Elisa plates

ELISA plates (Sarstedt, Nümbrecht, Germany) were coated overnight with 2 μg/ml purified and refolded GlyRαl or α2 ECD. ELISA plates were washed 5x with H₂O_dest. and blocked with 10% BSA diluted in PBS containing 0.05% Tween 20 for 1 h at 37°C. Wells were incubated with patient serum (1:100 in blocking solution) or GlyR pan-α antibody MAb4a (1:500) for 1 h at 37°C. The supernatants were transferred three times into fresh coated wells subsequent to incubation with GlyRα1/α2 and GFP co-transfected HEK293 cells or primary spinal cord neurons. The ELISA plates were further processed with washing steps in PBS and secondary antibody goat anti-mouse (1:20,000 in blocking, 115–035-146, RRID:AB_2307392, Dianova, Hamburg, Germany) or goat anti-human HRP (1:20,000 in blocking, 109–035-088, RRID:AB_2337584, Dianova, Hamburg, Germany) incubation for 1 h at 37°C. TMB solution (00–4,201–56, Thermo Fisher Scientific, Waltham, MA, United States) was added and reaction was stopped after 15 min with 1 M H₃PO₄. Absorbance was read with a Wallac 1,420 Victor2 Microplate Reader (Perkin Elmer, Waltham, MA, United States) at 450 nm. As controls, uncoated ELISA plates have been used with the same experimental procedure to test for specificity. Absorbance data upon binding to plates without the target protein have been subtracted from values exhibited from ELISA plates with GlyR ECDs bound.

ELISA plates (SARSTEDT) were coated overnight at 37°C with 250 ng/well of mis-disordered purified tau 151-391/4R. Peptide competitors (>95% purity; EZBiolab, Carmel, IN, USA) were dissolved in PBS at a final concentration of 1 mM. A 200 μM solution of peptides in PBS/Tween 20 were filled in wells of polypropylene microtitre plates (Greiner Bio-One). The mAb DC8E8 was diluted to a concentration of 0.6 μg/ml (3.8 nM) in PBS, and 60 μl of the diluted antibody solution was mixed with 40 μl of peptide solution in the polypropylene plate. The antibody/peptide mixtures were incubated for 1 hour at 25°C. Subsequently, 50 μl/well of antibody/peptide mixtures were transferred onto mis-disordered tau 151-391/4R-coated ELISA plates (in duplicates) and incubated for 1 hour at 25°C. Bound DC8E8 was detected using polyclonal goat anti-mouse Ig/HRP (Dako) with the chromogenic substrate o-phenylenediamine (Sigma-Aldrich).