Innotest enzyme linked immunosorbent assay elisa

CSF samples were collected between 8:00 a.m. and 2:00 p.m. without requiring fasting. This window was chosen because CSF Aβ42 levels during these times represent approximately 95–105% of average Aβ42 over time [32 (link)]. CSF was immediately aliquotted after collection and before freezing, and otherwise we used the ADNI biofluid protocols, including the use of 24-gauge Sprotte needles, aspiration syringes, and transfer into 15-ml polypropylene tubes. To avoid measurement bias related to well positions, sample positions were randomized for each biomarker assay.
CSF biomarkers were measured following the manufacturers’ protocols: Aβ42, t-tau, and p-tau₁₈ levels were measured using the INNO-BIA AlzBio3 immunoassay (Fujirebio, Malvern, PA, USA) in a Luminex 200 platform (Luminex, Austin, TX, USA) [33 (link)] (average interplate coefficients of variation of 13% for Aβ42, 10% for t-tau, and 11% for p-tau₁₈₁), Aβ40 levels using the INNOTEST® enzyme-linked immunosorbent assay (ELISA) (Fujirebio), α-synuclein levels using the Novex® ELISA (Thermo Fisher Scientific, Waltham, MA, USA), NfL levels using the NF-light® ELISA (Uman Diagnostics, Umeå, Sweden), and sICAM-1 and sVCAM-1 levels using a commercial multiplex kit (MILLIPLEX® MAP Human Neurodegenerative Magnetic Bead Panel 3 [HNDG3MAG-36 K]; EMD Millipore, Billerica, MA, USA).

The concentrations of tau and NfL in plasma were measured using the NF-light and Total Tau 2.0 kits using the Simoa platform (Quanterix, Billerica, MA) as previously described in detail [20 (link)]. Calibrators were run in duplicates, while samples were run in singlicates with a 4 fold dilution. Two quality control (QC) samples from plasma were run in duplicates in the beginning and the end of each run. For NfL, between-run precision was 6.0% at 8.5 pg/mL and 5.1% at 121 pg/mL, while for T-tau, between-run precision was 7.3% at 32.2 pg/mL and 7.0% at 7.5 pg/mL. Plasma NSE, serum S100B and CSF S100B and NSE concentrations were measured as singlicates on the cobas Elecsys platform, according to the manufacturer’s recommendations. CSF T-tau was measured using the INNOTEST enzyme-linked immunosorbent assay (ELISA, Fujirebio Europe, Ghent, Belgium), while CSF NfL was measured using an in-house ELISA as described previously in detail [21 (link)]. Elecsys- and ELISA-based methods were chosen for biomarkers for which Simoa ultrasensitivity was not needed. Between-run precision was <10% for all these assays. All measurements of CSF and plasma/serum concentrations were performed in one round of experiments using a single batch of reagents for each assay by board-certified laboratory technicians who were blinded to clinical data.