Alpha-synuclein detection assay
AssigneeThe Univ. Court of the Univ. of Edinburgh
InventorsAlison Green, Graham Fairfoul
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Abstract
A method of detecting the presence of alpha-synuclein aggregation in a biological sample is provided whereby a 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 alpha-synuclein or a fragment or variant thereof to form a reaction mixture, wherein a significant increase in the fluorescence of the reaction mixture during incubation is indicative of the presence of aggregates of alpha-synuclein in the biological sample.
Claims32
A method of detecting the presence of alpha-synuclein aggregation in a biological sample, the method comprising the steps:
- (i) providing a biological sample;
- (ii) providing a reaction sample comprising:
- (a) a population of beads comprising glass or zirconia oxide and having a mean diameter of the beads of 1 mm±10%;
- (b) a fluorophore that binds to protein aggregates and increases fluorescence when bound to protein aggregates;
- and
- (c) alpha-synuclein or a fragment or variant thereof;
- (iii) combining the biological sample and the reaction sample to form a reaction mixture;
- (iv) incubating the reaction mixture with intermittent agitation cycles;
- (v) illuminating the reaction mixture with a wavelength of light that excites the fluorophore;
- and
- (vi) determining the level of fluorescence of the reaction mixture during incubation,
- wherein steps (iv) to (vi) are carried out at the same time, and a significant increase in the fluorescence of the reaction mixture during steps (iv) to (vi) is indicative of the presence of aggregates of alpha-synuclein in the reaction mixture, and wherein the presence of aggregates of alpha-synuclein in the reaction mixture is indicative of the presence of aggregates of alpha-synuclein in the biological sample.
A method of diagnosing alpha-synucleinopathies, the method comprising the steps:
- (i) providing a biological sample from a subject;
- (ii) providing a reaction sample comprising:
- (a) a population of beads, wherein the population of beads comprise glass or zirconia oxide and have a mean diameter of the beads of 1 mm±10%;
- (b) a fluorophore that binds to protein aggregates and increases fluorescence when bound to protein aggregates;
- and
- (c) alpha-synuclein or a fragment or variant thereof;
- (iii) combining the biological sample and the reaction sample to form a reaction mixture;
- (iv) incubating the reaction mixture with intermittent agitation cycles;
- (v) illuminating the reaction mixture with a wavelength of light that excites the fluorophore;
- and
- (vi) determining the level of fluorescence of the reaction mixture during incubation,
- wherein steps (iv) to (vi) are carried out at the same time, and a significant increase in the fluorescence of the reaction mixture during steps (iv) to (vi) is indicative of the subject having an alpha-synucleinopathy.
A kit, comprising an aqueous buffered solution, the aqueous buffered solution comprising:
- (i) a population of beads comprising glass or zirconia oxide and having a have a mean diameter of the beads of 1 mm±10%;
- (ii) Thioflavin T;
- and
- (iii) alpha-synuclein or a fragment or variant thereof.
A method for detecting the presence of alpha-synuclein aggregation in a biological sample comprising cerebrospinal fluid for a non-diagnostic purpose, the method comprising the steps:
- (i) providing the biological sample;
- (ii) providing a reaction sample comprising:
- (a) a population of beads comprising zirconia/silica, zirconia oxide, or glass, wherein the population of beads have a mean diameter of the beads of 1 mm, and wherein the population of beads have a standard deviation of diameters of plus or minus 10% from the mean diameter,
- (b) thioflavin T;
- and
- (c) alpha-synuclein,
- (iii) combining the biological sample and the reaction sample to form a reaction mixture;
- (iv) incubating the reaction mixture with intermittent agitation cycles;
- (v) illuminating the reaction mixture with a wavelength of light that excites the thioflavin T;
- and
- (vi) determining the level of fluorescence of the reaction mixture during incubation,
- wherein steps (iv) to (vi) are carried out at the same time, and a significant increase in the fluorescence of the reaction mixture during steps (iv) to (vi) is indicative of the presence of aggregates of alpha-synuclein in the reaction mixture, and wherein the presence of aggregates of alpha-synuclein in the reaction mixture is indicative of the presence of aggregates of alpha-synuclein in the biological sample.
Dependencies are inferred from the claim text.