alpha synuclein aggregate antibody  (StressMarq)

Journal: Cell Reports Medicine

Article Title: Single extracellular vesicle detection assay identifies membrane-associated α-synuclein as an early-stage biomarker in Parkinson’s disease

doi: 10.1016/j.xcrm.2025.101999

Figure Lengend Snippet: Identification of α-synuclein as a serum EV membrane-associated protein (A) Representative images and quantification of membrane-associated α-synuclein (α-Syn) and the internal EV marker syntenin-1 on CD9+ EVs or L1EVs isolated from serum using the droplet assay. Anti-α-Syn antibody clone A17183A, which recognizes mainly the aggregated forms of α-Syn under native conditions (Ab1), or clone LB509, which recognizes a C-terminal region spanning amino acid 115–125 of total α-Syn (Ab2), was used; n = 5 individuals with PD tested; scale bar, 100 μm. (B) Assay calibration of L1EV membrane-associated α-Syn using Ab1 or Ab2 at various EV concentrations as measured by NTA; n = 3 individuals with PD tested. LOD and LOQ values are summarized in Table S1 . (C) SEC illustration, MSD electrochemiluminescence detection of α-Syn and the EV marker CD81, and immunoblotting of 10 eluted fractions (F) from 1 mL of serum. (D and E) (D) MSD electrochemiluminescence detection of α-Syn in neat serum and pooled SEC F3+F4 and (E) detection of α-Syn on L1EVs immunocaptured from neat serum or pooled SEC F3+F4 using the droplet assay; also see Figure S7 ; n = 3 individuals with PD tested. (F) Immunocapture using anti-CD9, anti-L1CAM, and anti-α-Syn from F3+F4 followed by immunoblotting, with beads alone (BA) used as a control. (G) Representative image of a single EV obtained with dSTORM and corresponding quantification showing the % colocalization of L1CAM and α-Syn on Tetraspanin Trio+ (Tetra Trio+) EVs from SEC F3+F4; pooled serum from 5 individuals with PD was tested; scale bar, 100 nm. Data are represented as mean ± SD. Statistical significance was determined by one-way ANOVA (A) or student’s two-tailed t test (D).

Article Snippet: Anti-aggregated-α-synuclein antibody (5G4) , Millipore , Cat# MABN389; RRID: AB_2716647.

Techniques: Membrane, Marker, Isolation, Electrochemiluminescence, Western Blot, Control, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Single extracellular vesicle detection assay identifies membrane-associated α-synuclein as an early-stage biomarker in Parkinson’s disease

doi: 10.1016/j.xcrm.2025.101999

Figure Lengend Snippet: L1EV membrane-associated α-synuclein is increased under pathological conditions (A) Immunoblotting of RIPA-soluble fraction from SH-SY5Y cells expressing PD-associated α-synuclein (α-Syn) mutations A53T, E46K, G51D, and A30P. (B) A53T and E46K mutant α-Syn increased the L1EV membrane-associated α-Syn/CD81 ratio compared to non-transduced control (NTC), wild-type (WT), G51D, and A30P as measured by the droplet assay. Confirmation of CM L1EV immunocapture is shown in Figure S8 . (C) Expression of 3K mutant α-Syn in SH-SY5Y cells increased the membrane (memb)-to-cytosol (cyto) ratio compared to the expression of the 3D mutant α-Syn; n = 5 independent experiments. (D) 3K mutant α-Syn increased the L1EV membrane association and α-Syn/CD81 ratio compared to WT and 3D, whereas expression of 3D mutant α-Syn decreased L1EV membrane association as measured by the droplet assay. (E) Quantification by dSTORM of membrane-associated α-Syn on individual EVs using antibody clone 5G4, which recognizes mainly the aggregated forms of α-Syn under native conditions. (F) Confocal images and corresponding quantification of iPSC-derived SNCA ISO and SNCA TRIP dopaminergic neurons stained with MAP2 and α-Syn, showing an approximately 2-fold increase in α-Syn immunofluorescence in the SNCA TRIP line; scale bar, 5 μm. (G) Immunoblotting showed an approximately 2-fold increase in endogenous α-Syn expression when normalized to GAPDH in SNCA TRIP neuronal lysates comparted to isogenic controls. (H) NTA of EVs in the neuronal CM showed that the total EV concentration is similar between the two lines and is not affected by pathological α-Syn expression. (I) Representative images of membrane-associated α-Syn and CD81 detection on L1EVs from neuronal CM as measured by the droplet assay; scale bar, 100 μm. (J) Membrane-associated α-Syn was increased in L1EVs isolated from SNCA TRIP neurons compared to SNCA ISO control L1EVs as measured by the droplet assay. (K) No difference in membrane-associated CD81 in L1EVs from SNCA TRIP neurons compared to SNCA ISO control L1EVs as measured by the droplet assay, in agreement with the NTA results. (L) L1EV membrane-associated α-Syn/CD81 ratio was increased by approximately 2-fold in SNCA TRIP when compared to SNCA ISO control L1EVs as measured by the droplet assay. n = 3 independent experiments (SH-SY5Y cells) unless stated otherwise or n = 3 independent differentiations (iPSC-derived neurons). Data are represented as mean ± SD. Statistical significance was determined by Kruskal-Wallis test (C), one-way ANOVA (B, D, and E), or student’s two-tailed t test (F–J and L). See also Figure S9 for further analysis.

