing metal nanoparticles (e.g., gold nanoparticles) to detect the presence or absence of fibrils. When fibrils are present in a solution containing metal nanoparticles (e.g., gold nanoparticles), the metal nanoparticles lack any change in absorption. For example, when fibrils are present in a solution containing metal nanoparticles (e.g., gold nanoparticles), the solution can appear red based on its absorption spectrum. When fibrils are not present in a solution containing metal nanoparticles (e.g., gold nanoparticles), the metal nanoparticles aggregate thereby causing a detectable change in absorption. For example, when fibrils are not present in a solution containing metal nanoparticles (e.g., gold nanoparticles), the solution can appear blue based on its absorption spectrum.
In some cases, a sample (e.g., a biological sample or an environmental sample) can be amplified such that misfolded polypeptides present in the sample form fibrils and/or globular polypeptide aggregates, and the amplified sample can be contacted with a solution containing one or more organic dyes (e.g., Congo Red) to detect the presence or absence of fibrils and/or polypeptide aggregates (e.g., globular polypeptide aggregates). Organic dyes (e.g., Congo Red) used in the methods described herein can have colloidal particles that provide unique optical properties. For example, when light interacts with a solution containing an organic dye in a suspension, it causes misfolded proteins can cause the organic dye to turn blue. When misfolded proteins within a solution aggregate, the absorption of the organic dye solution shifts. Accordingly, solutions containing one or more organic dyes (e.g., Congo Red) can be used to detect the presence or absence of misfolded polypeptides. For example, a sample suspected of containing misfolded polypeptides can be amplified such that misfolded polypeptides, when present, can aggregate to form fibrils (e.g., aggregates of two or more misfolded polypeptides), and the amplified sample can be contacted with a solution containing one or more organic dyes (e.g., Congo Red) to detect the presence or absence of fibrils. When fibrils are present in a solution containing one or more organic dyes (e.g., Congo Red), the organic dye(s) cause a detectable change in absorption. For example, when fibrils are present in a solution containing one or more organic dyes (e.g., Congo Red), the solution can appear blue (e.g., as compared to a solution that lacks fibrils) based on its absorption spectrum. When fibrils are not present in a solution containing one or more organic dyes (e.g., Congo Red), the organic dye(s) lack any change in absorption. For example, when fibrils are not present in a solution containing one or more organic dyes (e.g., Congo Red), the solution can appear red (e.g., as compared to a solution that contains fibrils) based on its absorption spectrum.
In some cases, the methods described herein (e.g., the methods for detecting the presence or absence of a misfolded polypeptide) can be used to detect the presence or absence of one or more aggregates of misfolded polypeptides. For example, the methods described herein can be used to detect the presence or absence of amyloid plaques. For example, the methods described herein can be used to detect the presence or absence of tau tangles.
In some cases, the methods described herein (e.g., the methods for detecting the presence or absence of a misfolded polypeptide) can be used to determine if a mammal (e.g., a human) has a proteinopathy based, at least in part, in the presence or absence of misfolded polypeptides in a sample obtained from the mammal. For example, a sample obtained from a mammal can be amplified such that misfolded polypeptides present in the sample can aggregate to form fibrils, the amplified sample can be contacted with a solution containing nanoparticles (e.g., metal nanoparticles such as gold nanoparticles) to detect the presence or absence of fibrils, and the mammal can be classified as having a proteinopathy if the presence of fibrils is detected. For example, a sample obtained from a mammal can be amplified such that misfolded polypeptides present in the sample can aggregate to form fibrils, the amplified sample can be contacted with a solution containing nanoparticles (e.g., metal nanoparticles such as gold nanoparticles) to detect the presence or absence of fibrils, and the mammal can be classified as not having a proteinopathy if the absence of fibrils is detected. In another example, a sample obtained from a mammal can be amplified such that misfolded polypeptides present in the sample can aggregate to form fibrils, the amplified sample can be contacted with a solution containing one or more organic dyes (e.g., Congo Red) to detect the presence or absence of fibrils, and the mammal can be classified as having a proteinopathy if the presence of fibrils is detected. For example, a sample obtained from a mammal can be amplified such that misfolded polypeptides present in the sample can aggregate to form fibrils, the amplified sample can be contacted with a solution containing one or more organic dyes (e.g., Congo Red) to detect the presence or absence of fibrils, and the mammal can be classified as not having a proteinopathy if the absence of fibrils is detected. In some cases, this document provides methods and materials for treating a mammal (e.g., a human) identified as having a proteinopathy as described herein (e.g., based, at least in part, on the presence of fibrils formed from misfolded polypeptides).
Any appropriate method can be used to amplify a sample (e.g., a sample suspected of containing misfolded polypeptides). In some cases, a sample can be amplified by shaking. A sample can be shaken for any appropriate amount of time. For example, a sample can be shaken for from about 3 hours to about 40 hours (e.g., from about 3 hours to about 36 hours, from about 3 hours to about 32 hours, from about 3 hours to about 24 hours, from about 3 hours to about 18 hours, from about 3 hours to about 15 hours, from about 3 hours to about 12 hours, from about 3 hours to about 8 hours, from about 5 hours to about 40 hours, from about 8 hours to about 40 hours, from about 12 hours to about 40 hours, from about 18 hours to about 40 hours, from about 22 hours to about 40 hours, from about 24 hours to about 40 hours, from about 28 hours to about 40 hours, from about 32 hours to about 40 hours, from about 6 hours to about 32 hours, from about 12 hours to about 28 hours, from about 15 hours to about 22 hours, from about 6 hours to about 18 hours, from about 12 hours to about 24 hours, from about 15 hours to about 28 hours, or from about 18 hours to about 32 hours). In some cases, a sample can be shaken for about 24 hours. A sample can be shaken at any appropriate speed. For example, a sample can be shaken at from about 200 RPM to about 1000 RPM (e.g., from about 200 RPM to about 800 RPM, from about 200 RPM to about 600 RPM, from about 200 RPM to about 400 RPM, from about 400 RPM to about 1000 RPM, from about 600 RPM to about 1000 RPM, from about 800 RPM to about 1000 RPM, from about 400 RPM to about 800 RPM, from about 500 RPM to about 700 RPM, from about 200 RPM to about 400 RPM, from about 400 RPM to about 600 RPM, or from about 600 RPM to about 800 RPM). In some cases, a sample can be shaken at from about 600 RPM to about 700 RPM. A sample can be shaken at any appropriate temperature. For example, a sample can be shaken at from about 30° C. to about 65° C. (e.g., from about 30° C. to about 60° C., from about 30° C. to about 55° C., from about 30° C. to about 50° C., from about 30° C. to about 45° C., from about 30° C. to about 40° C., from about 30° C. to about 35° C., from about 35° C. to about 65° C., from about 40° C. to about 65° C., from about 45° C. to about 65° C., from about 50° C. to about 65° C., from about 55° C. to about 65° C., from about 33° C. to about 60° C., from about 40° C. to about 55° C., from about 40° C. to about 50° C., or from about 50° C. to about 60° C.). In some cases, a sample can be shaken at about 42° C.
