1. A method for determining a presence of a soluble, misfolded \u03b1S protein in a sample, comprising:
contacting the sample with a monomeric, folded \u03b1S protein to form an incubation mixture;
conducting an incubation cycle two or more times on the incubation mixture effective to form an amplified portion of misfolded \u03b1S protein from the monomeric, folded \u03b1S protein, each incubation cycle comprising:
incubating the incubation mixture effective to cause misfolding andor aggregation of at least a portion of the monomeric, folded \u03b1S protein in the presence of the soluble, misfolded \u03b1S protein;
physically disrupting the incubation mixture effective to at least partly de-aggregate at least a portion of a misfolded \u03b1S aggregate present; and
determining the presence of the soluble, misfolded \u03b1S protein in the sample by detecting at least a portion of the amplified portion of misfolded \u03b1S protein,
the soluble, misfolded \u03b1S protein comprising one or more of: a soluble, misfolded \u03b1S monomer and a soluble, misfolded \u03b1S aggregate; and
the amplified portion of misfolded \u03b1S protein comprising one or more of: an amplified portion of the soluble, misfolded \u03b1S monomer, an amplified portion of the soluble, misfolded \u03b1S aggregate, and an insoluble, misfolded \u03b1S aggregate.
2. The method of claim 1:
further comprising contacting an indicator of misfolded \u03b1S protein to the incubation mixture, the indicator of misfolded \u03b1S protein being characterized by an indicating state in the presence of the misfolded \u03b1S protein and a non-indicating state in the absence of the misfolded \u03b1S protein; and
wherein the determining the presence of the soluble, misfolded \u03b1S protein in the sample comprises detecting the indicating state of the indicator of misfolded \u03b1S protein.
3. The method of claim 2, the indicator of misfolded \u03b1S protein comprising one or more of: Thioflavin T, Congo Red, m-I-Stilbene, Chrysamine G, PIB, BF-227, X-34, TZDM, FDDNP, MeO-X-04, IMPY, NIAD-4, luminescent conjugated polythiophenes, a fluorescent protein, and derivatives thereof.
4. The method of claim 1, the determining the presence of the soluble, misfolded \u03b1S protein in the sample comprising one or more of:
determining an amount of the soluble, misfolded \u03b1S protein in the sample;
detecting the amount of the soluble, misfolded \u03b1S protein in the sample at a sensitivity of at least about 80%;
detecting the amount of the soluble, misfolded \u03b1S protein in the sample at less than about 100 nmol;
detecting the amount of the soluble, misfolded \u03b1S protein in the sample in a molar ratio to monomeric, folded \u03b1S protein comprised by the sample, the molar ratio being less than about 1:100;
detecting the soluble, misfolded \u03b1S protein in the sample with a specificity of at least about 80%; and
determining an amount of the soluble, misfolded \u03b1S protein in the sample compared to a control sample.
5. The method of claim 1, the incubating comprising incubating the incubation mixture at one or more of: between about 4\xb0 C. and about 35\xb0 C.
6. The method of claim 1, the detecting the soluble, misfolded \u03b1S protein comprising one or more of: a Western Blot assay, a dot blot assay, an enzyme-linked immunosorbent assay (ELISA), a thioflavin T binding assay, a Congo Red binding assay, a sedimentation assay, electron microscopy, atomic force microscopy, surface plasmon resonance, and spectroscopy.
7. The method of claim 1, further comprising providing the monomeric, folded \u03b1S protein in labeled form, the detecting the soluble, misfolded \u03b1S protein comprising detecting the monomeric, folded \u03b1S protein in labeled form as incorporated into the amplified portion of misfolded \u03b1S protein.
