WO2024089232A1 - Alpha-synuclein detection assay comprising zinc ions in the reaction mixture and method for diagnosing alpha-synucleinopathies - Google Patents
Alpha-synuclein detection assay comprising zinc ions in the reaction mixture and method for diagnosing alpha-synucleinopathies Download PDFInfo
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- WO2024089232A1 WO2024089232A1 PCT/EP2023/080042 EP2023080042W WO2024089232A1 WO 2024089232 A1 WO2024089232 A1 WO 2024089232A1 EP 2023080042 W EP2023080042 W EP 2023080042W WO 2024089232 A1 WO2024089232 A1 WO 2024089232A1
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- Prior art keywords
- syn
- misfolded
- biological sample
- incubation mixture
- monomers
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
- G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/28—Neurological disorders
- G01N2800/2835—Movement disorders, e.g. Parkinson, Huntington, Tourette
Definitions
- the present invention relates to a method for detecting misfolded alpha- synuclein (a-syn) in a biological sample.
- the invention further relates to a kit of parts for detecting misfolded a-syn species in a biological sample.
- a-syn is a 140 amino acid long protein that is particularly abundant in the presynaptic nerve terminals in the central nervous system, a-syn is intrinsically disordered and exists in various forms. Under normal physiological conditions, a-syn is an unfolded soluble monomeric protein, whereas under pathological conditions (in synucleinopathies) a-syn undergoes misfolding and aggregates into oligomers, amyloid fibrils and eventually into Lewy bodies.
- Accumulation of aggregates comprising misfolded a-syn is associated with neurodegenerative diseases, such as Parkinson’s disease, dementia with Lewy bodies and multiple system atrophy.
- neurodegenerative diseases such as Parkinson’s disease, dementia with Lewy bodies and multiple system atrophy.
- Parkinson’s disease dementia with Lewy bodies and multiple system atrophy.
- the above neurodegenerative diseases are incurable and highly prevalent.
- Methods for detecting a presence or an absence of misfolded a-syn in a biological sample comprise seed amplification assays (SAAs), including real-time quaking-induced conversion (RT-QuIC) and protein misfolding cyclic amplification (PMCA).
- SAAs seed amplification assays
- RT-QuIC real-time quaking-induced conversion
- PMCA protein misfolding cyclic amplification
- WO 2018/007817 relates to a method of detecting the presence of a-syn aggregation in a biological sample, wherein the biological sample is mixed with a reaction sample comprising a population of beads, a fluorophore adapted to bind to protein aggregates and to increase fluorescence when bound to protein aggregates, and a-syn or a fragment or variant thereof to form a reaction mixture, the reaction mixture is illuminated and at the same time incubated with intermittent agitation cycles, wherein a significant increase in the fluorescence of the reaction mixture during incubation is indicative of the presence of aggregates of a-syn in the biological sample.
- a problem with known in the art methods for detecting a presence or an absence of misfolded a-syn in a biological sample is that the above methods are known for accounting for false positive results due to occurrence of spontaneous a- syn aggregation events.
- Another drawback of many methods known in the art is that they take a long time to perform, e. g. some methods may take as long as 5-12 days. Therefore, there is a need for improved biochemical tests for detecting a presence or an absence of misfolded a-syn in a biological sample.
- Said invention alleviates at least part of the above-discussed problems and at least partially addresses one or more of the above-mentioned needs.
- an ex vivo method for detecting a presence or an absence of misfolded a-syn in a biological sample, more precisely the ex vivo method comprising the steps of a) providing monomers of a-syn, a biological sample, the biological sample being suspected to contain misfolded a-syn, and a zinc ion carrier, b) preparing an incubation mixture comprising at least monomers of a-syn, a biological sample, the biological sample being suspected to contain misfolded a-syn, and a zinc ion carrier, c) incubating the incubation mixture, d) subjecting the incubation mixture to a process for physical disruption, e) repeating steps c)-d) one or more times, f) detecting a presence or an absence of aggregates comprising misfolded a-syn.
- a use of the zinc ion carrier in an ex vivo method for preventing or reducing an inhibitory effect of a biological matrix of a biological sample on a-syn aggregation, more precisely in the ex vivo method comprising steps of a) providing monomers of a-syn, a biological sample, the biological sample being suspected to contain misfolded a-syn, and a zinc ion carrier, b) preparing an incubation mixture comprising at least monomers of a-syn, a biological sample, the biological sample being suspected to contain misfolded a-syn, and a zinc ion carrier, c) incubating the incubation mixture, d) subjecting the incubation mixture to a process for physical disruption, e) repeating steps c)-d) one or more times, f) detecting a presence or an absence of aggregates comprising misfolded a-syn.
- an ex vivo method for diagnosing synucleinopathy, the ex vivo method comprising the steps of a) providing monomers of a-syn, a biological sample, the biological sample being suspected to contain misfolded a-syn, and a zinc ion carrier, b) preparing an incubation mixture comprising at least monomers of a-syn, a biological sample, the biological sample being suspected to contain misfolded a-syn, and a zinc ion carrier, c) incubating the incubation mixture, d) subjecting the incubation mixture to a process for physical disruption, e) repeating steps c)-d) one or more times, f) detecting a presence or an absence of aggregates comprising misfolded a-syn.
- an ex vivo method for prognosing synucleinopathy, the ex vivo method comprising the steps of a) providing monomers of a-syn, a biological sample, the biological sample being suspected to contain misfolded a-syn, and a zinc ion carrier, b) preparing an incubation mixture comprising at least monomers of a-syn, a biological sample, the biological sample being suspected to contain misfolded a-syn, and a zinc ion carrier, c) incubating the incubation mixture, d) subjecting the incubation mixture to a process for physical disruption, e) repeating steps c)-d) one or more times, f) detecting a presence or an absence of aggregates comprising misfolded a-syn.
- kits of parts comprising monomers of a-syn, a zinc ion carrier and instructions for detecting a presence of misfolded a-syn species in a biological sample is provided.
- Fig. 1 schematically illustrates an overview of a method for detecting a presence or an absence of misfolded a-syn in a biological sample.
- Fig. 2 schematically illustrates an overview of a method for detecting a presence or an absence of misfolded a-syn in a biological sample, wherein the method being part of a real-time quaking-induced conversion (RT-QuIC).
- RT-QuIC real-time quaking-induced conversion
- Fig. 3A diagrammatically illustrates a method for detecting a presence or an absence of misfolded a-syn in a biological sample comprising cerebrospinal fluid (CSF) under normal physiological (NC) conditions and Parkinson’s disease (PD) conditions, respectively, wherein 2.5 mM ZnSO4 is comprised within the incubation mixture, and wherein the method is a part of a RT-QuIC assay.
- CSF cerebrospinal fluid
- NC normal physiological
- PD Parkinson’s disease
- Fig. 3B diagrammatically illustrates a method for detecting a presence or an absence of misfolded a-syn in a biological sample comprising CSF under NC and PD conditions, respectively, wherein 5 mM ZnSO4 is comprised within the incubation mixture, and wherein the method is a part of a RT-QuIC assay.
- Fig. 4A diagrammatically illustrates a method for detecting a presence or an absence of misfolded a-syn in a biological sample comprising CSF under NC and PD conditions, respectively, wherein 0 mM ZnSO4 is comprised within the incubation mixture, and wherein the method is being part of a RT-QuIC assay.
- Fig. 4B diagrammatically illustrates a method for detecting a presence or an absence of misfolded a-syn in a biological sample comprising CSF under NC and PD conditions, respectively, wherein 5 mM ZnSO4 is comprised within the incubation mixture, and wherein the method is being part of RT-QuIC assay.
- a protein includes one or more proteins
- a zinc ion carrier includes one or more zinc ion carriers
- an inhibitory effect includes one or more inhibitory effects and the like.
- biological sample is understood within the scope of the invention to refer to any biological material obtained from a living or diseased animal, including a mammal; the biological material is suitable for being employed in the method for detecting a presence of misfolded a-syn.
- animal includes representatives of the kingdom Animalia, such as a worm, a mouse, a hamster, a monkey, a human.
- mammal includes humans.
- a-syn each of the terms “a-syn”, ’’recombinant a-syn”, ’’misfolded a-syn”, “soluble misfolded a-syn”, “non-monomeric misfolded a-syn” and the like is understood within the scope of the invention to refer to the population of proteins designated by the term as a whole.
