aβ 1 42 amyloid fibrils  (StressMarq)

Journal: bioRxiv

Article Title: Epitope Sequence and Modification Fingerprints of Anti-Aβ Antibodies

doi: 10.1101/2025.02.26.640323

Figure Lengend Snippet: (a) shows the titration of Aducanumab bs at different concentrations to achieve high selectivity for phosphorylated pSer8-Aβ1-15 over unphosphorylated Aβ1-15. The signals were internally normalized and fitted as a sigmoidal curve (Boltzmann). The phospho signal for pSer8-Aβ1-15 is shown in red (r 2 0.98) and the signal for unphosphorylated Aβ1-15 is shown in blue (r 2 0.96). (b) BLI steady-state analysis revealed KDs of 1.4nM and 8.7nM for phosphorylated and unphosphorylated Aβ1-15, respectively (1:1 model) (c) The Biolayer interferometry dose-response curve of Aducanumab binding to the NTA-loaded DAEFRHDSGYEVHHQ-6xHis-amide peptide is shown. (d) The Biolayer interferometry dose-response curve of Aducanumab binding to the NTA-loaded DAEFRHDpSGYEVHHQ-6xHis-amide peptide is shown.

Article Snippet: For the assessment of antibody binding to different Aβ aggregation states, we used Aβ1-42 monomers (Cat. No SPR-485B), Aβ1-42 oligomers (Cat. No SPR-488B) and preformed Aβ1-42 fibrils (Cat. No SPR-487B) (StressMarq biosciences, Victoria, BC, Canada).

Techniques: Titration, Binding Assay

Journal: bioRxiv

Article Title: Epitope Sequence and Modification Fingerprints of Anti-Aβ Antibodies

doi: 10.1101/2025.02.26.640323

Figure Lengend Snippet: (A) HFIP-treated Aβ1-42 monomer (m), oligomer (o) and fibril preparations (f) (1.44 µg each) were separated by 4-12% Bistris SDS-PAGE, blotted onto nitrocellulose and probed with Aducanumab bs (5 min exposure, F 0.84, image display: High: 65535; Low: 0; Gamma: 0.85). Following image recording the blot membrane was reprobed without prior stripping (no removal of primary and secondary antibodies) with mAb4G8 (1 min exposure, image display: High: 54971; Low: 0; Gamma: 0.99). That way the 4G8 signals were essentially added on top of the initial Aducanumab bs signals and background artifacts. The positions of pre-stained protein marker bands on the blot membrane are shown on the left-hand side. (B) Parallel sections from an AD patient were stained with Aducanumab bs and Aducanumab bs that had been pre-adsorbed with either Aβ1-42 monomers (Aβ1-42m) or Aβ1-42 fibrils (Aβ1-42f). Scale bar: 200 µm.

Article Snippet: For the assessment of antibody binding to different Aβ aggregation states, we used Aβ1-42 monomers (Cat. No SPR-485B), Aβ1-42 oligomers (Cat. No SPR-488B) and preformed Aβ1-42 fibrils (Cat. No SPR-487B) (StressMarq biosciences, Victoria, BC, Canada).

Techniques: SDS Page, Membrane, Stripping Membranes, Staining, Marker

Journal: bioRxiv

Article Title: Epitope Sequence and Modification Fingerprints of Anti-Aβ Antibodies

