Simoa kit

Cerebrospinal fluid (CSF) and plasma samples were collected in the morning after overnight fasting. After centrifugation, the samples were divided into 200 μL aliquots in polypropylene tubes and stored at −80°C until measurements were taken. CSF and plasma Aβ40, Aβ42, P-tau181, and total tau were measured on the HD-X analyzer (Quanterix) using Simoa kits (Quanterix, 103714, 101195). CSF albumin levels were measured using commercial assay kits (Thermo Fisher Scientific). Serum albumin levels were determined using a bromocresol green dye-binding assay (ADVIA 1800; Siemens, Berlin, Germany). The Qalb value was calculated using the following formula: (CSF albumin/serum albumin) × 1,000.

Lumbar CSF sampling and analysis were conducted according to the Alzheimer’s Association Flow Chart.^{37 (link)}
As previously described, the analysis of P-tau217 assays was performed using the Meso Scale Discovery (MSD) platform.^{38 (link)}
Total tau (T-tau) was quantified using Innotest® immunoassay (Fujirebio; Gent, Belgium). CSF Aβ42 and Aβ40 levels were measured using MSD or Fujirebio Lumipulse assays according to the manufacturer’s instructions, and the ratio values were subsequently calculated. NfL was measured using a Simoa kit (Quanterix; Billerica, MA). NPTX2 was measured in the facilities of ADx (Gent, Belgium) using a research grade sandwich ELISA. The number of available data per group for each of the CSF and imaging biomarkers together with the corresponding mean ± SD values are provided in Supplementary Table 3.

Plasma p-tau217 concentration was measured according to the published protocols using immunoassay on a Mesoscale Discovery platform developed by Lilly Research Laboratories.^{17 (link),30 (link)} Briefly, biotinylated-IBA493 was used as a capture antibody and SULFO-TAG-4G10-E2 (anti-Tau) as the detector, and samples were diluted 1:2. The assay was calibrated with a synthetic p-tau217 peptide. Plasma GFAP concentration was quantified using a Simoa kit (Quanterix) according to the manufacturer’s instructions. Plasma Aβ42/Aβ40, NfL, and t-tau were analyzed with a Simoa 4-plex kit (Quanterix) in plasma samples from 258 participants with DS and 28 non-DS siblings as previously described.^{12 (link)} Plasma sampling and analysis are further described in the eMethods in the Supplement. All samples were analyzed by staff who were blinded to the clinical and imaging data.

All clinical samples were analyzed in parallel using the Simoa Aβ_1–42 assay described above and the commercial Simoa kit from Quanterix that employs different antibody reagents for Aβ_1–42 capture (specific to the N-terminus of Aβ_1–42) and detection (specific to the C-terminus of Aβ_1–42). The Aβ_1–42 concentrations quantified from both assays were compared for all 84 samples tested. Additionally, on the basis of the results of clinical sample analysis, theoretical projections were made of each assay’s ability to quantify decreasing concentrations of Aβ_1–42 resulting from treatment with Aβ-lowering therapeutics.

Analysis of NfL concentration was performed with the NfL single molecule array (Simoa) kit (Quanterix^®, Billerica, USA) according to the manufacturer’s instructions. In brief, all samples were spun at 10 000×g for 5 minutes to precipitate debris and transferred to wells in 96-well Quanterix^® plates for duplicate tests and to run with on-board 4 × dilution (serum) and 2 × dilution (urine). Analysis in urine was performed in FTD and PPD (n = 38) to assess if NfL was detectable in urine and inter-assay variation was monitored using internal quality controls (serum) at two levels.