In some cases, a sample can be amplified by sonication. A sample can be sonicated for any appropriate amount of time. For example, a sample can be sonicated for from about 5 seconds to about 30 seconds (e.g., from about 5 seconds to about 25 seconds, from about 5 seconds to about 20 seconds, from about 5 seconds to about 15 seconds, from about 5 seconds to about 10 seconds, from about 10 seconds to about 30 seconds, from about 15 seconds to about 30 seconds, from about 20 seconds to about 30 seconds, from about 25 seconds to about 30 seconds, from about 10 seconds to about 25 seconds, from about 15 seconds to about 20 seconds, from about 10 seconds to about 15 seconds, or from about 20 seconds to about 25 seconds). A sonication step can be performed any number of times. For example, a sample can be sonicated from about 2 times to about 10 times (e.g., from about 2 times to about 8 times, from about 2 times to about 6 times, from about 2 times to about 4 times, from about 4 times to about 10 times, from about 6 times to about 10 times, from about 8 times to about 10 times, from about 4 times to about 8 times, from about 4 times to about 6 times, or from about 6 times to about 8 times). A sonication step can be performed any number of times. A sonication step can be performed at any temperature. For example, a sample can be sonicated at from about 30° C. to about 65° C.
In some cases, an amplified sample can be contacted with a solution containing nanoparticles (e.g., metal nanoparticles such as gold nanoparticles) or one or more organic dyes (e.g., Congo Red) immediately after amplification. For example, an amplified sample can be contacted with a solution containing nanoparticles (e.g., metal nanoparticles such as gold nanoparticles) or one or more organic dyes (e.g., Congo Red) from about 1 minute to about 60 minutes (e.g., from about 1 minute to about 45 minutes, from about 1 minute to about 30 minutes, from about 1 minute to about 20 minutes, from about 1 minute to about 10 minutes, from about 10 minutes to about 60 minutes, from about 20 minutes to about 60 minutes, from about 30 minutes to about 60 minutes, from about 45 minutes to about 60 minutes, from about 10 minutes to about 45 minutes, from about 20 minutes to about 30 minutes, from about 10 minutes to about 30 minutes, or from about 30 minutes to about 45 minutes) after amplification.
In some cases, an amplified sample can be stored (e.g., at 4° C. for an indefinite amount of time prior to being contacted with a solution containing nanoparticles (e.g., metal nanoparticles such as gold nanoparticles) or one or more organic dyes (e.g., Congo Red).
An amplified sample can be contacted with any appropriate solution containing nanoparticles (e.g., metal nanoparticles such as gold nanoparticles) or one or more organic dyes (e.g., Congo Red). In some cases, a solution can include disodium phosphate (Na2HPO4). For example, a solution can include from about 5 mM to about 15 mM disodium phosphate (e.g., from about 5 mM to about 12 mM, from about 5 mM to about 10 mM, from about 5 mM to about 8 mM, from about 7 mM to about 15 mM, from about 10 mM to about 15 mM, from about 12 mM to about 15 mM, from about 8 mM to about 12 mM, from about 7 mM to about 10 mM, from about 10 mM to about 12 mM, or about 10 mM disodium phosphate). In some cases, a solution can include potassium chloride (KCl). For example, a solution can include from about 1 mM to about 4 mM potassium chloride (e.g., from about 1 mM to about 3 mM, from about 1 mM to about 2 mM, from about 2 mM to about 4 mM, from about 3 mM to about 4 mM, from about 2 mM to about 3 mM, or about 2.7 mM potassium chloride). In some cases, a solution can include monopotassium phosphate (KH2PO4). For example, a solution can include from about 1 mM to about 3 mM monopotassium phosphate (e.g., from about 1 mM to about 2 mM, from about 2 mM to about 3 mM, or about 1.8 mM monopotassium phosphate). In some cases, a solution can have a pH of from about 5 to about 9 (e.g., from about 5 to about 8, from about 5 to about 7, from about 5 to about 6, from about 6 to about 9, from about 7 to about 9, from about 8 to about 9, from about 6 to about 8, from about 6 to about 7, from about 7 to about 8, or a pH of about 7.41).
An amplified sample can be contacted with a solution containing any appropriate nanoparticles. In some cases, a nanoparticle can be a metal nanoparticle. A metal nanoparticle can be made from any appropriate metal. Examples of metals that can be used to make a metal nanoparticle include, without limitation, gold, silver, copper, platinum, iron, and alloys thereof. In some cases, a metal nanoparticle can be a gold nanoparticle. In some cases, a nanoparticle can be a quantum dot. Examples of quantum dots that can be used that can be used as a nanopoarticle in the methods described herein include, without limitation, CdS, CdSe, CdTe, ZnS, ZnSe, PbS, and InP. A nanoparticle can be any appropriate size (e.g., can have any appropriate longest dimension such as a diameter). In some cases, a nanoparticle can have a diameter of from about 1 nm to about 100 nm (e.g., from about 1 nm to about 80 nm, from about 1 nm to about 60 nm, from about 1 nm to about 40 nm, from about 1 nm to about 20 nm, from about 20 nm to about 100 nm, from about 40 nm to about 100 nm, from about 60 nm to about 100 nm, from about 80 nm to about 100 nm, from about 20 nm to about 80 nm, from about 40 nm to about 60 nm, from about 20 nm to about 40 nm, from about 30 nm to about 50 nm, from about 40 nm to about 60 nm, or from about 5 nm to about 70 nm). A nanoparticle can be any shape (e.g., a sphere, a rod, a nanowire, a shell, a cube, and a star).
