Innotest elisa

CSF was collected at the time of MCI diagnosis in all patient groups. CSF was obtained by LP following a standard procedure at both centers. Part of the CSF was processed for routine AD biomarker analysis, and the remainder was divided into aliquots and stored according to international CSF biobanking procedures^{24 (link)} until RT-QuIC analysis.
At ISNB, CSF t-tau, p-tau, Aβ₄₂, and Aβ₄₀ were measured by automated chemiluminescent enzyme immunoassay on the Lumipulse G600 platform (Fujirebio, Ghent, Belgium). The Aβ₄₂:Aβ₄₀ ratio was calculated as previously described.^{25 (link)} At VUmc, CSF Aβ₄₂, p-tau, and t-tau concentrations were determined with Innotest ELISA (Fujirebio) or Elecsys assays (Roche Diagnostics, GmbH, Mannheim, Germany) run on the Cobas e601 analyzer (Roche Diagnostics, Basel, Switzerland).^{26 (link)} Pathologic values for the AD core markers were determined according to internally validated cutoff values at both centers.^{26 (link)- (link)28 (link)}

In the CSF samples collected for the study, Aβ42, t-tau, and p-tau levels were previously measured by using INNOTEST ELISA (Fujirebio Europe, Gent, Belgium) at the Laboratory of Clinical Neurochemistry, Department of Medicine and Surgery, University of Perugia (Perugia, Italy). Depending on the lumbar puncture date, CSF samples were classified as A+ for Aβ42 ≤ 800 (2011–2016) or ≤ 1200 (2008–2010) pg/mL, T+ for p-tau ≥ 60 pg/mL and N+ for t-tau ≥ 400 (2011–2016) or ≥ 200 (2008–2010) pg/mL.

In both PREVENT-AD and ADNI, CSF was obtained by lumbar puncture following an overnight fast. Levels of Aβ-42, p-Tau, and t-Tau were measured by the Innotest^® ELISA (enzyme-linked immunosorbent assays, Fujirebio) [40 (link)] and the Roche Elecsys CSF immunoassays (data file UPENNBIOMK9_04_19_17.csv) [41 (link),42 (link)], for PREVENT-AD and ADNI, respectively. Of note, the Elecsys Aβ-42 CSF immunoassay is currently under development for investigational use only and has an upper technical limit of 1700 pg/mL. Values above this limit are based on extrapolation of the calibration curve, and the performance of these values has not been formally established. These are still included in this study. In PREVENT-AD, Aβ-40 levels were further assessed by the MSD^® MULTI-SPOT Assay System (V-PLEX Plus Aβ Peptide Panel 1 (6E10) Kit, MesoScale, Rockville, USA).

CSF Aβ_1-42 and t-tau were measured locally with INNOTEST ELISA or INNOBIA AlzBio3 (Fujirebio, Ghent, Belgium). Cut-offs for Aβ_1-42 and t-tau were cohort-specific in EMIF-AD MBD [12 (link)]. Aβ_1-42 cut-offs were determined for each cohort using Gaussian mixture modelling [20 (link)]. We measured neurogranin, neurofilament-light, YKL-40, Aβ₃₈ and Aβ₄₀ by ELISA [2 (link)]. We performed untargeted mass spectrometry using tandem mass tag (TMT) with 10 + 1 plexing as previously described, using high-pH reverse phase HPLC for peptide prefractionation [20 (link), 21 (link)]. Peptides were mapped to 2535 proteins using the software ProteomeDiscoverer v.2.2. (Thermo Scientific), using Mascot (MatrixScience) for protein identification (precursor Dm tolerance 15 ppm, fragment tolerance 0.05 Da, max missed cleavage sites 2), searching the human subset of the UniProtKB SwissProt database (www.uniprot.org). Percolator (MatrixScience) was used for scoring peptide specific matches, and a strict 1% false discovery rate (FDR) was set as threshold for identification. For reporter ion quantification the following settings were used: Integration tolerance 20 ppm; Integration Method Most Confident Centroid; normalize on the reference protein average. The median (IQR) CV for these proteins was 5.6 (3.8, 8.0).

CSF data were available for participants with signs of cognitive decline who had been referred to the Memory Clinic at Skåne University Hospital for further investigation. With lumbar puncture, CSF was collected between 1995 and 2015 according to a structured protocol.^{19 (link)} CSF concentrations of Aβ42 were measured using INNOTEST ELISA (Fujirebio Europe, Ghent, Belgium).
Cut-off values for CSF Aβ42 were established using mixture modeling.^{17 (link)- 22 (link)} Because of a slight, assay-dependent drift in levels of CSF Aβ42 during the collection period 1995–2015, 2 different cutoffs were established for the period 1995–2003 (Aβ42 < 484.8 pg/mL) and 2004–2015 (Aβ42 < 577.1 pg/mL). This drift in INNOTEST CSF Aβ42 values during this period is well known.^{19 (link)}