Journal: Translational Neurodegeneration
Article Title: Ultrasensitive detection of aggregated α-synuclein using quiescent seed amplification assay for the diagnosis of Parkinson’s disease
doi: 10.1186/s40035-024-00426-9
Figure Lengend Snippet: The detection limit of QSAA. a Schematic of the detection of quiescent amplification products by fluorescence. b , c Direct observation of amplification products under a fluorescence microscope. hPFFs/mPFFs (0.01 μg/ml) were incubated with 1 mg/ml human monomer (HM)/ mouse monomer (MM) and 40 μM ThT. QSAA was carried out at 70℃ for 24 h in the presence or absence of 10% AS. d , e TEM was performed to evaluate the fibrillar structure of the amplification products in ( b , c ). f QSAA using different seeds (100 fg αSyn hPFFs, 100 pg βSyn PFFs, 100 pg γSyn PFFs, 100 pg Aβ PFFs, 100 pg ζ306 (2R) PFFs, 100 pg K19CFh (3R) PFFs, 100 pg mixture of ζ306 PFFs and K19CFh PFFs). Data are presented as mean ± SD ( n = 4). g Normalized maximum ThT fluorescence values for QSAA in ( f ). h The lag phase data from ( g ) were used to calculate the PAR (1/h) for both SAA and QSAA reactions. For reactions that did not produce ThT fluorescence surpassing the threshold (assigned a lag phase of 36 hours), the rate was established at 0.027. i , k Serial dilutions of the hPFFs/mPFFs (equivalent to 1 ng, 10 pg, 1 pg, and 100 fg PFFs) were added to the quiescent incubation system, together with 1 mg/ml HM/MM, 10 mM Tris-HCl pH 7.5, 50 mM NaCl, and 40 μM ThT in a total volume of 20 μl. Fluorescence images were collected at 24 h of QSAA reaction. Panels below the fluorescence images are schematic diagrams illustrating the amplification products. j , l Calculation of the relative fluorescence area of filamentous or lamellar amplification products within circular droplets. Data presented as mean ± SD ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 by one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test
Article Snippet: Human recombinant β-synuclein (βSyn) PFFs (Type 1) (Cat# SPR-457), human recombinant γ-synuclein (γSyn) PFFs (Type 1) (Cat# SPR-459), and human synthetic amyloid β 1-42 (Aβ 1-42 ) PFFs (Cat# SPR-487) were obtained from StressMarq Biosciences (British Columbia, Canada).
Techniques: Amplification, Fluorescence, Microscopy, Incubation, Comparison
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