- a-syn include any a-syn protein irrespectively of the length, conformation and post-translational modification thereof.
- a-syn is understood within the scope of the invention to refer to any a-syn protein population comprising at least one of a full-length a-syn and a truncated a-syn protein.
- the term “a-syn” includes any a-syn protein population comprising at least one of a wild-type a-syn protein and a mutated a-syn protein.
- the wild-type a- syn protein may be a recombinant protein.
- the mutated a-syn protein may be a recombinant protein.
- a-syn is understood within the scope of the invention to refer to soluble monomers of a-syn capable of assembly into aggregates.
- recombinant a-syn is understood within the scope of the invention to refer to any a-syn produced using gene technology, as opposed to wild-type a-syn, i. e. a-syn obtained by isolation from a biological source which natively produces a- syn.
- a-syn aggregation is understood within the scope of the invention to refer to aggregation of monomers of a-syn in the presence of soluble misfolded a- syn to form at least one of a-syn oligomers, a-syn amyloid fibrils and Lewy bodies.
- spontaneous a-syn aggregation is understood within the scope of the invention to refer to aggregation of monomers of a-syn in the absence of soluble misfolded a-syn to form at least one of a-syn oligomers, a-syn amyloid fibrils and Lewy bodies.
- misfolded a-syn is understood within the scope of the invention to refer to misfolded a-syn as a whole.
- the “non-monomeric misfolded a-syn” may include at least one of a-syn oligomers, a-syn amyloid fibrils and Lewy bodies.
- misfolded a-syn species is understood within the scope of the invention to refer to non-monomeric a-syn species formed due to misfolding of at least a portion of the monomers of a-syn in the presence of soluble misfolded a-syn.
- the soluble misfolded a-syn may be present in the biological sample provided at the step a) in the method of the invention.
- the soluble misfolded a-syn may be formed in the course of physical disruption of at least a portion of the aggregates comprising misfolded a-syn generated at the step d) in the method of the invention.
- aggregates comprising misfolded a-syn is understood within the scope of the invention to refer to the aggregates comprising a-syn which may be generated at the step d) of the method of the invention; the aggregates may be assembled from at least one of at least a portion of the monomers of a-syn, at least a portion of the misfolded a-syn species and at least a portion of the misfolded a-syn.
- the present invention relates to an ex vivo method for detecting a presence or an absence of misfolded alpha-synuclein (a-syn) in a biological sample, the method comprising the steps of a. providing monomers of a-syn, a biological sample suspected of comprising misfolded a-syn and a zinc ion carrier, b. preparing an incubation mixture from the monomers of a-syn, biological sample and zinc ion carrier provided in step a, c. incubating the incubation mixture prepared in step b, d. subjecting the mixture incubated according to step c to a process for physical disruption, e. repeating steps c)-d) one or more times, f. detecting a presence or an absence of aggregates comprising misfolded a-syn.
- a-syn alpha-synuclein
- an ex vivo method for detecting a presence or an absence of misfolded a-syn in a biological sample, more precisely the method comprising the steps of a) providing monomers of a-syn, a biological sample, the biological sample being suspected to contain misfolded a-syn, and a zinc ion carrier, b) preparing an incubation mixture comprising at least the monomers of a- syn, the biological sample and the zinc ion carrier, c) incubating the incubation mixture, d) subjecting the incubation mixture to a process for physical disruption, e) repeating steps c)-d) one or more times, f) detecting a presence or an absence of aggregates comprising misfolded a-syn.
- the present invention is not intended for being carried out on a human or animal body.
- the invention does not relate to a method for treatment of the human or animal body by surgery or therapy and diagnostic methods practised on the human or animal body.
- the method of the invention is an ex vivo method, i. e. a method performed entirely extracorporeal ly, outside of the human or animal body.
- the method may further comprise the step c') detecting a presence or an absence of aggregates comprising misfolded a-syn in the incubation mixture, wherein c') follows c) and precedes d).
- the wording “repeating steps c)-d)” includes repeating step c), c') and step d”.
- the incubating may be effective in the presence of soluble misfolded a-syn to cause conversion of at least a portion of the monomers of a-syn into misfolded a-syn species and subsequent recruitment of at least a portion of the misfolded a-syn species into aggregates comprising misfolded a-syn.
- the incubating the incubation mixture may include incubating at a temperature from about 30 °C to about 42 °C, including 37 °C.
- the physical disruption may be effective to cause breaking of aggregates comprising misfolded a-syn and providing soluble misfolded a-syn.
- the monomers of a-syn, the biological sample and the zinc ion carrier may be in a buffer solution.
- the buffer solution may be any buffer solution as long as the zinc carrier is substantially soluble therein.
- the buffer solution may have a pH value from about 4 to about 8.5, from about 5 to about 6.5, from about 5.5 to about 6.0.
- the buffer solution may have various compositions.
- the buffer solutions may contain at least one of MES, sodium phosphate, ammonium acetate, HEPES and Tris-CI.
- the buffer solution may contain MES at a final concentration of between from about 15 mM to about 500 mM, from about 15 mM to about 300 mM, from about 25 mM to about 200 mM, from about 50 mM to about 100 mM.
- the buffer solution may contain phosphate at a final concentration of between from about 15 mM to about 300 mM, from about 25 mM to about 200 mM, from about 50 mM to about 100 mM.
- the composition of the buffer solution is not limited to the above examples, and it may be chosen in such a way that its ionic strength is similar to that of the above-mentioned buffer solutions.
- the zinc ion carrier may be any zinc salt as long as it remains soluble under assay conditions and that the anion does not interfere with the assay.
- the zinc ion carrier may be at least one of zinc sulphate, zinc chloride and zinc nitrate.
- the zinc ions may be present at a concentration sufficient for counteracting the inhibitory effect of the matrix of the biological sample on the aggregation of a-syn without provoking spontaneous aggregation of a-syn in the absence of misfolded a- syn in the biological sample.
- the more zinc ions are added the more inhibition may be counter-acted.
- spontaneous aggregation of a-syn may be provoked.
- the above spontaneous aggregation of a-syn in the absence of misfolded a-syn being present in the biological sample may account for false positive results.
- lower or higher zinc ion concentrations may be chosen depending on the inhibitory effect of the matrix.
- the zinc ions may be present at a concentration from 0.5 mM to about 100 mM, from about 0.5 mM to about 50 mM, from about 0.5 mM to about 20 mM, from about 2 mM to about 10 mM, from about 2.5 mM to about 5 mM. It is appreciated that a person skilled in the art will be able to choose a suitable concentration of the zinc ions in view of e. g. the strength of the inhibitory effect of the matrix of the biological sample on the aggregation process. A matrix with a lower inhibitory effect on the aggregation of a-syn may require less zinc ions to be used in the method of the invention.
- a matrix with a higher inhibitory effect on the aggregation of a-syn may require more zinc ions to be used in the method of the invention.
- a lower amount of the biological sample, and hence less amount of matrix thereof, may require less zinc ions to be used in the method of the invention.
- the zinc ion concentration may be adjusted in relation to the amounts of other constituents of the incubation mixture including the biological sample and the monomers of a-syn provided.
- the concentration of zinc ions in the incubation mixture may need to be decreased; if the amount of the biological sample is increased, the concentration of zinc ions in the incubation mixture may need to be increased.
- the method of the invention may optionally comprise a step of treating the biological sample prior to preparing the incubation mixture.
- the simplifying the biological sample matrix may allow to increase the concentration of misfolded a-syn in the biological sample.
- the biological sample may be treated in various ways, e. g. by precipitating and subsequently removing at least a portion of the proteins contained therein. Further, other components of the sample matrix may be removed, such as lipids, salts etc.
- the incubation mixture may further comprise an indicator for detecting aggregates comprising misfolded a-syn therein.
- the indicator for detecting the above aggregates may be a fluorophore that is adapted to bind the aggregates and to increase fluorescence when bound to the aggregates.
- a suitable fluorophore may be selected in view of e. g. the structure of a- syn fibrils formed, the structure thereof being dependent on e. g. the amount of misfolded a-syn in the biological sample and the disease background (e. g. Parkinson’s disease, dementia with Lewy bodies or multiple system atrophy).
