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Comment
. 2023 Nov 2;146(11):e95-e97.
doi: 10.1093/brain/awad169.

Methodological considerations in neuronal extracellular vesicle isolation for α-synuclein biomarkers

Shijun Yan  1   2 , Cheng Jiang  1   2 , Jason J Davis  3 , George K Tofaris  1   2
Affiliations
Comment

Methodological considerations in neuronal extracellular vesicle isolation for α-synuclein biomarkers

Shijun Yan et al. Brain. .
No abstract available

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Conflict of interest statement

The authors report no competing interests.

Figures

Figure 1
Figure 1
α-Synuclein concentration in all extracellular vesicles isolated from serum by size exclusion chromatography. (A) Size exclusion chromatography spectra of 10 eluted fractions (F) from 1 ml of serum. (B) Extracellular vesicles were measured in each fraction using nanoparticle tracking analysis. (C) Immunoblotting for syntenin-1 (Synt-1) and CD9 in each fraction after total protein precipitation using methanol-chloroform extraction. (D) Profiling of each unprocessed fraction with duplexed MSD assay demonstrates that CD81 is detected in early fractions, which also contain very low levels of α-synuclein (<50 pg/ml) whereas later fractions are enriched in free α-synuclein (up to ∼3000 pg/ml).
Figure 2
Figure 2
α-Synuclein concentration in the L1CAM-positive serum extracellular vesicle subpopulation. (A) Immunocapture using anti-L1CAM antibodies from Fractions (F) 2 to 7 covering the two identified peaks on the size exclusion chromatography (SEC) spectra shows that full-length L1CAM was present in all fractions but co-immunoprecipitated with EV proteins syntenin-1 and CD9 only in F3 and F4 as shown by immunoblotting. (B) L1CAM immunocaptured EVs from Fractions 2 to 4 visualized with transmission electron microscopy (left). No EVs were detected with anti-HA antibodies used as control (right). (C) Nanoparticle tracking analysis of EVs eluted following L1CAM immunocapture from Fractions 2 to 4. (D) α-Synuclein concentration in the EV subpopulation immunocaptured with anti-L1CAM antibodies from pooled Fractions 2 to 4 (n = 4 healthy volunteers tested, two replicates for each individual).
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