Article Snippet: Anti-aggregated-α-synuclein antibody (5G4) , Millipore , Cat# MABN389; RRID: AB_2716647.

Techniques: Membrane, Western Blot, Expressing, Mutagenesis, Control, Derivative Assay, Staining, Immunofluorescence, Concentration Assay, Isolation, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Single extracellular vesicle detection assay identifies membrane-associated α-synuclein as an early-stage biomarker in Parkinson’s disease

doi: 10.1016/j.xcrm.2025.101999

Figure Lengend Snippet: Serum L1EV membrane-associated-α-synuclein is increased in individuals with iRBD or PD (A and B) (A) Boxplot and (B) corresponding receiver operating characteristic (ROC) curves of L1EV membrane-associated α-synuclein (α-Syn) measurements using the LB509 antibody for healthy controls (HCs) and individuals with iRBD or PD. (C) Boxplot of L1EV membrane-associated CD81 measurements for HCs and individuals with iRBD or PD. (D and E) (D) Boxplot and (E) corresponding ROC curves of L1EV membrane-associated α-Syn/CD81 ratio for HCs and individuals with iRBD or PD. (F) Correlation between total α-Syn levels in serum L1EV lysates as measured by MSD electrochemiluminescence and L1EV membrane-associated α-Syn/CD81 ratio as measured by the droplet-based immunoassay. n = 20 individuals per group. The midline of the boxplots indicates the median, and the box indicates the 25th and 75th percentiles (A, C, and D). Statistical significance was determined by Kruskal-Wallis test (A, C, and D). Least-squares regression line with 95% confidence interval (CI) was plotted, and Spearman coefficient was reported (F). Data on assay reproducibility are shown in Figure S10 .

Article Snippet: Anti-aggregated-α-synuclein antibody (5G4) , Millipore , Cat# MABN389; RRID: AB_2716647.

Techniques: Membrane, Electrochemiluminescence

Journal: Cell Reports Medicine

Article Title: Single extracellular vesicle detection assay identifies membrane-associated α-synuclein as an early-stage biomarker in Parkinson’s disease

doi: 10.1016/j.xcrm.2025.101999

Figure Lengend Snippet:

Article Snippet: Anti-aggregated-α-synuclein antibody (5G4) , Millipore , Cat# MABN389; RRID: AB_2716647.

Techniques: Virus, Recombinant, Electron Microscopy, Bicinchoninic Acid Protein Assay, Software, Membrane, Polymer, Lysis, Extraction, Western Blot, Conjugation Assay

Journal: Brain Communications

Article Title: Single-molecule characterization of salivary protein aggregates from Parkinson’s disease patients: a pilot study

doi: 10.1093/braincomms/fcae178

Figure Lengend Snippet: Illustration of the SiMPull method. ( A ) Prepared glass coverslip with NeutrAvidin bound to its surface and F-127 passivation. ( B ) Application of biotinylated capture antibodies, either LB509 or 6E10 antibody is used for the detection of α-synuclein- or Aβ-containing aggregates, respectively. ( C ) Application of saliva biofluid to the coverslip; Aβ- or α-synuclein-containing aggregates are specifically captured by the relevant immobilized antibodies on the coverslip surface. ( D ) Alexa-647-labelled LB509 and 6E10 antibodies are applied to the surface for aggregate detection. ( E ) Representative diffraction-limited image captured using Total Internal Reflection Fluorescence (TIRF) microscope set-up. ( F ) Representative super-resolved aggregates.

Article Snippet: All capture antibodies were diluted to 10 nM concentration in PBS. α-Synuclein aggregates were detected using Alexa 647–labelled LB509 antibodies (Santa Cruz, sc-58480, 647).