8. The method of claim 1, the sample being taken from a subject, further comprising determining or diagnosing one or more of:
the presence of PD in the subject according to the presence of the soluble, misfolded \u03b1S protein in the sample;
the presence of a disease associated with alpha-synuclein homeostasis in the subject according to the presence of the soluble, misfolded \u03b1S protein in the sample;
the presence of Multiple System Atrophy in the subject according to the presence of the soluble, misfolded \u03b1S protein in the sample;
the presence of PD in the subject according to the presence of the soluble, misfolded \u03b1S protein in the sample compared to a control sample taken from a control subject;
the presence of PD in the subject by comparing an amount of the soluble, misfolded \u03b1S protein in the sample to a predetermined threshold amount, the detecting comprising detecting the amount of the soluble, misfolded \u03b1S protein in the sample.
the presence of PD in the subject according to the presence of the soluble, misfolded \u03b1S protein in the sample, the subject exhibiting no clinical signs of dementia according to cognitive testing;
the presence of PD as a contributing factor to one or more clinical signs of dementia in the subject according to the presence of the soluble, misfolded \u03b1S protein in the sample, the subject exhibiting the one or more clinical signs of dementia according to cognitive testing; and
a progression or homeostasis of PD in the subject by comparing the amount of the soluble, misfolded \u03b1S protein in the sample to an amount of the soluble, misfolded \u03b1S protein in a comparison sample taken from the subject at a different time compared to the sample.
9. The method of claim 1, further comprising obtaining the sample from a subject,
the sample comprising one or more of: amniotic fluid; bile; blood; cerebrospinal fluid; cerumen; skin; exudate; feces; gastric fluid; lymph; milk; mucus; mucosal membrane; nasal secretions; peritoneal fluid; plasma; pleural fluid; pus; saliva; sebum; semen; sweat; synovial fluid; tears; and urine; and
the subject being one of a: human, mouse, rat, dog, cat, cattle, horse, deer, elk, sheep, goat, pig, and non-human primate.
10. The method of claim 1, the sample being taken from a subject, the subject being treated with one or more of:
an \u03b1S modulating therapy, further comprising:
comparing the amount of the soluble, misfolded \u03b1S protein in the sample to an amount of the soluble, misfolded \u03b1S protein in a comparison sample, the sample and the comparison sample being taken from the subject at different times over a period of time under the \u03b1S modulating therapy; and
determining the subject is one of: responsive to the \u03b1S modulating therapy according to a change in the soluble, misfolded \u03b1S protein over the period of time, and non-responsive to the \u03b1S modulating therapy according to homeostasis of the soluble, misfolded \u03b1S protein over the period of time; and
a disease-modifying therapy for PD, further comprising:
comparing the amount of the soluble, misfolded \u03b1S protein in the sample to an amount of the soluble, misfolded \u03b1S protein in a comparison sample, the sample and the comparison sample being taken from the subject at different times over a period of time under the disease-modifying therapy for PD; and
determining the subject is one of: responsive to the disease-modifying therapy for PD according to a change in the soluble, misfolded \u03b1S protein over the period of time, and non-responsive to the disease-modifying therapy for PD according to homeostasis of the soluble, misfolded \u03b1S protein over the period of time.
11. The method of claim 10, further comprising treating one or more of the subject determined to be responsive to the \u03b1S modulating therapy with the \u03b1S modulating therapy and the subject determined to be responsive to the disease-modifying therapy for PD with the disease-modifying therapy for PD.
12. The method of claim 1, the sample being taken from a subject, the subject being treated with an \u03b1S modulating therapy to inhibit the production of \u03b1S or to inhibit the aggregation of \u03b1S.
13. The method of claim 1, the physically disrupting the incubation mixture comprising one or more of: sonication, stirring, shaking, freezingthawing, laser irradiation, autoclave incubation, high pressure, homogenization, and cyclic agitation.
14. The method of claim 1, the monomeric, folded \u03b1S protein andor the soluble, misfolded \u03b1S protein comprising one or more of: one or more peptides formed via proteolytic cleavage of \u03b1S-140 protein; \u03b1S-140; \u03b1S-126; and \u03b1S-112.