In some cases, at least one nanoparticle (e.g., at least one metal nanoparticle) in a solution containing metal nanoparticles can be conjugated to another molecule. Examples of molecules that a nanoparticle in a solution containing nanoparticles can be conjugated to include, without limitation, a nanoparticle (e.g., a different nanoparticle such as a nanoparticle functionalized with citrate, cetyltrimethylammonium bromide, carboxylic Acid, poly(allylamine) hydrochloride, polyvinylpyrrolidone, poly(acrylic acid), polyethylene glycol, and/or polyethylenimine), nanobodies, biotinylated biomolecules, ligands and polypeptides such as antibodies.
An amplified sample can be contacted with a solution containing any appropriate organic dye(s). In some cases, an organic dye can be an azo dye. Examples of organic dyes that can be used as described herein include, without limitation, Congo Red, Nile Red, acridine orange, Trypan Blue, Evans Blue, Sirius Red f3b, primuline, X-34, 1,4-Bis(3-carboxy-4-hydroxyphenylethenyl)benzene, (trans,trans)-1-bromo-2,5-bis-(3-hydroxycarbonyl-4-hydroxy)styrylbenzene (BSB); BF-168, and (6-2-Fluoroethoxy)-2-[2-(4-methylaminophenil)ethenyl]benzoxazole. In some case, an organic dye that can be used as described herein can be as described elsewhere (see, e.g., Mishra et al., Mol. BioSyst., 7:1232-1240 (2011)).
In some cases, when an amplified sample is contacted with a solution containing nanoparticles (e.g., metal nanoparticles such as gold nanoparticles) or one or more organic dyes (e.g., Congo Red), the amplified sample and the solution containing nanoparticles (e.g., metal nanoparticles such as gold nanoparticles) or one or more organic dyes (e.g., Congo Red) can be incubated together (e.g., can be maintained in contact for an extended period of time) prior to detecting the presence or absence of fibrils. For example, an amplified sample and a solution containing nanoparticles (e.g., metal nanoparticles such as gold nanoparticles) or one or more organic dyes (e.g., Congo Red) can be incubated together for from about 2 minutes to about 60 minutes (e.g., from about 2 minutes to about 50 minutes, from about 2 minutes to about 40 minutes, from about 2 minutes to about 30 minutes, from about 2 minutes to about 20 minutes, from about 2 minutes to about 10 minutes, from about 2 minutes to about 5 minutes, from about 5 minutes to about 60 minutes, from about 10 minutes to about 60 minutes, from about 20 minutes to about 60 minutes, from about 30 minutes to about 60 minutes, from about 40 minutes to about 60 minutes, from about 50 minutes to about 60 minutes, from about 10 minutes to about 50 minutes, from about 20 minutes to about 40 minutes, from about 20 minutes to about 30 minutes, from about 30 minutes to about 40 minutes, or about 30 minutes).
Any appropriate method can be used to detect the presence or absence of fibrils and/or polypeptide aggregates (e.g., fibrils and/or polypeptide aggregates formed from misfolded polypeptides). In some cases, the presence or absence of fibrils in a solution containing nanoparticles (e.g., metal nanoparticles such as gold nanoparticles) or one or more organic dyes (e.g., Congo Red) that was contacted with an amplified sample can be detecting (e.g., visually detected) using color. For example, a solution containing nanoparticles (e.g., metal nanoparticles such as gold nanoparticles) that was contacted with an amplified sample containing fibrils can appear red (e.g., can appear red to the naked eye). For example, a solution containing nanoparticles (e.g., metal nanoparticles such as gold nanoparticles) that was contacted with an amplified sample lacking fibrils can appear blue (e.g., can appear blue to the naked eye). In some cases, the presence or absence of fibrils in a solution containing nanoparticles (e.g., metal nanoparticles such as gold nanoparticles) that was contacted with an amplified sample can be detecting using absorbance (e.g., absorbance of light such as visible light or near-infrared light). For example, a solution containing nanoparticles (e.g., metal nanoparticles such as gold nanoparticles) that was contacted with an amplified sample containing fibrils can have a wavelength of from about 510 nm to about 525 nm (e.g., about 516 nm or about 521 nm). In another example, a solution containing nanoparticles (e.g., metal nanoparticles such as gold nanoparticles) that was contacted with an amplified sample containing fibrils can have a wavelength of from about 510 nm to about 521 nm (e.g., about 516 nm or about 517 nm). For example, a solution containing nanoparticles (e.g., metal nanoparticles such as gold nanoparticles) that was contacted with an amplified sample lacking fibrils can have a wavelength of from about 530 nm to about 600 nm (e.g., from about 530 nm to about 575 nm, from about 530 nm to about 550 nm, from about 550 nm to about 600 nm, from about 575 nm to about 600 nm, or from about 550 nm to about 575 nm). In another example, a solution containing nanoparticles (e.g., metal nanoparticles such as gold nanoparticles) that was contacted with an amplified sample lacking fibrils can have a wavelength of from about 525 nm to about 600 nm (e.g., from about 530 nm to about 575 nm, from about 530 nm to about 550 nm, from about 550 nm to about 600 nm, from about 575 nm to about 600 nm, or from about 550 nm to about 575 nm).
For example, a solution containing one or more organic dyes (e.g., Congo Red) that was contacted with an amplified sample containing fibrils can appear blue (e.g., can appear blue to the naked eye). For example, a solution containing one or more organic dyes (e.g., Congo Red) that was contacted with an amplified sample lacking fibrils can appear red (e.g., can appear red to the naked eye). In some cases, the presence or absence of fibrils in a solution containing one or more organic dyes (e.g., Congo Red) that was contacted with an amplified sample can be detecting using absorbance (e.g., absorbance of light such as visible light or near-infrared light). For example, a solution containing one or more organic dyes (e.g., Congo Red) that was contacted with an amplified sample containing fibrils can have a wavelength of from about 494 nm to about 550 nm (e.g., from about 494 nm to about 525, from about 505 nm to about 550 nm, from about 515 nm to about 535 nm, from about 500 nm to about 550 nm, from about 525 nm to about 550 nm). For example, a solution containing one or more organic dyes (e.g., Congo Red) that was contacted with an amplified sample lacking fibrils can have a wavelength of from about 450 nm to about 493 nm (e.g., about 490 nm).
In some cases, the methods provided herein are not antibody-based methods. For example, the methods provided herein can be performed in the absence of antibody-based techniques.
In some cases, the methods provided herein can be performed in the absence of any stimulus. For example, the methods provided herein can be performed in the absence of electrochemical stimulus.