doi: 10.1101/2025.02.26.640323

Figure Lengend Snippet: (A) shows an example of CIEF-immunoassay results for Lecanemab bs displayed as lane view (Western blot simulation). The indicated Aβ peptides were separated by isoelectric focusing in microcapillaries on a Peggy Sue device, immobilized photochemically to the inner capillary wall and subjected to immunological detection. Under the tested conditions, Lecanemab bs detected Aβ1-40 (pI 5.31), Aβ2-40 (pI 5.98), Aβ3-40 (pI 5.97), Aβ-3-40 (pI 6.04), Aβ1-42 (pI approx. 5.3) and pSer8Aβ1-40 (amide) (pI 5.13-5.14). (B) Synthetic Aβ2-40 (361 ng), and monomerized (m), oligomeric (o) and fibrillar (f) preparations of Aβ1-42 (361 ng of each) were separated by 4-12% Bistris SDS-PAGE, blotted onto nitrocellulose and probed with Lecanemab bs (0.5 µg/mL). Exposure: 2 min 7.5 sec; F 0.84; image display: high: 47545; low: 0; gamma: 1.0. After blot development, the blot membrane was reprobed with mAb 4G8 without prior stripping. That way the 4G8 signals were essentially added on top of the initial Lecanemab bs signals and background artifacts. Exposure: 1 min F 0.84; image display: high: 46277; low: 0; gamma: 1.0. (C) The indicated Aβ peptides (50 ng of each) were separated by Bicine Tris (peptide) SDS-PAGE, blotted on PVDF, and probed with Lecanemab bs . Exposure: 5 min F 0.84, image display: high: 65535; low: 0; gamma: 0.85. (D) To confirm loading and successful blotting of all Aβ peptides, the blot membrane was re-probed with mAb 4G8 without prior stripping (see above). Exposure: 5 min F 0.84; image display: high: 65535; low: 0; gamma: 1.0. (E) Parallel sections from an AD patient were stained with Lecanemab bs and Lecanemab bs that had been pre-adsorbed with either Aβ1-42 monomers (Aβ1-42m), Aβ1-42 oligomers (Aβ1-42o) or Aβ1-42 fibrils (Aβ1-42f). Scale bar: 200 µm.

Article Snippet: For the assessment of antibody binding to different Aβ aggregation states, we used Aβ1-42 monomers (Cat. No SPR-485B), Aβ1-42 oligomers (Cat. No SPR-488B) and preformed Aβ1-42 fibrils (Cat. No SPR-487B) (StressMarq biosciences, Victoria, BC, Canada).

Techniques: Western Blot, SDS Page, Membrane, Stripping Membranes, Staining

Journal: bioRxiv

Article Title: Epitope Sequence and Modification Fingerprints of Anti-Aβ Antibodies

doi: 10.1101/2025.02.26.640323

Figure Lengend Snippet: The upper panel shows lane views (Western blots simulation) of a CIEF-immunoassay for assessing the detection of different Aβ variants by (A) mAb 4G8 (positive control antibody) and (B) Donanemab bs . The indicated Aβ variants were separated by isoelectric focusing in microcapillaries on a Peggy-Sue device, immobilized photochemically to the inner capillary wall and subjected to immunological detection by (A) mAb 4G8 and (B) Donanemab bs . (C) Synthetic Aβ peptides (50 ng per lane) were separated by Bicine Tris (peptide) SDS-PAGE, blotted on PVDF and probed with Donanemab bs (0.5 µg/mL) (upper image). Exposure: 5 min F 0.84; image display: high: 59406; low: 0; Gamma: 0.65. To confirm that all Aβ variants were loaded and blotted, the blot membrane was reprobed with mAb 4G8 without prior stripping. That way, the 4G8 signals were essentially added on top of the initial Donanemab bs signals and background artifacts (lower image). Exposure: 5 min F 0.84; image display: high: 65535; low: 0; gamma: 0.77. (D) AβpE3-40 and Aβ1-42 monomer (m), oligomer (o) and fibril preparations (f) were separated by 4-12% Bistris SDS-PAGE, blotted onto nitrocellulose and probed with Donanemab bs (image display: High: 64091; Low: 0; Gamma: 0.75), followed by reprobing without prior stripping with mAb4G8 (image display: High: 46984; Low: 0; Gamma: 1.0). The positions of prestained protein marker bands on the blot membrane are shown on the left-hand side. (E) A pre-adsorption experiment employing synthetic Aβ peptides in human AD brain tissue showed that the immunoreactivity of Donanemab bs was not suppressed by pre-adsorption of the antibody with excess amounts of synthetic Aβ1-40 and Aβ3-40 peptides. In contrast, a pre-incubation with AβpE3-40 effectively attenuated the antibody signal in extracellular Aβ deposits as well as in the vasculature.

Article Snippet: For the assessment of antibody binding to different Aβ aggregation states, we used Aβ1-42 monomers (Cat. No SPR-485B), Aβ1-42 oligomers (Cat. No SPR-488B) and preformed Aβ1-42 fibrils (Cat. No SPR-487B) (StressMarq biosciences, Victoria, BC, Canada).

Techniques: Western Blot, Positive Control, SDS Page, Membrane, Stripping Membranes, Marker, Adsorption, Incubation

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