Techniques: Fluorescence, Microscopy

Journal: Brain Communications

Article Title: Single-molecule characterization of salivary protein aggregates from Parkinson’s disease patients: a pilot study

doi: 10.1093/braincomms/fcae178

Figure Lengend Snippet: Analysis of diffraction-limited single-aggregate counting for Subgroup 1. Each FoV is 2500 µm 2 and 16 fields of view were measured for each participant, Parkinson’s disease n = 10 and control n = 10. ( A ) A non-significant increase in the number of α-synuclein-containing aggregates was observed in the saliva of people with Parkinson’s disease compared with controls ( W = 75, P = 0.063), and there was no difference in the number of Aβ-containing aggregates ( W = 45, P = 0.74) ( B ). ( C ) The ratio of the number of α-synuclein to Aβ aggregates is significantly increased 2.2-fold in people with Parkinson’s disease compared with controls ( W = 79, P = 0.029, r = 0.49). ( D ) ROC analysis of ratio values (AUC = 0.79). αS, α-synuclein; Aβ, amyloid-β; AUC, area under the curve; PD, Parkinson’s disease.

Article Snippet: All capture antibodies were diluted to 10 nM concentration in PBS. α-Synuclein aggregates were detected using Alexa 647–labelled LB509 antibodies (Santa Cruz, sc-58480, 647).

Techniques: Control

Journal: Brain Communications

Article Title: Single-molecule characterization of salivary protein aggregates from Parkinson’s disease patients: a pilot study

doi: 10.1093/braincomms/fcae178

Figure Lengend Snippet: Morphological analysis of α-synuclein and Aβ-containing aggregates from Subgroup 1 using dSTORM super-resolution imaging. For both types of aggregates, we compare the cumulative frequency curves for the morphological feature of interest. The two distributions are subtracted from each other to demonstrate how the two curves differ; the point of maximum difference is then used as a threshold to distinguish groups of morphologically distinct aggregates. ( A–H ) α-Synuclein. We find no difference in the size (area, A, B, E ) or shape (circularity, C, D, G ) of α-synuclein-containing aggregates (Parkinson’s disease n = 9, control n = 9). For Aβ-containing aggregates ( I–P , Parkinson’s disease n = 9, control n = 8), we show that the area distribution differs between the two groups and that the two groups maximally differ from each other at 0.03 µm 2 ( I, J, M ). Using this value as a cut-off, we show that saliva from patient with Parkinson’s disease contains a greater proportion of aggregates >0.03 µm 2 ( t (9.75) = 2.43, P = 0.036, d = 1.15). ROC analysis demonstrates that aggregate size can distinguish between Parkinson’s disease and controls ( N , AUC = 0.76). Shape data analysis shows a visible difference between circularity distributions ( K , L , O ), we find that the two distributions are maximally different from each other at a circularity value of 0.4 ( t (15) = 2.48, P = 0.025, d = 1.23), and ROC analysis shows that shape data can distinguish Parkinson’s disease from controls ( P , AUC = 0.76). αS, α-synuclein; Aβ, amyloid-β; AUC, area under the curve; PD, Parkinson’s disease.

Article Snippet: All capture antibodies were diluted to 10 nM concentration in PBS. α-Synuclein aggregates were detected using Alexa 647–labelled LB509 antibodies (Santa Cruz, sc-58480, 647).

Techniques: Imaging, Control

Journal: Brain Communications

Article Title: Single-molecule characterization of salivary protein aggregates from Parkinson’s disease patients: a pilot study

doi: 10.1093/braincomms/fcae178

Figure Lengend Snippet: Analysis of the combined single-aggregate count and super-resolution morphological data for Subgroup 1. A combined discriminator is calculated for each participant by multiplying the α-synuclein/Aβ aggregate ratio by the proportion of Aβ-containing aggregates satisfying both morphological feature thresholds (area >0.03 µm 2 and circularity <0.4, Parkinson’s disease n = 9, control n = 8). ( A ) The combined discriminator is significantly higher in the PD group (4.3-fold increase, t (8.83) = 2.88, P = 0.018, d = 1.36), ( B ) applying ROC analysis demonstrates that this metric can accurately distinguish between Parkinson’s disease and control participants (AUC = 0.89). AUC, area under the curve; PD, Parkinson’s disease.

Article Snippet: All capture antibodies were diluted to 10 nM concentration in PBS. α-Synuclein aggregates were detected using Alexa 647–labelled LB509 antibodies (Santa Cruz, sc-58480, 647).