- Each fluorophore may be expected to have a preference for one or more selected from a group consisting of a-syn oligomers, a-syn amyloid fibrils and Lewy bodies.
- the fluorophore may be at least one of thioflavin T, the derivate of Congo Red X-34, Curcumin, the derivative of Curcumin CRANAD-3, Nile Red, pFTAA and the like.
- the method according to the first aspect of the invention may further comprise bringing into contact the incubation mixture and an indicator for detecting aggregates comprising misfolded a-syn prior to the incubating the incubation mixture.
- the detecting a presence or an absence of aggregates comprising misfolded a-syn may be conducted by at least one selected from the group consisting of fluorescence assay, immunoassay (such as ELISA and the like), absorbance assay, electron microscopy, size exclusion chromatography, western blot and dot blot.
- the process for physical disruption may include at least one of shaking, homogenisation, stirring, sonication and the like.
- An a-syn protein is normally divided into mainly three regions: the N-terminus (amino acids 1-65 of an a-syn, with reference to wild-type human a-syn) that forms an a-helix upon binding to membranes and where most of the Parkinson’s disease mutations may be located, the NAC core (amino acids 66-95 of an a-syn, with reference to wild-type human a-syn), and the C-terminus (amino acids 96-140 of an a-syn, with reference to wild-type human a-syn) that contains most of post- translational modifications and critical for aggregation as well as important for the chaperone activity of the a-syn protein.
- the a-syn may be a wild-type protein or a mutated protein; the mutated protein may include point mutations that affect the protein aggregation ability compared to that of the wild-type protein.
- An example of such mutated a-syn protein is K23Q.
- the monomers of a-syn may comprise monomers of at least one of a full- length a-syn and a truncated a-syn, advantageously a full-length a-syn.
- the monomers of a-syn may comprise monomers of at least one of a full-length human a- syn or a part thereof comprising at least a region containing amino acids 10-130 (with reference to wild-type human a-syn), a region containing amino acids 10-100 (with reference to wild-type human a-syn), a region containing amino acids 10-95 (with reference to wild-type human a-syn) or a sequence showing at least 50 % identity, at least 60 % identity, at least 70 % identity, at least 80 % identity, at least 90 % identity, at least 95 % identity, or at least 99 % identity to any said region.
- the final concentration of the provided monomers of a-syn in the buffer solution may be from about 0.05 to about 1 mg/ml, from about 0.05 to about 0.5 mg/ml, from about 0.1 to about 0.3 mg/ml, including 0.1 mg/ml.
- Incubation mixtures of various volumes may be used. The volume of the incubation mixture may be adjusted depending on the consumable used for holding the incubation mixture, such as the type of a plate. For a 384-well plate the volume of the incubation mixture can be of from about 25 pl to about 100 pl. For a 96-plate of the incubation mixture can be of from about 50 pl to about 400 pl.
- the biological sample may comprise at least one of cerebrospinal fluid (CSF), blood, eye liquid, tears, brain tissue, olfactory mucosa, saliva, nasopharyngeal swab fluid, urine, intestinal fluid, skin, stem cells, faeces and a liquid preparation thereof.
- CSF cerebrospinal fluid
- the blood may comprise at least one of plasma, serum, red blood cells, white blood cells, blood platelets, exosomes and the like.
- the biological sample may be subjected to at least one of processing, concentrating and diluting in any conventional way.
- the incubation mixture may further contain one or more beads.
- the beads can be made of any suitable material, e. g. silica, glass and zirconium.
- the bead diameter may be from about 0.2 mm to about 1 mm, from about 0.4 mm to about 0,8 mm, or from about 0.5 mm to about 0,6 mm. Distinctly larger beads, e. g. 3 mm or greater in diameter, are not suitable for use in the method according to the invention.
- the person skilled in the art realises that the number and diameter of the beads could be chosen in such a way that the reaction becomes sufficiently facilitated (cf. the reaction when no beads are used) while the fluorescent measurement remains essentially unhindered (cf. the reaction when no beads are used).
- the method may be a part of a seed amplification assay.
- the principles of the seed amplification assays are well known to the person skilled in the art, and equipment, conditions and the like may easily be adapted to each relevant situation.
- the seed amplification assay may be one selected from the group consisting of PMCA and RT-QulC.
- a use of the zinc ion carrier for preventing or reducing an inhibitory effect of a biological matrix of a biological sample on a-syn aggregation in an ex vivo method comprising steps of a) providing monomers of a-syn, a biological sample, the biological sample being suspected to contain misfolded a-syn, and a zinc ion carrier, b) preparing an incubation mixture comprising at least the monomers of a- syn, the biological sample and the zinc ion carrier, c) incubating the incubation mixture, d) subjecting the incubation mixture to a process for physical disruption, e) repeating steps c)-d) one or more times, f) detecting a presence or an absence of aggregates comprising misfolded a-syn.
- the biological sample may comprise at least one of cerebrospinal fluid (CSF), blood, eye liquid, tears, brain tissue, olfactory mucosa, saliva, nasopharyngeal swab fluid, urine, intestinal fluid, skin, stem cells, faeces and a liquid preparation thereof.
- the blood may comprise at least one of plasma, serum, red blood cells, white blood cells, blood platelets, exosomes and the like.
- the biological matrix of the biological sample may inhibit the a-syn seeding reaction, wherein small a-syn oligomers provide a template for the assembly of soluble monomers of a-syn into aggregates comprising misfolded a-syn.
- an ex vivo method for diagnosing synucleinopathy, the ex vivo method comprising the steps of a) providing monomers of a-syn, a biological sample, the biological sample being suspected to contain misfolded a-syn, and a zinc ion carrier, b) preparing an incubation mixture comprising at least the monomers of a- syn, the biological sample and the zinc ion carrier, c) incubating the incubation mixture, d) subjecting the incubation mixture to a process for physical disruption, e) repeating steps c)-d) one or more times, f) detecting a presence or an absence of aggregates comprising misfolded a-syn.
- the synucleinopathy may comprise at least one of Parkinson’s disease, dementia with Lewy bodies and multiple system atrophy.
- the method may be used for diagnosing synucleinopathy in an animal, including a mammal, including a human.
- a use of the method discussed in the foregoing comprising the steps of a) providing monomers of a-syn, a biological sample, the biological sample being suspected to contain misfolded a-syn, and a zinc ion carrier, b) preparing an incubation mixture comprising at least the monomers of a- syn, the biological sample and the zinc ion carrier, c) incubating the incubation mixture, d) subjecting the incubation mixture to a process for physical disruption, e) repeating steps c)-d) one or more times, f) detecting a presence or an absence of aggregates comprising misfolded a-syn.
- the synucleinopathy may be at least one of Parkinson’s disease, dementia with Lewy bodies and multiple system atrophy.
- the method may be used for prognosing synucleinopathy in an animal, including a mammal, including a human.
- kits of parts comprising at least monomers of a-syn, a zinc ion carrier and instructions for detecting a presence of misfolded a-syn species in a biological sample is provided.
- the kit of parts may additionally include at least one selected from a group comprising a buffer solution, a size exclusion filter, beads and a plate.
- the size exclusion filter may be a filter that allows each molecule of a molecular weight of less than 150 kDa, less than 120 kDa, less than 100 kDa, less than 80 kDa, less than 75 kDa, less than 70 kDa, less than 65 kDa or less than 60 kDa or less than 50kDa to pass through the filter while preventing each molecule of a molecular weight that is greater than said molecular weight from passing through the filter.
- the plate may be of any suitable size, such as a 96-well plate or a 384-well plate.
- the method for detecting a presence or an absence of misfolded a-syn in a biological sample is a part of a RT-QuIC (Fig. 2).
- RT- QuIC is a cyclic process. Each cycle lasts for approximately four minutes (a 3 min elongation and a 1 min shaking). The cycle is continuously repeated for 48 hours. At the end of every fourth cycle fluorescence is measured with fluorescence intensity being proportional to the amount of generated aggregates.
- aggregation-prone misfolded a-syn seeds
- recombinant monomeric a-syn undergoes a conversion to adapt a misfolded state, and aggregation occurs.