15. The method of claim 1, comprising:
contacting the sample with Thioflavin T and a molar excess of the monomeric, folded \u03b1S protein to form the incubation mixture, the molar excess being greater than an amount of \u03b1S protein monomer included in the soluble, misfolded \u03b1S protein in the sample;
conducting the incubation cycle two or more times to form the incubation mixture into the detection mixture comprising the amplified portion of misfolded \u03b1S protein, each incubation cycle comprising:
incubating the incubation mixture effective to cause misfolding andor aggregation of at least the portion of the monomeric, folded \u03b1S protein in the presence of the soluble, misfolded \u03b1S protein;
shaking the incubation mixture effective to at least partly de-aggregate at least the portion of the misfolded \u03b1S aggregate present; and
determining the presence of the soluble, misfolded \u03b1S protein in the sample by detecting a fluorescence of the Thioflavin T corresponding to the soluble, misfolded \u03b1S protein, the soluble, misfolded \u03b1S protein comprising the soluble, misfolded \u03b1S protein and the amplified portion of misfolded \u03b1S protein.
16. A method for determining a presence of a soluble, misfolded \u03b1S protein in a sample, comprising:
capturing a soluble, misfolded \u03b1S protein from the sample to form a captured soluble, misfolded \u03b1S protein;
contacting the captured soluble, misfolded \u03b1S protein with a molar excess of monomeric, folded \u03b1S protein to form an incubation mixture, the molar excess being greater than an amount of \u03b1S protein monomer included in the captured soluble, misfolded \u03b1S protein;
conducting an incubation cycle two or more times on the incubation mixture effective to form an amplified portion of misfolded \u03b1S protein from the monomeric, folded \u03b1S protein, each incubation cycle comprising:
incubating the incubation mixture effective to cause misfolding andor aggregation of at least a portion of the monomeric, folded \u03b1S protein in the presence of the captured soluble, misfolded \u03b1S protein;
physically disrupting the incubation mixture effective to at least partly de-aggregate at least a portion of a misfolded \u03b1S aggregate present; and
determining the presence of the soluble, misfolded \u03b1S protein in the sample by detecting at least a portion of the soluble, misfolded \u03b1S protein,
the soluble, misfolded \u03b1S protein comprising one or more of: a soluble, misfolded \u03b1S monomer and a soluble, misfolded \u03b1S aggregate;
the captured, soluble, misfolded \u03b1S protein comprising one or more of: a captured, soluble, misfolded \u03b1S monomer and a captured, soluble, misfolded \u03b1S aggregate; and
the amplified portion of misfolded \u03b1S protein comprising one or more of: an amplified portion of the soluble, misfolded \u03b1S monomer, an amplified portion of the soluble, misfolded \u03b1S aggregate, and an insoluble, misfolded \u03b1S aggregate.
17. The method of claim 16, the capturing comprising selectively concentrating the soluble, misfolded \u03b1S protein in one or more of the sample, the incubation mixture, and the detection mixture.
18. The method of claim 17, the selectively concentrating the soluble, misfolded \u03b1S protein comprising one or more of: pre-treating the sample prior to forming the incubation mixture; pre-treating the incubation mixture prior to incubating the incubation mixture; and contacting one or more \u03b1S specific antibodies to the soluble, misfolded \u03b1S protein to form a captured soluble, misfolded \u03b1S protein.
19. The method of claim 18, the one or more \u03b1S specific antibodies comprising one or more of: \u03b1\u03b2-synuclein N-19; \u03b1-synuclein C-20-R; \u03b1-synuclein 211; \u03b1-synuclein Syn 204; \u03b1-synuclein 2B2D1; \u03b1-synuclein LB 509; \u03b1-synuclein SPM451; \u03b1-synuclein 3G282; \u03b1-synuclein 3H2897; \u03b1\u03b2-synuclein Syn 202; \u03b1\u03b2-synuclein 3B6; \u03b1\u03b2\u03b3-synuclein FL-140; an antibody specific for an amino acid sequence of \u03b1S; and an antibody specific for a conformation of the soluble, misfolded \u03b1S protein.