In some cases, the methods provided herein can be performed in the absence of any sensor. For example, the methods provided herein can be performed in the absence of any colorimetric sensor. For example, the methods provided herein can be performed in the absence of any electrochemical sensor.
The methods described herein (e.g., the methods for detecting the presence or absence of a misfolded polypeptide) can be used to detect the presence or absence of any misfolded polypeptide. In some cases, a misfolded polypeptide can be associated with a disease. Examples of polypeptides that can be misfolded, and where the misfolded polypeptide can be detected as described herein include, without limitation, prion protein (PrP) polypeptides, tau polypeptides, amyloid ß polypeptides, α-synuclein polypeptides, and TDP-43 polypeptides.
The methods described herein (e.g., the methods for detecting the presence or absence of a misfolded polypeptide) can be used to detect the presence or absence of a misfolded polypeptide associated with any proteinopathy. As used herein, a proteinopathy is any disease associated with misfolding and, optionally, aggregation of one or more of the misfolded polypeptides. In some cases, a proteinopathy can be a transmissible spongiform encephalopathy (TES). In some cases, a proteinopathy can be a protein-misfolding disease (PMD). In some cases, a proteinopathy can be a tauopathy. In some cases, a proteinopathy can be an α-synucleinopathy. Examples of proteinopathies associated polypeptides that can be misfolded, and where the misfolded polypeptide can be detected as described herein include, without limitation, chronic wasting disease (CWD), Cruzefeldt-Jakob Disease, transmissible mink encephalopathy, feline spongiform encephalopathy, ungulate spongiform encephalopathy, bovine-spongiform encephalapothy, camilid spongiform encephalopathy, pituitary pars intermedia dysfunction (PPID), Alzheimer's Disease (AD), Parkinson's Disease (PD), Pick's disease, Lewy body dementia (LBD), amyotrophic lateral sclerosis (ALS), multiple systems atrophies, progressive supranuclear palsies, corticobasal degenerations, and chronic traumatic encephalopathies.
The methods described herein (e.g., the methods for detecting the presence or absence of a misfolded polypeptide) can be used to detect the presence or absence of misfolded polypeptides in any appropriate sample. In some cases, a sample can be a biological sample (e.g., a sample obtained from a mammal). In some cases, a sample can be an environmental sample. A sample can be a fresh sample or a fixed sample (e.g., a formaldehyde-fixed sample or a formalin-fixed sample). In some cases, a sample can be a processed sample. For example, a processed sample can be homogenized. For example, a processed sample can be diluted (e.g., can be diluted in a buffer such as phosphate buffered saline (PBS)). In some cases, one or more biological molecules (e.g., polypeptides) can be isolated from a sample. For example, polypeptides can be isolated from a sample and can be enriched or concentrated prior to being amplified as described herein.
When the methods described herein (e.g., the methods for detecting the presence or absence of a misfolded polypeptide) are used to detect the presence or absence of misfolded polypeptides in a biological sample, the biological sample can be obtained from any appropriate mammal. In some cases, a sample can be obtained from a living mammal. In some cases, a sample can be obtained from a mammal post-mortem sample. For example, a post-mortem sample can be a mammalian tissue or byproduct intended for consumption by another mammal (e.g., a human) such as beef or venison. In some cases, a mammal can be a cervid (e.g., can be a member of the (′ervidae family). Examples of mammals that a sample can be obtained from and where the sample can be assessed for the presence or absence or misfolded polypeptides as described herein (e.g., by amplifying the sample and contacting the amplified sample with a solution containing nanoparticles (e.g., metal nanoparticles such as gold nanoparticles) or one or more organic dyes (e.g., Congo Red)) include, without limitation, humans, non-human primates (e.g., monkeys), camels, horses, mink, cats, cows, sheep, mice, rats, hamsters, brocket, chital, elk, fallow deer, marsh deer, mule deer, muntjac, moose, pampas deer, red deer, reindeer, roe deer, sambar deer, sika, white-tailed deer, antelope, and goats.
When the methods described herein (e.g., the methods for detecting the presence or absence of a misfolded polypeptide) are used to detect the presence or absence of misfolded polypeptides in a biological sample, the biological sample can be any type of biological sample. Examples of biological samples that can be assessed for the presence or absence or misfolded polypeptides as described herein (e.g., by amplifying the sample and contacting the amplified sample with a solution containing nanoparticles (e.g., metal nanoparticles such as gold nanoparticles) or one or more organic dyes (e.g., Congo Red)) include, without limitation, lymph tissue, muscle tissue, tonsil tissue, skin tissue, brain tissue, brain-stem tissue, blood, cerebrospinal fluid, urine, feces, saliva, mucus, liver tissue, heart tissue, intestinal tissue, spleen tissue, and eye tissue.
When the methods described herein (e.g., the methods for detecting the presence or absence of a misfolded polypeptide) are used to detect the presence or absence of misfolded polypeptides in an environmental sample, the environmental sample can be obtained from any appropriate source. Examples of sources that a sample can be obtained from and where the sample can be assessed for the presence or absence of misfolded polypeptides as described herein (e.g., by amplifying the sample and contacting the amplified sample with a solution containing nanoparticles (e.g., metal nanoparticles such as gold nanoparticles) or one or more organic dyes (e.g., Congo Red)) include, without limitation, soil, water, dust, and plants.
When the methods described herein (e.g., the methods for detecting the presence or absence of a misfolded polypeptide) are used to detect the presence or absence of misfolded polypeptides in an environmental sample, the environmental sample can be obtained by any appropriate method. Examples of methods that can be used to obtain an environmental sample that can be assessed for the presence or absence of misfolded polypeptides as described herein (e.g., by amplifying the sample and contacting the amplified sample with a solution containing nanoparticles (e.g., metal nanoparticles such as gold nanoparticles) or one or more organic dyes (e.g., Congo Red)) include, without limitation, swabs and filters (e.g., air filtration system filters).