- Each well of a black 96-well plate (# 655906, Greiner Bio-One, Germany) was prepared to contain in a total volume of 100 pl six visually inspected spherical silica beads (#BMBG 800-200- 1 , OPS Diagnostics, USA), 50 mM 2-(N-morpholino)ethanesulfonic acid (MES) (pH 5.5) (#M1503, Duchefa Biochemie, Netherlands), 10 pM Thioflavin-T (#ab120751 , Abeam, UK), a suitable amount of zinc salt, 0.1 mg/ml filtered recombinant a-syn and 15 pl CSF.
- MES 2-(N-morpholino)ethanesulfonic acid
- MES 2-(N-morpholino)ethanesulfonic acid
- 10 pM Thioflavin-T (#ab120751 , Abeam, UK)
- a suitable amount of zinc salt 0.1 mg/ml filtered
- Samples were analysed in triplicates in a blind experiment. Positive and negative control samples were included for each assay plate. A replicate was defined positive if a threshold of 4000 RFU had been passed within 48 h. The test result for a sample was defined as positive if two or three replicates were positive, and as negative if no replicate was positive. Analysis of samples with one positive replicate out of three was repeated, and the test result was defined as positive when two or more out of then six replicates were positive and else as negative.
- the zinc salt used was ZnSO4 (#1 .08883, Sigma-Aldrich, UK).
- ZnSO4 was tested for both Normal control (NC) conditions and Parkinson’s disease (PD) conditions (Table 1 and Figures 3A and 3B).
- N Normal control
- PD Parkinson’s disease
- Addition of 5 mM ZnSO4 was the most optimal one due to the elevated fluorescence and the above 48 hour-difference in lag time between the NC and the PD (Table 1 ).
- Table 1 RT-QuIC lag times at 4000 RFU upon addition of 2.5 mM ZnSO4 and 5 mM ZnSO4 to the Normal Control (NC) sample and the Parkinson’s disease (PD) sample, respectively
- Table 2 RT-QuIC lag times at 4000 RFU upon addition of 0 mM ZnSO4 and 5 mM ZnSO4 to Normal Control (NC) sample and Parkinson’s disease (PD) sample, respectively not determined
- a non-zinc ion carrier has been used in the RT-QuIC assay.
- the non-zinc ion carriers tested include NaCI, MgCl2, AICI3, CuCl2, CaCl2, MnCh For each of NaCI, MgCl2, AICI3, CaCl2 and CuCl2, no aggregation/fibrillation was observed under the NC conditions nor under the PD conditions (data are not shown).
- MnCl2 aggregation/fibrillation occurs under both the NC conditions and the PD conditions.
- the difference between the lag time for aggregation of a-syn under the NC conditions and that under the PD conditions is too small to be able to reliably distinguish the normal condition from the PD condition (data are not shown).
- the foregoing shows the matrix of the biological sample - CSF - inhibits aggregation/fibrillation of a-syn in the RT-QuiC assay.
- the a-syn seed aggregation reaction that forms the basis of the assay is inhibited by the biological sample matrix.
- the addition of zinc ions facilitates the reaction so that it will take place even in the presence of biological sample matrix.
- the above examples illustrate the importance of zinc ion carrier for counteracting the inhibitory effect of the matrix of the biological sample on the aggregation of a-syn without provoking spontaneous aggregation of a-syn in the absence of misfolded a-syn in the biological sample. This effect provides for reliable detection of the presence of misfolded a-syn in the biological sample and thereby allows diagnosing synucleinopathy and/or prognosing synucleinopathy with high sensitivity and high specificity.
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Abstract
A method for detecting a presence or an absence of misfolded alpha-synuclein (α-syn) in a biological sample, the method comprising the steps of a) providing monomers of α-syn, a biological sample, the biological sample being suspected to contain misfolded α-syn, and a zinc ion carrier, b) preparing an incubation mixture comprising the monomers of α-syn, the biological sample and the zinc ion carrier, c) incubating the incubation mixture, d) subjecting the incubation mixture to a process for physical disruption, e) repeating steps c)-d) one or more times, f) detecting a presence or an absence of aggregates comprising misfolded α-syn, is provided. A kit of parts which comprises monomers of α-syn, a zinc ion carrier and instructions for detecting a presence or absence of misfolded α-syn species in a biological sample is additionally provided.
Description
ALPHA-SYNUCLEIN DETECTION ASSAY COMPRISING ZINC IONS IN THE REACTION MIXTURE AND METHOD FOR DIAGNOSING ALPHA-SYNUCLEINOPATHIES
TECHNICAL FIELD
The present invention relates to a method for detecting misfolded alpha- synuclein (a-syn) in a biological sample. The invention further relates to a kit of parts for detecting misfolded a-syn species in a biological sample.
BACKGROUND a-syn is a 140 amino acid long protein that is particularly abundant in the presynaptic nerve terminals in the central nervous system, a-syn is intrinsically disordered and exists in various forms. Under normal physiological conditions, a-syn is an unfolded soluble monomeric protein, whereas under pathological conditions (in synucleinopathies) a-syn undergoes misfolding and aggregates into oligomers, amyloid fibrils and eventually into Lewy bodies.
Accumulation of aggregates comprising misfolded a-syn is associated with neurodegenerative diseases, such as Parkinson’s disease, dementia with Lewy bodies and multiple system atrophy. The above neurodegenerative diseases are incurable and highly prevalent.
Methods for detecting a presence or an absence of misfolded a-syn in a biological sample comprise seed amplification assays (SAAs), including real-time quaking-induced conversion (RT-QuIC) and protein misfolding cyclic amplification (PMCA). The above methods have demonstrated adequate sensitivity and specificity in detecting misfolded a-syn across a range of biological samples.
WO 2018/007817 relates to a method of detecting the presence of a-syn aggregation in a biological sample, wherein the biological sample is mixed with a reaction sample comprising a population of beads, a fluorophore adapted to bind to protein aggregates and to increase fluorescence when bound to protein aggregates, and a-syn or a fragment or variant thereof to form a reaction mixture, the reaction mixture is illuminated and at the same time incubated with intermittent agitation cycles, wherein a significant increase in the fluorescence of the reaction mixture during incubation is indicative of the presence of aggregates of a-syn in the biological sample.
A problem with known in the art methods for detecting a presence or an absence of misfolded a-syn in a biological sample is that the above methods are
known for accounting for false positive results due to occurrence of spontaneous a- syn aggregation events. Another drawback of many methods known in the art is that they take a long time to perform, e. g. some methods may take as long as 5-12 days. Therefore, there is a need for improved biochemical tests for detecting a presence or an absence of misfolded a-syn in a biological sample.
SUMMARY
In the light of the above, it is an object of the present invention to provide a method for detecting a presence or an absence of misfolded a-syn in a biological sample, a use of a zinc ion carrier for preventing an inhibitory effect of a biological matrix of a biological sample on a-syn aggregation in said method and a kit of parts comprising monomers of a-syn, a zinc ion carrier and instructions for detecting a presence or an absence of misfolded a-syn species in a biological sample. Said invention alleviates at least part of the above-discussed problems and at least partially addresses one or more of the above-mentioned needs.
According to a first aspect of the invention an ex vivo method is provided for detecting a presence or an absence of misfolded a-syn in a biological sample, more precisely the ex vivo method comprising the steps of a) providing monomers of a-syn, a biological sample, the biological sample being suspected to contain misfolded a-syn, and a zinc ion carrier, b) preparing an incubation mixture comprising at least monomers of a-syn, a biological sample, the biological sample being suspected to contain misfolded a-syn, and a zinc ion carrier, c) incubating the incubation mixture, d) subjecting the incubation mixture to a process for physical disruption, e) repeating steps c)-d) one or more times, f) detecting a presence or an absence of aggregates comprising misfolded a-syn.
According to another aspect of the invention a use of the zinc ion carrier in an ex vivo method is provided for preventing or reducing an inhibitory effect of a biological matrix of a biological sample on a-syn aggregation, more precisely in the ex vivo method comprising steps of
a) providing monomers of a-syn, a biological sample, the biological sample being suspected to contain misfolded a-syn, and a zinc ion carrier, b) preparing an incubation mixture comprising at least monomers of a-syn, a biological sample, the biological sample being suspected to contain misfolded a-syn, and a zinc ion carrier, c) incubating the incubation mixture, d) subjecting the incubation mixture to a process for physical disruption, e) repeating steps c)-d) one or more times, f) detecting a presence or an absence of aggregates comprising misfolded a-syn.
