US 12,078,643 B2
Alpha-synuclein detection assay and method for diagnosing alpha-synucleinopathies
Alison Green, Midlothian (GB); and Graham Fairfoul, Midlothian (GB)
Assigned to The University Court of the University of Edinburgh, Edinburgh (GB)
Filed by The University Court of the University of Edinburgh, Midlothian (GB)
Filed on May 17, 2021, as Appl. No. 17/314,531.
Application 17/314,531 is a continuation of application No. 16/315,854, granted, now 11,099,197, previously published as PCT/GB2017/051988, filed on Jul. 6, 2017.
Claims priority of application No. 1611840 (GB), filed on Jul. 7, 2016.
Prior Publication US 2021/0263049 A1, Aug. 26, 2021
This patent is subject to a terminal disclaimer.
Int. Cl. G01N 33/68 (2006.01); G01N 21/64 (2006.01); G01N 33/58 (2006.01)
CPC G01N 33/6896 (2013.01) [G01N 21/6428 (2013.01); G01N 33/582 (2013.01); G01N 2021/6439 (2013.01); G01N 2333/4709 (2013.01); G01N 2800/2814 (2013.01); G01N 2800/2835 (2013.01); G01N 2800/7047 (2013.01)] 16 Claims
 
1. A method for detecting the presence of alpha-synuclein aggregation in a human cerebrospinal fluid (“CSF”) sample, the method comprising the steps:
(i) providing a human CSF sample;
(ii) providing a reaction sample comprising:
(a) a population of beads, wherein the population of beads have a mean diameter of the beads from 1 mm+10% to 0.001 mm+10%, and wherein the population of beads are at least one of: (1) exclusive of magnetic beads; and (2) chemically inert;
(b) a fluorophore adapted to bind to protein aggregates and to increase fluorescence when bound to protein aggregates; and
(c) alpha-synuclein or a fragment or variant thereof;
(iii) combining the human CSF 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 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 human CSF sample.