Techniques: Control

Journal: Brain Communications

Article Title: Single-molecule characterization of salivary protein aggregates from Parkinson’s disease patients: a pilot study

doi: 10.1093/braincomms/fcae178

Figure Lengend Snippet: Analysis of diffraction-limited single-molecule aggregate counting, super-resolution morphological data and combined discriminator data for Subgroup 2. For diffraction-limited data, each FoV is 2500 µm 2 and 16 fields of view a captured for each participant, n = 10 Parkinson’s disease and 10 control. ( A ) The ratio of the number of α-synuclein to Aβ aggregates is significantly higher in patients with Parkinson’s disease (1.9-fold increase, t (12.68) = 2.42, P = 0.031, d = 1.08). ( B ) ROC analysis of diffraction-limited ratio values (AUC = 0.77). For super-resolution data, analysis was completed as described in (Subgroup 2 Parkinson’s disease n = 9 and control n = 9). ( C ) For Aβ-containing aggregates, we show that there is a different area distribution between the two groups, applying the threshold values identified in Subgroup 1 and ( D ) we show that there is significantly more Aβ aggregates >0.03 µm 2 in the Parkinson’s disease group ( t (8.365) = 2.852, P = 0.02, d = 1.345). ( E ) For Aβ shape data, analysis shows a visible difference between circularity distributions, the Parkinsons disease group has more Aβ with a circularity <0.4 ( F , t (10.43) = 2.901, P = 0.015, d = 1.367). Full analysis, including α-synuclein data, is shown in . A combined discriminator was calculated as previously described in . ( G ) The combined discriminator is significantly higher in the Parkinson’s disease group (4-fold increase, t (10.43) = 2.901, P = 0.025, d = 1.367), ( H ) applying ROC analysis demonstrate that this metric can accurately distinguish between Parkinson’s disease and controls (AUC = 0.86). Aβ, amyloid-β; AUC, area under the curve; PD, Parkinson’s disease.

Article Snippet: All capture antibodies were diluted to 10 nM concentration in PBS. α-Synuclein aggregates were detected using Alexa 647–labelled LB509 antibodies (Santa Cruz, sc-58480, 647).

Techniques: Control

Journal: Brain Communications

Article Title: Single-molecule characterization of salivary protein aggregates from Parkinson’s disease patients: a pilot study

doi: 10.1093/braincomms/fcae178

Figure Lengend Snippet: Analysis of combined single-molecule aggregate count data and super-resolution morphological data for data combined across Subgroups 1 and 2. ( A ) The α-synuclein/Aβ aggregate ratio was significantly higher in when the subgroups were combined. ( B ) The combined discriminator was calculated, as previously described (Parkinson’s disease n = 18, control n = 17), the combined discriminator was increased 5-fold in the Parkinson’s disease group ( W = 41, P < 0.001). ( C ) Applying ROC analysis demonstrated that this metric can accurately distinguish between Parkinson’s disease and controls (AUC = 0.86). αS, α-synuclein; Aβ, amyloid-β; AUC, area under the curve; PD, Parkinson’s disease.

Article Snippet: All capture antibodies were diluted to 10 nM concentration in PBS. α-Synuclein aggregates were detected using Alexa 647–labelled LB509 antibodies (Santa Cruz, sc-58480, 647).

Techniques: Control

Journal: Brain Communications

Article Title: Single-molecule characterization of salivary protein aggregates from Parkinson’s disease patients: a pilot study

doi: 10.1093/braincomms/fcae178

Figure Lengend Snippet: Tests for correlation between patient demographic and clinical scores and measures of α-synuclein/Aβ ratio, proportion of Aβ aggregates meeting morphology thresholds (threshold Aβ) and the combined discriminator

Article Snippet: All capture antibodies were diluted to 10 nM concentration in PBS. α-Synuclein aggregates were detected using Alexa 647–labelled LB509 antibodies (Santa Cruz, sc-58480, 647).

Techniques: Control

Journal: STAR Protocols

Article Title: Protocol for screening α-synuclein PET tracer candidates in vitro and ex vivo

doi: 10.1016/j.xpro.2023.102788

Figure Lengend Snippet: Immunofluorescence of α -syn and Tau aggregates in cultured neurons Neurons were transduced with virus and Pffs as mentioned in the steps, the control group was only treated with AAV-hTau or AAV-α-Syn. Then neurons were fixed and immunostained with p-α -syn 129 (right) or AT8 (middle). Inclusions were shown as dense and dotted aggregates in cell bodies and neurites. The scale bar is 20 μm.

Article Snippet: Anti-aggregated α-synuclein antibody (clone 5G4) (1:200) , Millipore , RRID: AB_2716647.

Techniques: Immunofluorescence, Cell Culture, Transduction, Virus, Control

Journal: STAR Protocols

Article Title: Protocol for screening α-synuclein PET tracer candidates in vitro and ex vivo

doi: 10.1016/j.xpro.2023.102788

Figure Lengend Snippet:

Article Snippet: Anti-aggregated α-synuclein antibody (clone 5G4) (1:200) , Millipore , RRID: AB_2716647.

Techniques: Ubiquitin Proteomics, Virus, Plasmid Preparation, Recombinant, Bicinchoninic Acid Protein Assay, Modification, Suspension, In Vitro, Ex Vivo, Injection, Control, Software, Spectrophotometry, Fluorescence, Sterility, Ointment