According to yet another aspect of the invention a use of an ex vivo method is provided for diagnosing synucleinopathy, the ex vivo method comprising the steps of a) providing monomers of a-syn, a biological sample, the biological sample being suspected to contain misfolded a-syn, and a zinc ion carrier, b) preparing an incubation mixture comprising at least monomers of a-syn, a biological sample, the biological sample being suspected to contain misfolded a-syn, and a zinc ion carrier, c) incubating the incubation mixture, d) subjecting the incubation mixture to a process for physical disruption, e) repeating steps c)-d) one or more times, f) detecting a presence or an absence of aggregates comprising misfolded a-syn.
According to yet another aspect of the invention a use of an ex vivo method is provided for prognosing synucleinopathy, the ex vivo method comprising the steps of a) providing monomers of a-syn, a biological sample, the biological sample being suspected to contain misfolded a-syn, and a zinc ion carrier, b) preparing an incubation mixture comprising at least monomers of a-syn, a biological sample, the biological sample being suspected to contain misfolded a-syn, and a zinc ion carrier, c) incubating the incubation mixture, d) subjecting the incubation mixture to a process for physical disruption,
e) repeating steps c)-d) one or more times, f) detecting a presence or an absence of aggregates comprising misfolded a-syn.
According to yet another aspect of the invention a kit of parts comprising monomers of a-syn, a zinc ion carrier and instructions for detecting a presence of misfolded a-syn species in a biological sample is provided.
These and other aspects of the invention are apparent from and elucidated with reference to the embodiments described hereinafter.
BRIEF DESCRIPTION OF THE DRAWINGS
For exemplifying purposes, the invention will be described in closer detail in the following with reference to embodiments thereof illustrated in the attached drawings.
Fig. 1 schematically illustrates an overview of a method for detecting a presence or an absence of misfolded a-syn in a biological sample.
Fig. 2 schematically illustrates an overview of a method for detecting a presence or an absence of misfolded a-syn in a biological sample, wherein the method being part of a real-time quaking-induced conversion (RT-QuIC).
Fig. 3A diagrammatically illustrates a method for detecting a presence or an absence of misfolded a-syn in a biological sample comprising cerebrospinal fluid (CSF) under normal physiological (NC) conditions and Parkinson’s disease (PD) conditions, respectively, wherein 2.5 mM ZnSO4 is comprised within the incubation mixture, and wherein the method is a part of a RT-QuIC assay.
Fig. 3B diagrammatically illustrates a method for detecting a presence or an absence of misfolded a-syn in a biological sample comprising CSF under NC and PD conditions, respectively, wherein 5 mM ZnSO4 is comprised within the incubation mixture, and wherein the method is a part of a RT-QuIC assay.
Fig. 4A diagrammatically illustrates a method for detecting a presence or an absence of misfolded a-syn in a biological sample comprising CSF under NC and PD conditions, respectively, wherein 0 mM ZnSO4 is comprised within the incubation mixture, and wherein the method is being part of a RT-QuIC assay.
Fig. 4B diagrammatically illustrates a method for detecting a presence or an absence of misfolded a-syn in a biological sample comprising CSF under NC and PD conditions, respectively, wherein 5 mM ZnSO4 is comprised within the incubation
mixture, and wherein the method is being part of RT-QuIC assay. For better visibility moving average (n=10) curves are shown for the PD samples.
DETAILED DESCRIPTION OF THE INVENTION
In the following detailed description, technical terms and expressions are defined, and various ways of working the invention are described.
Generally, all terms and expressions used in the application text are to be interpreted according to the meaning commonly applied to them in the pertinent prior art unless explicitly defined otherwise herein.
As used in this specification and the appended claims, the singular forms ”a”, ”an” and ’’the” include plural referents unless the context clearly dictates otherwise. Thus, e. g. ”a protein” includes one or more proteins, “a zinc ion carrier” includes one or more zinc ion carriers, “an inhibitory effect” includes one or more inhibitory effects and the like.
The term “biological sample” is understood within the scope of the invention to refer to any biological material obtained from a living or diseased animal, including a mammal; the biological material is suitable for being employed in the method for detecting a presence of misfolded a-syn. The term “animal” includes representatives of the kingdom Animalia, such as a worm, a mouse, a hamster, a monkey, a human. The term “mammal” includes humans.
Each of the terms “a-syn”, ’’recombinant a-syn”, ’’misfolded a-syn”, “soluble misfolded a-syn”, “non-monomeric misfolded a-syn” and the like is understood within the scope of the invention to refer to the population of proteins designated by the term as a whole. E. g. “a-syn” include any a-syn protein irrespectively of the length, conformation and post-translational modification thereof.
The term “a-syn” is understood within the scope of the invention to refer to any a-syn protein population comprising at least one of a full-length a-syn and a truncated a-syn protein. The term “a-syn” includes any a-syn protein population comprising at least one of a wild-type a-syn protein and a mutated a-syn protein. The wild-type a- syn protein may be a recombinant protein. The mutated a-syn protein may be a recombinant protein.
The term ’’monomers of a-syn” is understood within the scope of the invention to refer to soluble monomers of a-syn capable of assembly into aggregates.
The term “recombinant a-syn” is understood within the scope of the invention to refer to any a-syn produced using gene technology, as opposed to wild-type a-syn, i. e. a-syn obtained by isolation from a biological source which natively produces a- syn.
The term “a-syn aggregation” is understood within the scope of the invention to refer to aggregation of monomers of a-syn in the presence of soluble misfolded a- syn to form at least one of a-syn oligomers, a-syn amyloid fibrils and Lewy bodies.
The term “spontaneous a-syn aggregation” is understood within the scope of the invention to refer to aggregation of monomers of a-syn in the absence of soluble misfolded a-syn to form at least one of a-syn oligomers, a-syn amyloid fibrils and Lewy bodies.
The term ’’misfolded a-syn” is understood within the scope of the invention to refer to misfolded a-syn as a whole. The “non-monomeric misfolded a-syn” may include at least one of a-syn oligomers, a-syn amyloid fibrils and Lewy bodies.
The term ’’misfolded a-syn species” is understood within the scope of the invention to refer to non-monomeric a-syn species formed due to misfolding of at least a portion of the monomers of a-syn in the presence of soluble misfolded a-syn. The soluble misfolded a-syn may be present in the biological sample provided at the step a) in the method of the invention. The soluble misfolded a-syn may be formed in the course of physical disruption of at least a portion of the aggregates comprising misfolded a-syn generated at the step d) in the method of the invention.
The term ’’aggregates comprising misfolded a-syn” is understood within the scope of the invention to refer to the aggregates comprising a-syn which may be generated at the step d) of the method of the invention; the aggregates may be assembled from at least one of at least a portion of the monomers of a-syn, at least a portion of the misfolded a-syn species and at least a portion of the misfolded a-syn.
The present invention relates to an ex vivo method for detecting a presence or an absence of misfolded alpha-synuclein (a-syn) in a biological sample, the method comprising the steps of a. providing monomers of a-syn, a biological sample suspected of comprising misfolded a-syn and a zinc ion carrier, b. preparing an incubation mixture from the monomers of a-syn, biological sample and zinc ion carrier provided in step a,
c. incubating the incubation mixture prepared in step b, d. subjecting the mixture incubated according to step c to a process for physical disruption, e. repeating steps c)-d) one or more times, f. detecting a presence or an absence of aggregates comprising misfolded a-syn.
The invention will be described in more detail below.
According to a first aspect of the invention an ex vivo method is provided for detecting a presence or an absence of misfolded a-syn in a biological sample, more precisely the method comprising the steps of a) providing monomers of a-syn, a biological sample, the biological sample being suspected to contain misfolded a-syn, and a zinc ion carrier, b) preparing an incubation mixture comprising at least the monomers of a- syn, the biological sample and the zinc ion carrier, c) incubating the incubation mixture, d) subjecting the incubation mixture to a process for physical disruption, e) repeating steps c)-d) one or more times, f) detecting a presence or an absence of aggregates comprising misfolded a-syn.
The present invention is not intended for being carried out on a human or animal body. In other words, the invention does not relate to a method for treatment of the human or animal body by surgery or therapy and diagnostic methods practised on the human or animal body. As appears from the present specification, the method of the invention is an ex vivo method, i. e. a method performed entirely extracorporeal ly, outside of the human or animal body.