20. The method of claim 19, the one or more \u03b1S specific antibodies being coupled to a solid phase.
21. The method of claim 20, the solid phase comprising one or more of a magnetic bead and a multiwell plate.
22. The method of claim 16, the sample being taken from a subject, the subject being treated with one or more of:
an \u03b1S modulating therapy, further comprising:
comparing the amount of the soluble, misfolded \u03b1S protein in the sample to an amount of the soluble, misfolded \u03b1S protein in a comparison sample, the sample and the comparison sample being taken from the subject at different times over a period of time under the \u03b1S modulating therapy; and
determining the subject is one of: responsive to the \u03b1S modulating therapy according to a change in the soluble, misfolded \u03b1S protein over the period of time, and non-responsive to the \u03b1S modulating therapy according to homeostasis of the soluble, misfolded \u03b1S protein over the period of time; and
a disease-modifying therapy for PD, further comprising:
comparing the amount of the soluble, misfolded \u03b1S protein in the sample to an amount of the soluble, misfolded \u03b1S protein in a comparison sample, the sample and the comparison sample being taken from the subject at different times over a period of time under the disease-modifying therapy for PD; and
determining the subject is one of: responsive to the disease-modifying therapy for PD according to a change in the soluble, misfolded \u03b1S protein over the period of time, and non-responsive to the disease-modifying therapy for PD according to homeostasis of the soluble, misfolded \u03b1S protein over the period of time.
23. The method of claim 1, the sample being taken from a subject, further comprising determining or diagnosing one or more of:
the presence of PD in the subject according to the presence of the soluble, misfolded \u03b1S protein in the sample;
the presence of a disease associated with alpha-synuclein homeostasis in the subject according to the presence of the soluble, misfolded \u03b1S protein in the sample;
the presence of Multiple System Atrophy in the subject according to the presence of the soluble, misfolded \u03b1S protein in the sample;
the presence of PD in the subject according to the presence of the soluble, misfolded \u03b1S protein in the sample compared to a control sample taken from a control subject;
the presence of PD in the subject by comparing an amount of the soluble, misfolded \u03b1S protein in the sample to a predetermined threshold amount, the detecting comprising detecting the amount of the soluble, misfolded \u03b1S protein in the sample.
the presence of PD in the subject according to the presence of the soluble, misfolded \u03b1S protein in the sample, the subject exhibiting no clinical signs of dementia according to cognitive testing;
the presence of PD as a contributing factor to one or more clinical signs of dementia in the subject according to the presence of the soluble, misfolded \u03b1S protein in the sample, the subject exhibiting the one or more clinical signs of dementia according to cognitive testing; and
a progression or homeostasis of PD in the subject by comparing the amount of the soluble, misfolded \u03b1S protein in the sample to an amount of the soluble, misfolded \u03b1S protein in a comparison sample taken from the subject at a different time compared to the sample.
24. A kit for determining a presence of a soluble, misfolded \u03b1S protein in a sample, comprising:
one or more of a known amount of a monomeric, folded \u03b1S protein and a known amount of an indicator of misfolded \u03b1S protein;
instructions, the instructions directing a user to:
contact the sample with one or more of the known amount of the monomeric, folded \u03b1S protein and the known amount of the indicator of misfolded \u03b1S protein to form an incubation mixture;
repeat an incubation cycle two or more times to form the incubation mixture into a detection mixture, each incubation cycle comprising:
incubating the incubation mixture effective to cause misfolding andor aggregation of at least a portion of the monomeric, folded \u03b1S protein in the presence of the soluble, misfolded \u03b1S protein to form an amplified portion of misfolded \u03b1S protein;
physically disrupting the incubation mixture effective to at least partly de-aggregate at least a portion of a misfolded \u03b1S aggregate present; and
determine the presence of the soluble, misfolded \u03b1S protein in the sample by detecting the soluble, misfolded \u03b1S protein,
the soluble, misfolded \u03b1S protein comprising one or more of: a soluble, misfolded \u03b1S monomer and a soluble, misfolded \u03b1S aggregate; and
the amplified portion of misfolded \u03b1S protein comprising one or more of: an amplified portion of the soluble, misfolded \u03b1S monomer, an amplified portion of the soluble, misfolded \u03b1S aggregate, and an insoluble, misfolded \u03b1S aggregate.
