Simoa plasma NfL measurements

Plasma sample NfL concentration was determined using the in-house Simoa NfL assay, which has been described in detail previously.⁷ Briefly, paramagnetic carboxylated beads (Quanterix, Boston, MA) were coated with a mouse anti-NfL antibody (UD1; UmanDiagnostics, Umeå, Sweden) and incubated for 35 minutes with sample and a biotinylated mouse anti-NfL antibody (UD2; UmanDiagnostics) in a Simoa HD-1 instrument (Quanterix). The bead-conjugated immunocomplex was thoroughly washed before incubation with streptavidin-conjugated β-galactosidase (Quanterix). After additional washes, resorufin β-d-galactopyranoside (Quanterix) was added and the immunocomplex was applied to a multiwell array designed to enable imaging of every single bead. The average number of enzymes per bead (AEB) of samples was interpolated onto the calibrator curve constructed by AEB measurements on bovine NfL (UmanDiagnostics) serially diluted in assay diluent. Samples were analyzed blind and in duplicate using one batch of reagents. The average repeatability coefficient of variation of a sample with the mean concentration 14.3 pg/mL was 7.5% and the interassay coefficient of variation was 12%, and for a sample with a mean concentration of 129.5 pg/mL this was 5.3% and 10.2%, respectively. The limit of detection, determined as the mean blank signal +3 SD for the Simoa NfL assay, was 0.3 pg/mL, and the lower limit of quantification (LLOQ), determined as the mean blank signal +10 SD, was 2.7 pg/mL when compensated for a 4-fold sample dilution. All samples analyzed were above the LLOQ.