Sensitive Measurement of Aβ Peptides in nEVs

Our pre-experiment results suggested that the electrochemiluminescence method was not sufficiently sensitive to detect Aβ and t-tau in nEVs (K15199E and K15121D, respectively; MSD; data not shown). Therefore, we used two single-molecule array kits (Simoa; Quanterix, Billerica, MA, USA): Neurology 4-Plex E and pTau-181 V2, to measure nEV proteins. Notably, compared to previous single-factor or tri-factor kits, the former was newly developed for highly specific and sensitive measurement of the concentrations of full-length Aβ_1–42 and Aβ_1–40 [27 (link)]. All assays were conducted in duplicate, and the quality control is described in Additional file 1: Supplementary material and shown in Additional file 1: Table S2. Unexpectedly, based on quality control, p-tau181 and neurofilament light (NFL) results were both excluded, and only Aβ₄₀ and Aβ₄₂ results were included in the subsequent analysis. We did not analyze glial fibrillary acidic protein, as this is an astrocytic marker.