Detecting Amyloid-like Structures on Immobilized IgG

The presence of amyloid-like structures on immobilized IgG was detected as described previously (5 (link)). Microtiter wells (Corning, 9018) were coated with 10 μg/ml of monomeric IgG (mono-IgG) or agg-IgG in TBS, pH 7.2, and incubated at 4°C overnight. The unreacted sites in the wells were blocked with TBS containing 0.5% gelatin at room temperature for 45 min. Both, polyclonal antibodies (Novus, NBP2-25093) and monoclonal antibodies (Novus, NBP2-13075) to amyloid-β peptide 1-42 (Aβ) were used to detect the amyloid-like structures formed on the mono-IgG and agg-IgG following their immobilization. Normal rabbit IgG and normal mouse IgG were used as controls for the antibodies. The antibodies (10 μg/ml) diluted in TBS containing 0.1% gelatin and 0.02% Tween 20 were added to the wells and incubated at 37°C for 1 h. After washing the wells, bound polyclonal anti-Aβ antibodies were detected by using HRP-conjugated donkey anti-rabbit IgG (GE Healthcare) and bound monoclonal anti-Aβ antibodies were detected by using HRP-conjugated goat anti-mouse IgG (Thermo Fisher Scientific). Color was developed with ABTS reagent and the OD was read at 405 nm in a microtiter plate reader (Molecular Devices). Immobilized Aβ peptide 1-42 (Bachem) was used as a control for immobilized IgG and for anti-Aβ antibodies.