The method may further comprise the step c') detecting a presence or an absence of aggregates comprising misfolded a-syn in the incubation mixture, wherein c') follows c) and precedes d). In this case the wording “repeating steps c)-d)” includes repeating step c), c') and step d”.
The incubating may be effective in the presence of soluble misfolded a-syn to cause conversion of at least a portion of the monomers of a-syn into misfolded a-syn species and subsequent recruitment of at least a portion of the misfolded a-syn species into aggregates comprising misfolded a-syn.
The incubating the incubation mixture may include incubating at a temperature from about 30 °C to about 42 °C, including 37 °C.
The physical disruption may be effective to cause breaking of aggregates comprising misfolded a-syn and providing soluble misfolded a-syn.
The monomers of a-syn, the biological sample and the zinc ion carrier may be in a buffer solution. The buffer solution may be any buffer solution as long as the zinc carrier is substantially soluble therein. The buffer solution may have a pH value from about 4 to about 8.5, from about 5 to about 6.5, from about 5.5 to about 6.0. The buffer solution may have various compositions. The buffer solutions may contain at least one of MES, sodium phosphate, ammonium acetate, HEPES and Tris-CI. In some embodiments the buffer solution may contain MES at a final concentration of between from about 15 mM to about 500 mM, from about 15 mM to about 300 mM, from about 25 mM to about 200 mM, from about 50 mM to about 100 mM. In some embodiments the buffer solution may contain phosphate at a final concentration of between from about 15 mM to about 300 mM, from about 25 mM to about 200 mM, from about 50 mM to about 100 mM. The person skilled in the art realises that the composition of the buffer solution is not limited to the above examples, and it may be chosen in such a way that its ionic strength is similar to that of the above-mentioned buffer solutions.
The zinc ion carrier may be any zinc salt as long as it remains soluble under assay conditions and that the anion does not interfere with the assay. The zinc ion carrier may be at least one of zinc sulphate, zinc chloride and zinc nitrate.
The zinc ions may be present at a concentration sufficient for counteracting the inhibitory effect of the matrix of the biological sample on the aggregation of a-syn without provoking spontaneous aggregation of a-syn in the absence of misfolded a- syn in the biological sample. The more zinc ions are added, the more inhibition may be counter-acted. However, if more zinc ions than necessary to counteract the inhibition are added, spontaneous aggregation of a-syn may be provoked. The above spontaneous aggregation of a-syn in the absence of misfolded a-syn being present in the biological sample may account for false positive results. Thus, depending on the inhibitory effect of the matrix lower or higher zinc ion concentrations may be chosen. The zinc ions may be present at a concentration from 0.5 mM to about 100 mM, from about 0.5 mM to about 50 mM, from about 0.5 mM to about 20 mM, from about 2 mM to about 10 mM, from about 2.5 mM to about 5 mM. It is appreciated that a person
skilled in the art will be able to choose a suitable concentration of the zinc ions in view of e. g. the strength of the inhibitory effect of the matrix of the biological sample on the aggregation process. A matrix with a lower inhibitory effect on the aggregation of a-syn may require less zinc ions to be used in the method of the invention. A matrix with a higher inhibitory effect on the aggregation of a-syn may require more zinc ions to be used in the method of the invention. E. g. when the biological sample blood, it is beneficial to use more zinc ions than what is used when the biological sample is CSF.
A lower amount of the biological sample, and hence less amount of matrix thereof, may require less zinc ions to be used in the method of the invention.
The person skilled in the art realises that the zinc ion concentration may be adjusted in relation to the amounts of other constituents of the incubation mixture including the biological sample and the monomers of a-syn provided. E. g. if the amount of the biological sample is decreased, the concentration of zinc ions in the incubation mixture may need to be decreased; if the amount of the biological sample is increased, the concentration of zinc ions in the incubation mixture may need to be increased.
With a view to simplifying the biological sample matrix, the method of the invention may optionally comprise a step of treating the biological sample prior to preparing the incubation mixture. The simplifying the biological sample matrix may allow to increase the concentration of misfolded a-syn in the biological sample. The biological sample may be treated in various ways, e. g. by precipitating and subsequently removing at least a portion of the proteins contained therein. Further, other components of the sample matrix may be removed, such as lipids, salts etc.
The incubation mixture may further comprise an indicator for detecting aggregates comprising misfolded a-syn therein. The indicator for detecting the above aggregates may be a fluorophore that is adapted to bind the aggregates and to increase fluorescence when bound to the aggregates. The person skilled in the art realises that a suitable fluorophore may be selected in view of e. g. the structure of a- syn fibrils formed, the structure thereof being dependent on e. g. the amount of misfolded a-syn in the biological sample and the disease background (e. g. Parkinson’s disease, dementia with Lewy bodies or multiple system atrophy). Each fluorophore may be expected to have a preference for one or more selected from a group consisting of a-syn oligomers, a-syn amyloid fibrils and Lewy bodies. The
fluorophore may be at least one of thioflavin T, the derivate of Congo Red X-34, Curcumin, the derivative of Curcumin CRANAD-3, Nile Red, pFTAA and the like.
The method according to the first aspect of the invention may further comprise bringing into contact the incubation mixture and an indicator for detecting aggregates comprising misfolded a-syn prior to the incubating the incubation mixture.
The detecting a presence or an absence of aggregates comprising misfolded a-syn may be conducted by at least one selected from the group consisting of fluorescence assay, immunoassay (such as ELISA and the like), absorbance assay, electron microscopy, size exclusion chromatography, western blot and dot blot.
The process for physical disruption may include at least one of shaking, homogenisation, stirring, sonication and the like.
An a-syn protein is normally divided into mainly three regions: the N-terminus (amino acids 1-65 of an a-syn, with reference to wild-type human a-syn) that forms an a-helix upon binding to membranes and where most of the Parkinson’s disease mutations may be located, the NAC core (amino acids 66-95 of an a-syn, with reference to wild-type human a-syn), and the C-terminus (amino acids 96-140 of an a-syn, with reference to wild-type human a-syn) that contains most of post- translational modifications and critical for aggregation as well as important for the chaperone activity of the a-syn protein.
The a-syn may be a wild-type protein or a mutated protein; the mutated protein may include point mutations that affect the protein aggregation ability compared to that of the wild-type protein. An example of such mutated a-syn protein is K23Q.
The monomers of a-syn may comprise monomers of at least one of a full- length a-syn and a truncated a-syn, advantageously a full-length a-syn. The monomers of a-syn may comprise monomers of at least one of a full-length human a- syn or a part thereof comprising at least a region containing amino acids 10-130 (with reference to wild-type human a-syn), a region containing amino acids 10-100 (with reference to wild-type human a-syn), a region containing amino acids 10-95 (with reference to wild-type human a-syn) or a sequence showing at least 50 % identity, at least 60 % identity, at least 70 % identity, at least 80 % identity, at least 90 % identity, at least 95 % identity, or at least 99 % identity to any said region.
The final concentration of the provided monomers of a-syn in the buffer solution may be from about 0.05 to about 1 mg/ml, from about 0.05 to about 0.5 mg/ml, from about 0.1 to about 0.3 mg/ml, including 0.1 mg/ml.
Incubation mixtures of various volumes may be used. The volume of the incubation mixture may be adjusted depending on the consumable used for holding the incubation mixture, such as the type of a plate. For a 384-well plate the volume of the incubation mixture can be of from about 25 pl to about 100 pl. For a 96-plate of the incubation mixture can be of from about 50 pl to about 400 pl.
The biological sample may comprise at least one of cerebrospinal fluid (CSF), blood, eye liquid, tears, brain tissue, olfactory mucosa, saliva, nasopharyngeal swab fluid, urine, intestinal fluid, skin, stem cells, faeces and a liquid preparation thereof. The blood may comprise at least one of plasma, serum, red blood cells, white blood cells, blood platelets, exosomes and the like. As it is appreciated by the person skilled in the art, the biological sample may be subjected to at least one of processing, concentrating and diluting in any conventional way.
The incubation mixture may further contain one or more beads. The beads can be made of any suitable material, e. g. silica, glass and zirconium. The bead diameter may be from about 0.2 mm to about 1 mm, from about 0.4 mm to about 0,8 mm, or from about 0.5 mm to about 0,6 mm. Distinctly larger beads, e. g. 3 mm or greater in diameter, are not suitable for use in the method according to the invention. The person skilled in the art realises that the number and diameter of the beads could be chosen in such a way that the reaction becomes sufficiently facilitated (cf. the reaction when no beads are used) while the fluorescent measurement remains essentially unhindered (cf. the reaction when no beads are used).
