Surface plasmon resonance (SPR)

The binding strength of the recombinant antibodies to Aβ protofibrils and monomers was further investigated by SPR using Biacore 8 K instrument (Cytiva, Uppsala, Sweden). Soluble Aβ1-42 protofibrils, prepared as described previously [31], were immobilized by amine coupling (NHS/EDC, GE kit #BR100633) on a biacore CM5 sensor chip (Cytiva, Uppsala, Sweden). Three-fold dilution series of the recombinant antibodies (ranging from 100 to 1.23 nM) in  PBS-P+ buffer (pH 7.4) (Cytiva, Uppsala, Sweden) were injected using a single cycle kinetic method at a flow rate of 30 μl/min with an association phase of 120 s, followed by a dissociation phase of 7200 s. Injection of buffer was used as a blank control. The binding data were fitted to a 1:1 kinetic model using Biacore Insight Evaluation. The dissociation rate constant was calculated as an average value from 2–5 measurements. Surfaces were regenerated using 3 M MgCl₂ between cycles. For binding to monomers, the recombinant antibodies were immobilized on the mentioned chips at a fixed concentration of 1 μg/ml. A single cycle kinetic method was used to inject a two-fold dilution series of Aβ1-40 monomers (ranging from 4000 to 250 nM) (Bachem, H-1194, Bubendorf, Switzerland). The steady-state kinetic model was used for analysis.