Neurology 4 plex e

Biomarker quantification was conducted using single molecule array (Simoa) HD‐X Analyzers from Quanterix at the Clinical Neurochemistry Laboratory of Sahlgrenska University Hospital in Sweden. A commercially available assay (Quanterix Neurology‐4 Plex E) was used to simultaneously quantify for Aβ42, Aβ40 (and Aβ42/Aβ40, consequently), NfL, and GFAP.
²⁷ (link) P‐tau231 and p‐tau181 were analyzed using Simoa assays developed at the University of Gothenburg, which have been validated as described elsewhere.
⁵ (link),
⁶ (link) To measure p‐tau217, a novel commercially available assay from ALZpath (ALZpathDX) was used.
⁴³ (link) All samples from the same participant were analyzed in the same analytical run, and each sample was quantified in duplicate. Internal quality controls (iQC) at three different concentrations, for each measurand, were analyzed in duplicate in the beginning and end of each run. Before analysis, blood samples were thawed, vortexed, and centrifuged at 4000 × g for 10 minutes as suggested in recent studies.
⁴⁴ (link),
⁴⁵ (link)

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.