The method may be a part of a seed amplification assay. The principles of the seed amplification assays are well known to the person skilled in the art, and equipment, conditions and the like may easily be adapted to each relevant situation. The seed amplification assay may be one selected from the group consisting of PMCA and RT-QulC.
According to another aspect of the invention a use of the zinc ion carrier is provided for preventing or reducing an inhibitory effect of a biological matrix of a biological sample on a-syn aggregation in an ex vivo method comprising steps of a) providing monomers of a-syn, a biological sample, the biological sample being suspected to contain misfolded a-syn, and a zinc ion carrier, b) preparing an incubation mixture comprising at least the monomers of a- syn, the biological sample and the zinc ion carrier, c) incubating the incubation mixture,
d) subjecting the incubation mixture to a process for physical disruption, e) repeating steps c)-d) one or more times, f) detecting a presence or an absence of aggregates comprising misfolded a-syn.
The above wording “preventing or reducing” is to be understood as alleviating an inhibitory effect at least to some extent.
The biological sample may comprise at least one of cerebrospinal fluid (CSF), blood, eye liquid, tears, brain tissue, olfactory mucosa, saliva, nasopharyngeal swab fluid, urine, intestinal fluid, skin, stem cells, faeces and a liquid preparation thereof. The blood may comprise at least one of plasma, serum, red blood cells, white blood cells, blood platelets, exosomes and the like. The biological matrix of the biological sample may inhibit the a-syn seeding reaction, wherein small a-syn oligomers provide a template for the assembly of soluble monomers of a-syn into aggregates comprising misfolded a-syn.
According to yet another aspect of the invention a use of an ex vivo method is provided for diagnosing synucleinopathy, the ex vivo method comprising the steps of a) providing monomers of a-syn, a biological sample, the biological sample being suspected to contain misfolded a-syn, and a zinc ion carrier, b) preparing an incubation mixture comprising at least the monomers of a- syn, the biological sample and the zinc ion carrier, c) incubating the incubation mixture, d) subjecting the incubation mixture to a process for physical disruption, e) repeating steps c)-d) one or more times, f) detecting a presence or an absence of aggregates comprising misfolded a-syn.
The synucleinopathy may comprise at least one of Parkinson’s disease, dementia with Lewy bodies and multiple system atrophy. The method may be used for diagnosing synucleinopathy in an animal, including a mammal, including a human.
According to yet another aspect of the invention a use of the method discussed in the foregoing is provided for prognosing synucleinopathy, the method comprising the steps of a) providing monomers of a-syn, a biological sample, the biological sample being suspected to contain misfolded a-syn, and a zinc ion carrier,
b) preparing an incubation mixture comprising at least the monomers of a- syn, the biological sample and the zinc ion carrier, c) incubating the incubation mixture, d) subjecting the incubation mixture to a process for physical disruption, e) repeating steps c)-d) one or more times, f) detecting a presence or an absence of aggregates comprising misfolded a-syn.
The synucleinopathy may be at least one of Parkinson’s disease, dementia with Lewy bodies and multiple system atrophy. The method may be used for prognosing synucleinopathy in an animal, including a mammal, including a human.
According to yet another aspect of the invention a kit of parts comprising at least monomers of a-syn, a zinc ion carrier and instructions for detecting a presence of misfolded a-syn species in a biological sample is provided.
The kit of parts may additionally include at least one selected from a group comprising a buffer solution, a size exclusion filter, beads and a plate.
The size exclusion filter may be a filter that allows each molecule of a molecular weight of less than 150 kDa, less than 120 kDa, less than 100 kDa, less than 80 kDa, less than 75 kDa, less than 70 kDa, less than 65 kDa or less than 60 kDa or less than 50kDa to pass through the filter while preventing each molecule of a molecular weight that is greater than said molecular weight from passing through the filter.
The plate may be of any suitable size, such as a 96-well plate or a 384-well plate.
It is appreciated that the above-mentioned embodiments of the first aspect of the present invention may also apply, even if not explicitly mentioned, to the further aspects of the present invention.
The invention has mainly been described above with reference to some embodiments. However, as it is readily appreciated by the person skilled in the art, other embodiments than the ones disclosed above are equally possible within the scope of the invention as defined by the appended claims.
Examples
The present examples are provided for illustrative purposes only and are not to be construed as limiting the scope of the present invention as defined by the appended claims.
In one example the method for detecting a presence or an absence of misfolded a-syn in a biological sample (Fig. 1 ) is a part of a RT-QuIC (Fig. 2). RT- QuIC is a cyclic process. Each cycle lasts for approximately four minutes (a 3 min elongation and a 1 min shaking). The cycle is continuously repeated for 48 hours. At the end of every fourth cycle fluorescence is measured with fluorescence intensity being proportional to the amount of generated aggregates. In the presence of aggregation-prone misfolded a-syn (seeds) recombinant monomeric a-syn undergoes a conversion to adapt a misfolded state, and aggregation occurs. At the start of each cycle continuous shaking for 1 min breaks up formed aggregates generating new seeds for the next cycle. In case seeds are continuously present in the sample, more and more monomeric a-syn gets converted into the misfolded a- syn, and fluorescence increases over time and eventually passes a threshold of 4000 relative fluorescence units (RFU). In samples devoid of seeds spontaneous aggregation might occur, but the threshold will not be reached before the end of the test.
On the day of the assay recombinant wild-type a-syn (# S-1001-2, rPeptide, USA) was passed through DNA fast flow Ultracel filtration units (#MRCFOR100, Merck Millipore, Ireland) using a centrifugation force of 1500 g. Each well of a black 96-well plate (# 655906, Greiner Bio-One, Germany) was prepared to contain in a total volume of 100 pl six visually inspected spherical silica beads (#BMBG 800-200- 1 , OPS Diagnostics, USA), 50 mM 2-(N-morpholino)ethanesulfonic acid (MES) (pH 5.5) (#M1503, Duchefa Biochemie, Netherlands), 10 pM Thioflavin-T (#ab120751 , Abeam, UK), a suitable amount of zinc salt, 0.1 mg/ml filtered recombinant a-syn and 15 pl CSF. Plates were sealed with an optic sealing film (#232701 , Thermo Scientific, USA) and incubated in a Synergi Him multimode reader at 37 °C with repeated shake and rest: 1 min double orbital shaking (3 mm, 425 cpm) followed by 3 min rest. Fluorescence (excitation: 435 nm, emission: 480 nm) was read at the end of every fourth cycle.
Samples were analysed in triplicates in a blind experiment. Positive and negative control samples were included for each assay plate. A replicate was defined positive if a threshold of 4000 RFU had been passed within 48 h. The test result for a
sample was defined as positive if two or three replicates were positive, and as negative if no replicate was positive. Analysis of samples with one positive replicate out of three was repeated, and the test result was defined as positive when two or more out of then six replicates were positive and else as negative.
In one example the zinc salt used was ZnSO4 (#1 .08883, Sigma-Aldrich, UK). To determine the optimal concentration of ZnSO4 for the RT-QuIC assay, various concentrations of ZnSO4 were tested for both Normal control (NC) conditions and Parkinson’s disease (PD) conditions (Table 1 and Figures 3A and 3B). Addition of 5 mM ZnSO4 was the most optimal one due to the elevated fluorescence and the above 48 hour-difference in lag time between the NC and the PD (Table 1 ).
Table 1 : RT-QuIC lag times at 4000 RFU upon addition of 2.5 mM ZnSO4 and 5 mM ZnSO4 to the Normal Control (NC) sample and the Parkinson’s disease (PD) sample, respectively
Difference between addition of 5 mM ZnSO4 and no addition of ZnSO4 under the NC conditions and the PD conditions, respectively: upon addition of 5 mM ZnSO4 the lag times become more uniform and are decreased compared to those in case of no addition of ZnSO4 (Table 2 and Figures 4A and 4B). Upon addition of 5 mM ZnSO4 no aggregation occurs under the NC conditions (lag time above 120 hours), whereas under the PD conditions the lag time is less than 15 hours (Table 2 and Figure 4B). The foregoing allows reliable detection of the presence of misfolded a- syn in the biological sample.