25. The kit of claim 24, further comprising one or more of:
a multiwell microtitre plate;
a microfluidic plate;
a shaking apparatus;
an incubating apparatus;
a buffer composition comprising one or more of: Tris-HCL, PBS, MES, PIPES, MOPS, BES, TES, and HEPES; the instructions further comprising directions to the user to prepare the incubation mixture comprising the buffer composition at a total concentration of between about 1 \u03bcm and about 1M; and
a salt composition, the instructions further comprising directions to the user to prepare the incubation mixture comprising the salt composition at a total concentration of between about 1 jam and about 500 mM.
26. The kit of claim 24, the indicator of misfolded \u03b1S protein comprising one or more of: Thioflavin T, Congo Red, m-I-Stilbene, Chrysamine G, PIB, BF-227, X-34, TZDM, FDDNP, MeO-X-04, IMPY, NIAD-4, luminescent conjugated polythiophenes, a fluorescent protein, and derivatives thereof.
27. The kit of claim 24, the instructions directing the user to detect the soluble, misfolded \u03b1S protein comprising directions to conduct one or more of: a Western Blot assay, a dot blot assay, an enzyme-linked immunosorbent assay (ELISA), a thioflavin T binding assay, a Congo Red binding assay, a sedimentation assay, electron microscopy, atomic force microscopy, surface plasmon resonance, and spectroscopy.
28. The kit of claim 24, the monomeric, folded \u03b1S protein comprising one or more of a covalently incorporated radioactive amino acid, a covalently incorporated, isotopically labeled amino acid, and a covalently incorporated fluorophore.
29. The kit of claim 24, the instructions further comprising directing the user to determine or diagnose, in a subject corresponding to the sample, one or more of:
the presence of PD in the subject according to the presence of the soluble, misfolded \u03b1S protein in the sample;
the presence of a disease associated with alpha-synuclein homeostasis in the subject according to the presence of the soluble, misfolded \u03b1S protein in the sample;
the presence of Multiple System Atrophy in the subject according to the presence of the soluble, misfolded \u03b1S protein in the sample;
the presence of PD in the subject according to the presence of the soluble, misfolded \u03b1S protein in the sample compared to a control sample taken from a control subject;
the presence of PD in the subject by comparing an amount of the soluble, misfolded \u03b1S protein in the sample to a predetermined threshold amount, the detecting comprising detecting the amount of the soluble, misfolded \u03b1S protein in the sample.
the presence of PD in the subject according to the presence of the soluble, misfolded \u03b1S protein in the sample, the subject exhibiting no clinical signs of dementia according to cognitive testing;
the presence of PD as a contributing factor to one or more clinical signs of dementia in the subject according to the presence of the soluble, misfolded \u03b1S protein in the sample, the subject exhibiting the one or more clinical signs of dementia according to cognitive testing; and
a progression or homeostasis of PD in the subject by comparing the amount of the soluble, misfolded \u03b1S protein in the sample to an amount of the soluble, misfolded \u03b1S protein in a comparison sample taken from the subject at a different time compared to the sample.
30. The kit of claim 24,
further comprising one or more \u03b1S specific antibodies, the one or more \u03b1S specific antibodies comprising one or more of: an antibody specific for an amino acid sequence of \u03b1S, an antibody specific for a conformation of the soluble, misfolded \u03b1S protein, \u03b1\u03b2-synuclein N-19; \u03b1-synuclein C-20-R; \u03b1-synuclein 211; \u03b1-synuclein Syn 204; \u03b1-synuclein 2B2D1; \u03b1-synuclein LB 509; \u03b1-synuclein SPM451; \u03b1-synuclein 3G282; \u03b1-synuclein 3H2897; \u03b1\u03b2-synuclein Syn 202; \u03b1\u03b2-synuclein 3B6; and \u03b1\u03b2\u03b3-synuclein FL-140; and
the instructions further comprising directing the user to selectively concentrate the soluble, misfolded \u03b1S protein by contacting the one or more \u03b1S specific antibodies to the soluble, misfolded \u03b1S protein to form a captured soluble, misfolded \u03b1S protein.
