Serum Neurofilament Light Chain Quantification

Serum samples were collected from each of the participants and then processed, divided into aliquots, and frozen at −80°C according to standardized procedures. Serum NfL concentrations were measured with the NF-Light assay from UmanDiagnostics (Umeå, Sweden) and transferred onto the Simoa platform with a home-brew kit (Quanterix Corp, Boston, MA). Detailed instructions can be found in the Simoa Homebrew Assay Development Guide (Quanterix). In short, paramagnetic carboxylated beads (catalog no. 100451, Quanterix) were activated by adding 5% (vol/vol) 10 mg/mL 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (catalog no. 100022, Quanterix) to a magnetic beads solution with 1.4×10⁶ beads/μL. After a 30-minute incubation at room temperature, the beads were washed with a magnetic separator, and an initial volume, i.e., 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide + bead solution volume in the previous step, of 0.3 mg/mL ice cold solution of the capture antibody (UD1, UmanDiagnostics) was added. After a 2-hour incubation on a mixer (2,000 rpm, Multi-Tube Vortexer, Allsheng, China) at room temperature, the beads were washed, and an initial reaction volume of blocking solution was added. After 3 washes, the conjugated beads were suspended and stored at 4°C pending analysis. Before analysis, the beads were diluted to 2,500 beads/μL in bead diluent. The detection antibody (1 mg/mL, UD2, UmanDiagnostics) was biotinylated by adding 3% (vol/vol) 3.4 mmol/L EZ‐Link NHS‐PEG4‐Biotin (Quanterix), followed by a 30-minute incubation at room temperature. Free biotin was removed with spin filtration (Amicon Ultra-2, 50 kDa, Sigma, St. Louis, MO), and the biotinylated antibody was stored at 4°C pending analysis. The serum samples were assayed in duplicate on a Simoa HD-1 instrument (Quanterix) using a 2-step assay dilution protocol that starts with an aspiration of the bead diluent from 100 μL conjugated beads (2,500 beads/μL), followed by the addition of 20 μL biotinylated antibody (0.1 μg/mL) and 100 μL of 4-fold diluted sample (or undiluted calibrator) to the bead pellet. For both samples and calibrator, the same diluent was used (phosphate-buffered saline; 0.1% Tween-20; 2% bovine serum albumin; 10 μg/mL TRU Block [Meridian Life Science, Inc, Memphis, TN]). After a 47-cadence incubation (1 cadence = 45 seconds), the beads were washed, followed by the addition of 100 μL streptavidin-conjugated β-galactosidase (150 pmol/L, catalog No. 100439, Quanterix). This was followed by a 7-cadence incubation and a wash. Before reading, 25 μL resorufin β−D-galactopyranoside (catalog No. 100017, Quanterix) was added. The calibrator curve was constructed by use of the standard from the NfL ELISA (NF-Light, UmanDiagnostics) in triplicate. The lower limits of detection and quantification, as defined by the concentration derived from the signal of blank samples (sample diluent) + 3 and 10 SD, were 0.97 and 2.93 pg/mL, respectively. To evaluate the linearity of the assay, 6 different samples were analyzed at 4- (default), 8-, and 16-fold dilution, and the average coefficient of variation for the concentration measured at the different dilutions was 11.5%. All samples were measured as duplicates. The mean coefficient of variation of duplicate concentrations was 4.3%. In addition, a quality control sample was measured in duplicate on each of the 7 runs used to complete the study. The intra-assay coefficient of variation for this sample was <10%. All measurements were performed by board-certified laboratory technicians in one round of experiments using one batch of reagents.