Abb 135

GAP-43 concentration was measured using an in house sandwich ELISA (Sandelius et al., 2018 ). Nunc-Immuno Polysorp microwell modules (Thermo Fisher Scientific) were coated with mouse anti-GAP-43 antibody (0.77 μg/mL NM4 ((Fujirebio, Ghent, Belgium)) in 50 mM sodium bicarbonate buffer pH 9.6, overnight at 4 °C. After washing, wells were blocked with PBS-T, 1x Casein (diluted from 10x casein blocking buffer, B6429, Sigma-Aldrich) for 1 h at RT. Thereafter, in house recombinant full-length GAP-43 calibrators (78 pg/mL – 50000 pg/mL), blanks, two folds pre-diluted control samples and media samples in assay diluent (PBS-T, 1% BSA) were co-incubated with a rabbit detector antibody (0.14 μg/mL ABB-135 (Nordic Biosite, Täby, Sweden)) overnight at 4 °C. After additional washes, plates were incubated with anti-rabbit HRP (1:30000, Promega) for 1.5 h. After subsequent washes, wells were incubated for 20 min with TMB (TMB, KemEnTech Diagnostics) in the dark. The color reaction was stopped by addition of 100 μL 0.2 M H₂SO₄ and the absorbance was read in a SunriseTM micropl ate absorbance reader (Tecan) at 450 nm (650 nm as reference value).

The ELISA was developed in house combining the mouse monoclonal GAP-43 antibody NM4 (Fujirebio) and a polyclonal GAP-43 antibody (ABB-135, Nordic Biosite, Täby, Sweden) with a C-terminal epitope. For detailed description, see Supplementary Material. The analyses were performed by board-certified laboratory technicians blinded to clinical information. During sample runs in the clinical evaluation study, the repeatability CV% of control samples, with the concentrations 3157 pg/mL and 857 pg/mL, was 5.5% versus 11% and the interassay CV% was 6.9% versus 15.6% across 18 ELISA plates.