When the methods described herein (e.g., the methods for detecting the presence or absence of a misfolded polypeptide) are used to detect the presence or absence of misfolded polypeptides in an environmental sample, the environmental sample can be obtained from any environmental setting. Examples of environmental settings that an environmental sample that can be assessed for the presence or absence of misfolded polypeptides as described herein (e.g., by amplifying the sample and contacting the amplified sample with a solution containing nanoparticles (e.g., metal nanoparticles such as gold nanoparticles) or one or more organic dyes (e.g., Congo Red)) can be obtained from include, without limitation, natural habitats, waterways, farms, food processing facilities (e.g., meat processing facilities), water-treatment facilities, and hospitals (e.g., human hospitals and veterinary hospitals). When an environmental sample is obtained from a food processing facility, the food processing facility can process food intended for mammalian (e.g., human) consumption. For example, an environmental sample can be obtained from a food processing facility that processes agricultural commodities (e.g., alfalfa, corn, beets, soybeans, oats, grasses, potatoes, straw, and related byproducts) for mammalian consumption. When an environmental sample is obtained from a hospital, the sample can be obtained from any surface (e.g., a stainless steel surface) in frequent contact with patients and/or biological fluids (e.g., blood, urine, and feces). For example, an environmental sample obtained from a hospital can be obtained from surgical tools, examination surfaces, and countertops.
In some cases, the presence or absence of misfolded polypeptides in a sample can be confirmed using one or more techniques traditionally used to detect the presence of the presence or absence of misfolded polypeptides in a sample. For example, ELISA, IHC, and/or RT-QuIC tests can be used to confirm the detection of the presence or absence of misfolded polypeptides in a sample.
This document also provides methods and materials for treating a mammal (e.g., a human) identified as having a proteinopathy as described herein (e.g., based, at least in part, on the presence of fibrils and/or polypeptide aggregates formed from misfolded polypeptides). For example, a mammal identified as having a proteinopathy based, at least in part, on the presence of fibrils formed from misfolded polypeptides in a sample obtained from the mammal can be administered one or more (e.g., one, two, three, four, five or more) agents that can be used to treat a proteinopathy. Examples of agents that can be used to treat a proteinopathy include, without limitation, agents (e.g., small molecules, oligonucleotides, peptides, and engineered immune cells) that can target the misfolded polypeptide, nanoparticle based delivery systems, and any combinations thereof. For example, a mammal identified as having a proteinopathy based, 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 a proteinopathy. Examples of therapies that can be used to treat a proteinopathy include, without limitation, physical therapy, occupational therapy, speech therapy, and any combinations thereof.
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
This Example describes the design of a method for detecting misfolded polypeptides that combines the incredible sensitivity of prion amplification methods with gold nanoparticle (AuNP)-based detection. The method described herein is also referred to as the Minnesota Quaking-induced Conversion (MN-QuIC) test (
Samples from 13 CWD detected (positive) and 13 CWD not-detected (negative) white-tailed deer lymphoid tissues were selected for independent EP-QuIC and MN-QuIC analyses (Table 1). White tailed deer lymphoid tissue was homogenized in PBS (10% w:v) using a BeadBug™ Homogenizer on max speed for 90 seconds. Tubes contained 1.5 mm zirconium beads. All positive and negative samples were selected based on independent ELISA, IHC, and/or RT-QuIC results.
EP-QuIC was performed using a modified RT-QuIC master mix. Specifically, the addition of ThT was excluded to avoid potential interference with downstream AuNP absorbance readings. A BMG FLUOStar® Omega plate reader and a ThermoMixer (Eppendorf) were utilized to perform EP-QuIC analyses. Plate reader reactions were performed at 420 and shaken at 700 RPM, double orbital for 57 seconds and then rested for 83 seconds for 24 hours. Each sample was run with 4 replicates and controls with 6 replicates. Thermomixer reactions were performed at 480 for 24 hours with 600 RPM. Each sample had a minimum of 3 replicates. Replicates were pooled at the end of the reaction for subsequent ThT fluorescence analysis. To ensure fibrillation of the recombinant substrate, 100 μL were taken from the pooled fractions and added to 1 μL of 1 mM ThT, and fluorescence was measured in RFUs. These values were compared to a negative control to determine relative amounts of fibrillation.
AuNPs. 15 nm gold nanospheres were used for all MN-QuIC experiments reported herein, although other particle shapes (rod, cube, star, etc.) and materials (silver, copper, etc.) have utility for the method. Nanoparticles were buffer exchanged by centrifuging 533 μL of nanoparticle solution (12,000 rpm; 10 minutes), removing 490 μL of supernatant, and resuspending in 320 μL of low NaCl PBS buffer (10 mM Na2HPO4, 2.7 mM KCl, 1.8 mM KH2PO4, pH to 7.4 with HCl). In this solution, the nanoparticles are stable for weeks.
Prion amplification. Recombinant hamster PrP (HaPrP90-231) was produced and filtered. 10% tissue homogenates were diluted in 0.1% SDS/1X PBS solution. 1X PBS, 1 mM EDTA, 170 mM NaCl, and 0.1 mg/mL HaPrPrP were mixed prior to the addition of 98 μL into wells on a black 96-well plate with clear bottoms. 2 μL of diluted 10% tissue homogenates were added in each well before the plate was sealed and shaken on either a plate reader or ThermoMixer C equipped with SmartBlock plate and Thermotop (Eppendorf). Plate reader reactions were performed at 42, for 24 hours with 700 RPM, and Thermomixer reactions were performed at 48, for 24 hours with 600 RPM. Each sample had a minimum of 3 replicates. The resultant products were visualized with the addition of gold nanoparticles (prepared as described below). Absorbance and visual color were recorded.
Visualization of post amplified material with nanoparticles was achieved by diluting individual wells to 50% in 1×PBS, 1 mM EDTA, 170 mM NaCl, and 1.2 mM sodium phosphate. 40 μL of these dilutions were then added to 360 μL of buffer exchanged AuNPs. This solution was then left to react for 30 minutes, although often the color differences between negative and positive samples became visible to the naked eye within the first two minutes. After the 30 minutes, photographs of the tubes were taken. Three replicates of 100 μl were taken from the 400 μl AuNP mixture and pipetted into three separate wells of a 96 well plate. The absorbance spectrum was then taken of each well at wavelengths 300-1000 nm.
Results from the EP-QuIC and MN-QuIC were grouped into either a positive or negative pool based on the known infection status from IHC, ELISA, and RT-QuIC data. A Mann-Whitney, non-parametric, two-tailed t-test was performed to determine statistical significance between the groups. Significance was determined using an alpha level of p<0.05.
