Surface plasmon resonance

Measurements of the affinity of aβComAb GW-23B7 to Aβ oligomeric conformers and monomeric Aβ were determined by surface plasmon resonance (SPR) using previously described methods [24]. Experiments were performed using a Reichert SR7000DC refractometer system (Reichert Technologies, Depew, NY, USA) equipped with a CM5 sensor chip. Acetate buffer was used for immobilization of the peptide Aβ₄₂ (ligand). We used Aβ₄₂ for these binding studies (not Aβ₄₀) because Aβ₄₂ species are the major constituents of amyloid plaques, with Aβ₄₀ species being more common in vascular amyloid [25, 26], although some reports suggest that Aβ₄₀ species overall can be the most abundant in the AD cortex [27, 28]. Importantly, Aβ₄₂ species are considered critical for seeding of amyloid deposits [4, 29]. Carboxymethyl dextran on a CM5 gold sensor chip was activated by injecting a solution of 10 mg/ml NHS and 40 mg/ml 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide over the sensor chip surface for 7 minutes at a flow rate of 20 μl/minute. Then the ligand, 50 μg/ml hexafluoroisopropanol-treated synthetic peptide Aβ₄₂ (monomeric Aβ), or glutaraldehyde-polymerized Aβ_1–42 in 10 mM sodium acetate (NaAc), pH 4.5, was immobilized by injection over the surface for 20 minutes. The unreacted sites on the sensor chip surface were blocked by injection of 1 M ethanolamine, pH 8.5, for 10 minutes. Analytes containing different concentrations of aβComAb GW-23B7 in PBS with 0.05% Tween 20, pH 7.4, were injected over the surface, and the association and dissociation reactions were monitored. After each binding cycle, the surface was regenerated by a short, fast injection of 10 mM hydrochloric acid. The binding dissociation constant (K_D) was determined using Scrubber software 2.0a (BioLogic Software, Campbell, Australia).