Table 2: RT-QuIC lag times at 4000 RFU upon addition of 0 mM ZnSO4 and 5 mM ZnSO4 to Normal Control (NC) sample and Parkinson’s disease (PD) sample, respectively
not determined
Difference between the NC conditions and the PD conditions: no aggregation/fibrillation is observed under the NC conditions (Table 2 and Figure 4B). A positive response is induced under the PD conditions as shown by aggregation/fibrillation in the RT-QuIC assay (Table 2 and Figure 4B).
In other examples a non-zinc ion carrier has been used in the RT-QuIC assay. The non-zinc ion carriers tested include NaCI, MgCl2, AICI3, CuCl2, CaCl2, MnCh For each of NaCI, MgCl2, AICI3, CaCl2 and CuCl2, no aggregation/fibrillation was observed under the NC conditions nor under the PD conditions (data are not shown). For MnCl2, aggregation/fibrillation occurs under both the NC conditions and the PD conditions. However, the difference between the lag time for aggregation of a-syn under the NC conditions and that under the PD conditions is too small to be able to reliably distinguish the normal condition from the PD condition (data are not shown).
In yet another example the effect of the matrix of a biological sample on the aggregation of a-syn was studied. When no CSF sample was added to an incubation mixture (the incubation mixture containing 5 mM Zn ions), aggregation/fibrillation was detected within 9 hours in the RT-QuIC assay (data are not shown). When a CSF sample, however, was part of an incubation mixture (the incubation mixture containing 5 mM Zn ions), aggregation/fibrillation was detected significantly later in time (within 15.5 hours) and only under the PD conditions, whereas no aggregation/fibrillation was at all detected under the NC conditions (data are not shown). The foregoing shows the matrix of the biological sample - CSF - inhibits aggregation/fibrillation of a-syn in the RT-QuiC assay.
To conclude, the a-syn seed aggregation reaction that forms the basis of the assay is inhibited by the biological sample matrix. The addition of zinc ions facilitates the reaction so that it will take place even in the presence of biological sample matrix. The above examples illustrate the importance of zinc ion carrier for counteracting the inhibitory effect of the matrix of the biological sample on the aggregation of a-syn without provoking spontaneous aggregation of a-syn in the absence of misfolded a-syn in the biological sample. This effect provides for reliable detection of the presence of misfolded a-syn in the biological sample and thereby allows diagnosing synucleinopathy and/or prognosing synucleinopathy with high sensitivity and high specificity.
Claims
CLAIMS An ex vivo method for detecting a presence or an absence of misfolded alpha- synuclein (a-syn) in a biological sample, the method comprising the steps of a. providing monomers of a-syn, a biological sample, the biological sample being suspected to contain misfolded a-syn, and a zinc ion carrier, b. preparing an incubation mixture comprising at least monomers of a-syn, a biological sample, the biological sample being suspected to contain misfolded a-syn, and a zinc ion carrier, c. incubating the incubation mixture, d. subjecting the incubation mixture to a process for physical disruption, e. repeating steps c)-d) one or more times, f. detecting a presence or an absence of aggregates comprising misfolded a-syn. The method according to claim 1 , wherein the method further comprises the step c') detecting a presence or an absence of aggregates comprising misfolded a-syn in the incubation mixture, wherein b') follows b) and precedes c). The method according to any one of the preceding claims, wherein the incubating being effective in the presence of soluble misfolded a-syn to cause conversion of at least a portion of the monomers of a-syn into misfolded a-syn species and subsequent recruitment of at least a portion of the misfolded a-syn species into aggregates. The method according to any one of the preceding claims, wherein the physical disruption being effective to cause breaking of aggregates comprising misfolded a-syn and providing soluble misfolded a-syn.
5. The method according to any one of the preceding claims, wherein in step b) the monomers of a-syn, the biological sample and the zinc ion carrier are in a buffer solution.
6. The method according to any one of the preceding claims, wherein the zinc ion carrier comprises at least one of zinc sulphate, zinc chloride and zinc nitrate.
7. The method according to any one of the preceding claims, wherein the incubation mixture further comprises an indicator for detecting aggregates comprising misfolded a-syn therein.
8. The method according to any one of the preceding claims, wherein the detecting a presence or an absence of aggregates comprising misfolded a-syn is conducted by at least one of fluorescence assay, immunoassay, absorbance assay, electron microscopy, size exclusion chromatography, western blot and dot blot.
9. The method according to claim 7, wherein the indicator for the detecting aggregates comprising misfolded a-syn comprises at least one of Thioflavin T, X-34, Curcumin, CRANAD-3, Nile Red and pFTAA.
10. The method according to any one of the preceding claims, wherein the process for physical disruption is at least one of shaking, homogenisation, stirring and sonication.
11. The method according to any one of the preceding claims, wherein the biological sample comprises at least one of cerebrospinal fluid (CSF), blood, eye liquid, brain tissue, olfactory mucosa, saliva, urine, skin biopsy and stem cells.
12. The method according to any one of the preceding claims, the method being part of a seed amplification assay.
The method according to claim 12, wherein the seed amplification assay is one of PMCA and RT-QulC. A use of the zinc ion carrier for preventing or reducing an inhibitory effect of a biological matrix of a biological sample on a-syn aggregation in an ex vivo method comprising the steps of a. providing monomers of a-syn, a biological sample, the biological sample being suspected to contain misfolded a-syn, and a zinc ion carrier, b. preparing an incubation mixture comprising at least monomers of a-syn, a biological sample, the biological sample being suspected to contain misfolded a-syn, and a zinc ion carrier, c. incubating the incubation mixture, d. subjecting the incubation mixture to a process for physical disruption, e. repeating steps c)-d) one or more times, f. detecting a presence or an absence of aggregates comprising misfolded a-syn. The use of the zinc ion carrier according to the claim 14, wherein the biological sample comprises is at least one of CSF, blood, eye liquid, brain tissue, olfactory mucosa, saliva, urine, skin biopsy and stem cells. A use of an ex vivo method for diagnosing synucleinopathy, the ex vivo method comprising the steps of a. providing monomers of a-syn, a biological sample, the biological sample being suspected to contain misfolded a-syn, and a zinc ion carrier, b. preparing an incubation mixture comprising at least monomers of a-syn, a biological sample, the biological sample being suspected to contain misfolded a-syn, and a zinc ion carrier, c. incubating the incubation mixture, d. subjecting the incubation mixture to a process for physical disruption, e. repeating steps c)-d) one or more times,
f. detecting a presence or an absence of aggregates comprising misfolded a-syn. A use of an ex vivo method for prognosing synucleinopathy, the ex vivo method comprising the steps of a. providing monomers of a-syn, a biological sample, the biological sample being suspected to contain misfolded a-syn, and a zinc ion carrier, b. preparing an incubation mixture comprising at least monomers of a-syn, a biological sample, the biological sample being suspected to contain misfolded a-syn, and a zinc ion carrier, c. incubating the incubation mixture, d. subjecting the incubation mixture to a process for physical disruption, e. repeating steps c)-d) one or more times, f. detecting a presence or an absence of aggregates comprising misfolded a-syn. The use of the method according to claim 16 and 17, wherein the synucleinopathy comprises at least one of Parkinson’s disease, dementia with Lewy bodies and multiple system atrophy. A kit of parts which comprises monomers of a-syn, a zinc ion carrier and instructions for detecting a presence or absence of misfolded a-syn species in a biological sample.
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| WO2018007817A1 (en) | 2016-07-07 | 2018-01-11 | The University Court Of The University Of Edinburgh | Alpha-synuclein detection assay and method for diagnosing alpha-synucleinopathies |
| JP2018091844A (en) * | 2016-11-29 | 2018-06-14 | 国立大学法人 長崎大学 | METHOD FOR DETECTING α-SYNUCLEIN |
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| WO2018007817A1 (en) | 2016-07-07 | 2018-01-11 | The University Court Of The University Of Edinburgh | Alpha-synuclein detection assay and method for diagnosing alpha-synucleinopathies |
| JP2018091844A (en) * | 2016-11-29 | 2018-06-14 | 国立大学法人 長崎大学 | METHOD FOR DETECTING α-SYNUCLEIN |
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