EP-QuIC analyses revealed successful prion amplification of CWD+ tissues (Table 1) as measured by ThT fluorescence versus negative controls. Relative fluorescence units of positive samples ranged from 50,122 RFU to 69,793 RFU, with the positive control at 99,639 RFU (
The optical properties of nanoparticles are extremely sensitive to their surrounding environment. While changes in the peak absorbed wavelength can be achieved by proteins simply absorbing onto the nanoparticles, very large peak absorbance shifts can come when nanoparticles aggregate together. These shifts due to aggregating can often be seen with the naked eye. After quaking and thermal treatment, if no fibrillation occurs in the prion substrate, prions added to AuNPs cause the AuNPs to aggregate compared to when no protein is present (
In some cases, MN-QuIC can be defined as the process where misfolded protein seeded amplification is performed using a combination of shaking and incubating with an appropriate substrate and results are immediately visualized using metallic nanoparticles. To show that MN-QuIC is comparable to RT-QuIC, the commonly used method for misfolded protein detection, 10% (w:v) lymphoid tissue homogenates from 5 positive and 5 negative animals were tested using both RT-QuIC and MN-QuIC. It was found that both absorbance of gold nanoparticles and visual color change from MN-QuIC accurately reflect results from standard RT-QuIC ThT reading (
Together these results demonstrate that AuNP-based analyses of amplified prion products can rapidly (e.g., in less than 24 hours) detect misfolded polypeptides. This light-based assay for the detection of misfolded polypeptides is cost-effective and portable, and the results are readily distinguishable by eye or using a standard spectrophotometer.
Amplify misfolded polypeptides
Shake sample for 24-48 hours.
Prepare metal nanoparticle solution
1A) Obtain 15 nm citrate capped AuNP in deionized (DI) water; concentration ˜ 2.7 nM.
2A) Aliquot 400 μL of AuNP solution into centrifuge tube.
3A) Spin at 12000 rpm for 10 minutes.
4A) Remove 360 μL leaving pelleted AuNP in 40 μL DI water.
5A) Add re-suspended pellet in 320 μL of low NaCl buffer.
Detect misfolded polypeptide
1B) Take sample directly from amplification, dilute to 25% protein v/v with End Point buffer.
2B) Add 40 μL 25% post amplification PrP solution to vial of 360 μL AuNP to bring the combined solution up to 400 μL.
3B) Let sit 30 minutes.
4B) Take picture of mixture and look for color change.
5B) Spin down mixture at 3000 rpm.
6B) Add 100 μL of mixture to three different wells on a 96-well plate.
7B). Insert plate into spectrometer and take the absorbance spectrum of each well.
Pictures taken at step 4B are shown in
In addition to the color shift, a change in the absolute absorbance between positive and negative samples was seen because the AuNPs aggregate in the negative samples. The centrifugation step forces the larger/heavier aggregates out of solution and leaves a smaller absorption peak in the absorbance spectrum (
The peak absorbance wavelength can also be used to determine the presence or absence of misfolded polypeptides.
The absorbance spectrum was taken and the peak absorbance wavelength was found for each sample. CWD positive samples had peak absorbance wavelengths around 516 nm. Samples that were CWD negative had peak absorbance wavelengths around 521 nm (
MN-QuIC is a method for detecting prionogenic fragments in a sample. It works by amplifying fibril formation through violent shaking and seeding with a sensitive form of prion, usually hamster PrP (HaPrP). The data is recorded in real-time.
This Example describes the design of a nanoparticle-based assay that combines the unique color properties of AuNPs and the methods of quaking-based prion protein fibril amplification to detect the presence or absence of PrPCWD using both visual and spectroscopic methods (
The results in this Example re-present and expand on at least some of the results provided in other Examples.
Comparison of Gold Nanoparticle Interaction with Recombinant Cellular Prions Vs Amplified Misfolded Fibrils
To investigate whether AuNPs can differentiate between misfolded fibrils from recombinant hamster prion protein (rHaPrP), two sets of reaction mixtures seeded with and without spontaneously misfolded rHaPrP prion fibrils were processed following modified RT-QuIC protocols without ThT. The presence of fibril formation was examined in all reaction mixtures by adding and quantifying ThT post hoc (
Understanding that pathogenic prions (PrpSc) can induce rHaPrP misfolding and amplification and thus influence AuNP aggregation (see above), the potential of MN-QuIC for CWD diagnostics using PrpCWD positive and negative white-tailed deer lymphoid tissues was investigated. Homogenates of independently confirmed CWD positive and negative white-tailed deer medial retropharyngeal lymph nodes (RPLN) were used (Table 2). Independent RT-QuIC analyses were performed on all tissues used for AuNP analyses (
Field deployment and higher throughput protocols To show the potential for a portable, field-deployable diagnostic proof of concept experiments were performed in a rural field-station. Pooled retropharyngeal lymph nodes, parotid lymph nodes and palatine tonsils tissues from 13 animals collected from the surrounding wild deer population were tested. Three of these animals (blinded to the field team) were CWD positive as determined by regulatory ELISA and IHC testing of medial retropharyngeal lymph nodes. Using a blinded testing approach, MN-QuIC successfully detected, via red AuNP solutions, all three CWD positive animals. Additionally we successfully detected, with purple AuNP solutions, 8 out of the 10 CWD negative (i.e., not detected by ELISA) animals. The field test gave false positive signals for 2 of the 10 CWD ELISA not detected samples. However, the retropharyngeal lymph nodes, parotid lymph nodes and palatine tonsils were later retested individually (not pooled) for the two false positive animals. It was found that when all three tissues for each animal were tested individually they all gave negative (purple AuNP solution) results via the MN-QuIC assay. These preliminary experiments demonstrate the potential utility of MN-QuIC as a portable, field-deployable diagnostic tool for researchers and agencies.
In order to demonstrate higher throughput protocols, palatine tonsil samples from a set of ten CWD negative and ten CWD positive white-tailed deer (Table 3) were tested. The status of these tissues was confirmed with RT-QuIC (
In light of the results described above, the potential mechanism underlying AuNP aggregation caused by rHaPrP solutions was examined. The theoretical isoelectric point (IP) of the rHaPrP is around pH 8.9 and because of this, rHaPrP is positively charged in the pH7.4 AuNP buffer, whereas citrate capped AuNPs are negatively charged even at pHs well below the buffer. Thus at pH 7.4, there exists an electrostatic attractive force between AuNPs and native rHaPrP that contributes to their interactions (aggregation and the color change). It was examined whether the charge on the protein would change when the pH of the environment is altered and that the interaction between AuNP and rHaPrP would be disrupted. As the pH of the AuNP solution was raised closer to the IP of rHaPrP, the absorbance peak shift from 515 nm of the AuNP-rHaPrP solution decreased (
Together these results demonstrate that AuNP-based analyses of amplified prion products can rapidly (e.g., in less than 24 hours) detect misfolded polypeptides as a portable, sensitive field test.