31. The kit of claim 30, comprising the one or more \u03b1S specific antibodies coupled to a solid phase.
32. The kit of claim 24, the instructions for physically disrupting the incubation mixture comprising directions to a user to employ one or more of: sonication, stirring, shaking, freezingthawing, laser irradiation, autoclave incubation, high pressure, homogenization, and cyclic agitation.
The claims below are in addition to those above.
All refrences to claim(s) which appear below refer to the numbering after this setence.
1. A device for the treatment of a damaged nerve comprising:
a solid, non-permeable tubular membrane having an outer surface and an inner surface; and
a textured surface on the outer surface and the inner surface of the solid tubular membrane, the textured surface including a plurality of pores having diameters between 1\u20133 \u03bcm and depths of at least 0.5 \u03bcm
whereby the device facilitates regeneration of the damaged nerve by diverting fibrous tissue overgrowth into the plurality of pores.
2. The device of claim 1 wherein the textured surface further includes a plurality of channels having widths between 1\u20133 \u03bcm and depths of at least 0.5 \u03bcm whereby the channels provide a guide for axons as the axons elongate during the nerve regeneration process.
3. The device of claim 2 wherein the tubular membrane comprises an inner surface and the textured surface is further provided on the inner surface of the tubular membrane.
4. The device of claim 1 wherein a fibrin matrix is embedded on the textured surface of the tubular membrane.
5. The device of claim 4 wherein the fibrin matrix contains a neurotrophic agent.
6. The device of claim 4 wherein the fibrin matrix contains an anti-inflammatory agent.
7. The device of claim 4 wherein the fibrin matrix contains an anti-bacterial agent.
8. A device for the treatment of a damaged nerve comprising a solid, non-permeable tubular titanium membrane having an inner surface and an outer surface, the inner surface and outer surface having a textured topography formed from a plurality of pores and a plurality of channels, the tubular membrane having a first end and a second end for receiving ends of a severed nerve, whereby the tubular titanium membrane facilitates regeneration of the damaged nerve by diverting fibrous tissue overgrowth into the plurality of pores.
9. The device of claim 8 wherein the pores have diameters between 1\u20133 \u03bcm and depths of at least 0.5 \u03bcm and the channels have widths between 1\u20133 \u03bcm and depths of at least 0.5 \u03bcm, whereby the channels provide a guide for axons as the axons elongate during the nerve regeneration process.
10. A tubular membrane for the treatment of a damaged nerve comprising:
a solid, non-permeable substrate formed of polymeric material; and
a metallized coating on the surface of the polymeric substrate, the metallized coating including a plurality of pores with diameters between 1\u20133 \u03bcm and depths of at least 0.5 \u03bcm
whereby the tubular membrane facilitates regeneration of the damaged nerve by diverting fibrous tissue overgrowth into the plurality of pores.
11. The tubular membrane of claim 10 wherein the metallized coating further includes a plurality of channels with widths between 1\u20133 \u03bcm and depths of at least 0.5 \u03bcm, whereby the channels provide a guide for axons as the axons elongate during the nerve regeneration process.
12. The tubular membrane of claim 11 further comprising a fibrin matrix embedded in the metallized coating.
13. The tubular membrane of claim 12 wherein the fibrin matrix contains a neurotrophic agent.
14. The device of claim 12 wherein the fibrin matrix contains an anti-inflammatory agent.
15. The device of claim 12 wherein the fibrin matrix contains an anti-bacterial agent.
16. A method of treating a damaged nerve comprising the steps of:
providing a solid, non-permeable tubular membrane with a textured surface on an outer and an inner surface, the textured surface comprises a plurality of pores having diameters between 1\u20133 \u03bcm and depths of at least 0.5 \u03bcm; and
placing a damaged nerve within the tubular membrane, whereby the tubular membrane facilitates regeneration of the damaged nerve by diverting fibrous tissue overgrowth into the plurality of pores.
17. The method of claim 16 wherein the textured surface on the inner surface further comprises a plurality of channels having widths between 1\u20133 \u03bcm and depths of at least 0.5 \u03bcm, whereby the channels provide a guide for axons as the axons elongate during the nerve regeneration process.