Human serum (Sigma-Aldrich, St. Louis, MO, USA) was depleted of
endogenous IgG by passage through a protein G-Sepharose column. Antibodies and
ADCs were incubated at a concentration of 40 μg/ml in IgG-depleted serum
for 0, 3, or 5 days at 37 °C followed by immunoprecipitation using
agarose beads coupled to goat anti-human Fc-specific antibody (Sigma-Aldrich).
Immunoprecipitated antibodies and ADCs were run on SDS-polyacrylamide gels and
transferred onto nitrocellulose membranes (0.45 μm pore size; Genesee
Scientific, San Diego, CA, USA). Immunoblotting was carried out using standard
methods with goat anti-human IgG (H + L) antibodies conjugated with HRP (Jackson
Immunoresearch, West Grove, PA, USA). HRP was detected using SuperSignal West
Pico Chemiluminescent Substrate (Thermo Fisher Scientific) followed by scanning
with a C-DiGit blot scanner (LI-COR Biosciences, Lincoln, NE, USA).
endogenous IgG by passage through a protein G-Sepharose column. Antibodies and
ADCs were incubated at a concentration of 40 μg/ml in IgG-depleted serum
for 0, 3, or 5 days at 37 °C followed by immunoprecipitation using
agarose beads coupled to goat anti-human Fc-specific antibody (Sigma-Aldrich).
Immunoprecipitated antibodies and ADCs were run on SDS-polyacrylamide gels and
transferred onto nitrocellulose membranes (0.45 μm pore size; Genesee
Scientific, San Diego, CA, USA). Immunoblotting was carried out using standard
methods with goat anti-human IgG (H + L) antibodies conjugated with HRP (Jackson
Immunoresearch, West Grove, PA, USA). HRP was detected using SuperSignal West
Pico Chemiluminescent Substrate (Thermo Fisher Scientific) followed by scanning
with a C-DiGit blot scanner (LI-COR Biosciences, Lincoln, NE, USA).