Twenty-eight white-tailed deer tissues (14 CWD-negative and 14 CWD-positive) were selected for RT-QuIC and MN-QuIC analyses. These samples were collected from white-tailed deer (Tables 2 and 3) and their CWD status was independently identified utilizing the Bio-Rad TeSeE Short Assay Protocol (SAP) Combo Kit (BioRad Laboratories Inc., Hercules, CA, USA). Positive RPLNs were confirmed by IHC. Metadata containing information of all specimens examined, including tissue type, are provided in Tables 2 and 3. White-tailed deer RPLNs and palatine tonsils were homogenized in PBS (10% w:v) in 2 mL tubes containing 1.5 mm zirconium beads with a BeadBug Homogenizer (Benchmark Scientific, Sayreville New Jersey, USA) on max speed for 90 seconds. All CWD positive and negative samples were selected based on independant ELISA, IHC, and/or RT-QuIC results and were subsampled using methods as described elsewhere (Schwabenlander et al., J. Wildl. Dis., doi: 10.7589/JWD-D-21-00033 (2021)).
Recombinant hamster PrP (HaPrP90-231) production and purification followed the methods described elsewhere (Schwabenlander et al., J. Wildl. Dis., doi: 10.7589/JWD-D-21-00033 (2021)). The substrate is derived from a truncated form (amino acids 90-231) of the Syrian hamster PRNP gene cloned into the pET41-a(+) expression vector and was expressed in Rosetta (DE3) E. coli.
For QuIC analysis, a master mix was made to the following specifications: 1X PBS, 1 mM ethylenediaminetetraacetic acid (EDTA), 170 mM NaCl, 10 μM thioflavin T (ThT), and 0.1 mg/mL rHaPrP. In instances where the end reaction would be analyzed using AuNPs, ThT could be excluded. The 10% tissue homogenates were further diluted 100-fold in 0.1% Sodium Dodecyl Sulfate (SDS) using methods described elsewhere (Schwabenlander et al., J. Wildl. Dis., doi: 10.7589/JWD-D-21-00033 (2021)) (final tissue dilution: 0.1%), and 2 μL of the diluent were added to each well containing 98 μL of RT-QuIC master mix. Spontaneous misfolding of recombinant prion protein was generated similarly but with unfiltered recombinant proteins and reagents. For these reactions, no infectious seed was necessary. The spontaneously misfolded material was used to seed reactions for the dynamic light scattering experiment, described below. Plates were amplified on a FLUOstar® Omega plate reader (BMG Labtech, Cary, North Carolina, USA; 42ºC, 700 rpm, double orbital, shake for 57 seconds, rest for 83 seconds). Fluorescent readings were taken at ˜45 minute increments.
A standard benchtop shaking incubator (thermomixer) was leveraged to produce QuIC-based prion amplifications as described elsewhere (Cheng, et al., J. Clin. Microbiol. 54:1751-1754 (2016); and Vendramelli et al., J. Clin. Microbiol. 56:e00423-18 (2018)), although with slight modifications. Plates which were made for amplification on the thermomixer were prepared identical to those amplified on the plate reader. Reactions were performed on a ThermoMixerR C equipped with SmartBlock plate and Thermotop (Eppendorf, Enfield, Connecticut, USA) at 48ºC for 24 hours at 600 RPM (60s shake and 60s rest). A 24 hour run time was selected based on independent RT-QuIC results for medial retropharyngeal lymph nodes and palatine tonsils from CWD+ white-tailed deer reported as described elsewhere (Schwabenlander et al., J. Wildl. Dis., doi: 10.7589/JWD-D-21-00033 (2021)), including those examined herein, showing significant seeding activity within 9 to 24 hours (
Post amplified material was visualized with 15 nm citrate capped gold nanoparticles purchased from usaNanopartz (Loveland, Colorado, USA) with stock concentrations ranging from 2.45 nM to 2.7 nM. AuNP protocols were modified from Springer et al. (Anal. Bioanal. Chem., 404:2869-2875 (2012)) and Zhang et al. (Talanta 89:401-406 (2012)). AuNPs were buffer exchanged using 530 μL of stock solution that was centrifuged in 1.6 mL tubes at 13,800 g for 10 minutes. 490 μL of supernatant was removed and the undisturbed pellet was resuspended with 320 μL of a low concentration phosphate buffer (PBSlow; PH 7.4 via addition of HCl) made of 10 mM Na2HPO+(anhydrous), 2.7 mM KCl, 1.8 mM KH2PO4 (monobasic). After the quaking/incubation steps, protein solutions were diluted to 50% in MN-QuIC buffer, consisting of 1×PBS with the addition of final concentrations of 1 mM EDTA, 170 mM NaCl, 1.266 mM sodium phosphate. 40 μL of the protein diluted 50% in MN-QuIC buffer was then added to the 360 μL AuNP solution with ample mixing (results shown in
The necessary MN-QuIC equipment was assembled as described above on two portable tables. Medial retropharyngeal lymph nodes, parotid lymph nodes and palatine tonsil were collected as described elsewhere (Schwabenlander et al., J. Wildl. Dis., doi: 10.7589/JWD-D-21-00033 (2021)), sampled and pooled together for each of the 13 animals tested. Tissues were subject to 24 hour MN-QuIC protocols as described above. Three replicates were performed for each of the 13 animals and, for field-based analyses, an animal was considered CWD positive if one or more replicates was red.
Spontaneously misfolded rHaPrP samples (described above) were produced from solutions of rHaPrP with no seed added. A 96-well RT-QuIC reaction was performed with half the wells consisting of native rHaPrP seeded with spontaneously misfolded protein, and half consisting of native rHaPrP with no seed. The 96-well plate was then amplified using QuIC protocols described above. Post amplification, seeded samples were confirmed to have beta-sheet fibrillation while the non-seeded samples were confirmed to not have fibrillation based on ThT binding (described above). Seeded and non-seeded samples were diluted to 50% in MN-QuIC buffer and 40 μL of these solutions were added to 360 μL of AuNPs in PBSlow. Additionally, a blank with no protein was produced by adding 40 μL of MN-QuIC buffer to 360 μL of AuNPs in PBSlow. For native rHaPrP samples, color change could start to be observed within 1 minute of protein addition. No color change was observed in spontaneously misfolded rHaPrP samples at any time length. Dynamic light scattering measurements of all samples were taken after 5 minutes of protein addition using a Microtract NanoFlex Dynamic Light Scattering Particle Analyzer (Verder Scientific, Montgomeryville, PA, USA) and measurement times were 60 seconds. Five measurements were taken for each sample and then averaged.
In order to test the effects of pH on protein interacting with AuNP, four different 10 mM tris-buffer solutions with pHs ranging from 7.2 to 8.6 were created. Tris was used to give buffering for the desired pH range. AuNPs were buffer exchanged as described above except tris-buffer was used instead of PBSlow. Protein solutions were added as previously described.
GraphPad Prism version 9.0 for Windows (GraphPad Software, San Diego, California USA, graphpad.com) was used for conducting statistical analysis. Three technical replicates were used to demonstrate the potential application of AuNP on spontaneously misfolded rHaPrP. For initial trials on RPLN tissues from eight (four positive and four negative) animals, four and three technical replicates used for RT-QuIC and AuNPs respectively. For plate-based protocols, palatine tonsils from ten positive and ten negative animals were tested using four and eight replicates for RT-QuIC and AuNPs, respectively. Unless specified in figures, rate of amyloid formation and maximum wavelength of samples were compared to negative controls on the respective plate. The one-tailed Mann-Whitney unpaired u-test (α=0.05) was used to test the average difference for all parameters of interests between samples.
MN-QuIC is a method for detecting prionogenic fragments in a sample. It works by amplifying fibril formation through shaking and seeding with a sensitive form of prion such as HaPrP. Amplified proteins are then added to gold nanoparticles for detection.
Filter each solution with a 0.22 μm Syringe filter.
The table gives the volumes for one well on a 96-well plate. Scale up as needed.
HaPrP concentration varies between batches. Make the ratio of the water and HaPrP such that the final concentration of HaPrP is 0.1 mg/mL.
Appendix B: PBS low NaCl recipe
1.) Mix the following chemicals in DI water:
2.) pH to 7.41 using HCl (about 4 drops of 3.7% HCl)
This Example describes the analysis of various parameters within a nanoparticle-based assay.
A solution rich in native (non-misfolded) prions (PrP) was created.
CWD positive or CWD negative samples were seeded into a solution of native (non-misfolded) prions (PrP).
A MN-QuIC protocol was performed as described in Example 5 except that shaking was performed for 20 hours or 24 hours.
Different sized nanoparticles (10 nm and 15 nm) were used in a 24 hour MN-QuIC assay. For both 10 nm and 15 nm AuNPs, the samples containing CWD positive tissue appeared red and samples containing CWD negative tissue appeared blue. The colors of these samples are reflected in the peak absorbance spectra. The 517/580 ratio (e.g., the ratio of the absorbance at 517 nm divided by the absorbance at 580 nm) is shown in
Different shaking times were used in another MN-QuIC assay. For this experiment, the shaking time was 20 hours. Samples containing CWD positive tissue appeared red, and samples containing CWD negative tissue appeared blue. The colors of these samples are reflected in their peak absorbance (
This Example describes the use of a nanoparticle-based assay that combines the unique color properties of AuNPs and the methods of quaking-based prion protein fibril amplification to detect the presence or absence of misfolded α-synuclein polypeptides (e.g., to diagnose Parkinson's disease) using both visual and spectroscopic methods.
A solution rich in native (non-misfolded) Recombinant hamster PrP (HaPrP) polypeptides was created.
Seeds of misfolded α-synuclein were put into a solution of native HaPrP as a positive sample (e.g., to mimic a proteinopathy associated with misfolded α-synuclein polypeptides such as Parkinson's disease). Seeds of native (non-misfolded) α-synuclein were also put into a solution of native (non-misfolded) HaPrP as a negative sample (mimicking a no disease).
A MN-QuIC protocol was performed largely as described in Example 5. After shaking, AuNP was applied to wells containing the positive and negative samples.
These experiments determine that misfolded α-synuclein polypeptides cross seed and induce misfolding in prion (PrP), thus allowing detection of misfolded α-synuclein polypeptides with an AuNP MN-QuIC assay.
Samples were contacted with AuNPs. Samples containing misfolded α-synuclein polypeptides appeared red. Samples containing native α-synuclein polypeptides appeared blue. These colors were also reflected in the peak absorbance spectra (
This Example describes the use of an organic dye-based assay that combines the unique color properties of organic dyes (Congo Red) and the methods of quaking-based protein fibril amplification to detect the presence or absence of misfolded α-synuclein polypeptides (e.g., to diagnose Parkinson's disease) using both visual and spectroscopic methods.
A solution rich in native (non-misfolded) α-synuclein polypeptides was created. Seeds of misfolded α-synuclein were put into a solution of native α-synuclein as a positive sample (e.g., to mimic a proteinopathy associated with misfolded α-synuclein polypeptides such as Parkinson's disease). Seeds of native (non-misfolded) α-synuclein were also put into a solution of native (non-misfolded) α-synuclein as a negative sample (mimicking a no disease).
A MN-QuIC protocol was performed similarly to the method as described in Example 5 (one notable difference is that α-synuclein was used as a substrate instead of PrP). After shaking, Congo Red was applied to wells containing the positive and negative samples. For samples contacted with Congo Red, samples were shaken on plate reader for 32 hours at 42° C.
Samples were contacted with different ratios of Congo red to polypeptide (15:1 and 5:1 ratios). A color difference was visibly observed between the misfolded α-synuclein polypeptides and the native (non-misfolded) α-synuclein polypeptides. Samples containing misfolded α-synuclein polypeptides looked bluer then samples containing native (non-misfolded) α-synuclein polypeptides (
These results demonstrate that a MN-QuIC assay can be used to detect misfolded polypeptides associated with human diseases. These results also demonstrate that organic dyes (e.g., Congo Red) can be used in a MN-QuIC Assay (e.g., as an alternative to AuNPs).
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
This application claims the benefit of U.S. Patent Application Ser. No. 63/176,114, filed on Apr. 16, 2021, and the benefit of U.S. Patent Application Ser. No. 63/277,999, filed on Nov. 10, 2021. The disclosure of the prior application is considered part of (and is incorporated by reference in) the disclosure of this application.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2022/025059 | 4/15/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63277999 | Nov 2021 | US | |
| 63176114 | Apr 2021 | US |
