Horseradish peroxidase conjugated anti human igg

Labelled proteins were diluted to a final concentration of 5 μg/ml in PBS and coated onto the wells of a 96-well polystyrene plate (Pierce, ThermoFisher Scientific, Waltham, MA) by incubating overnight at 4°C. The plate was washed three times with PBST (PBS with 0.1% Tween-20) followed by blocking with 5% skim milk in PBST for 3h at room temperature. The blocking solution was removed, and the plate was again washed twice with PBST. Proteins were probed with KZ52 antibody [26 (link),35 (link)] at a dilution of 1:1000 overnight at 4°C. The plate was then washed and incubated with horseradish peroxidase-conjugated anti-human IgG (Invitrogen, ThermoFisher Scientific, Waltham, MA) at a dilution of 1:2000 for 2h at room temperature. After washing the plate four times with PBST, TMB solution (3,3’,5,5’-tetramethylbenzidine; ThermoFisher Scientific, Waltham, MA) was added to each well, incubated for 15 min, followed by addition of an equal volume of 2M sulphuric acid. The optical density was immediately read at 450 nm in a Synergy H1 microplate reader (BioTek, Winooski, VT).

All proteins and pseudovirions were run on 4–20% denaturing polyacrylamide gels (Bio-Rad, Hercules, CA) and transferred to nitrocellulose membrane using Trans-blot Turbo (Bio-Rad, Hercules, CA). Membranes were rinsed with PBST, blocked with 5% skim milk in PBST for 1 h followed by probing of GP and p24 with monoclonal antibody (mAb) H3C8 at a dilution of 1:1000, [60 (link)] and mouse mAB B1217M at a dilution of 1:2000 (Genetex, Irvine, CA), respectively. The mAb H3C8 was humanized by cloning its variable heavy and light chain fragments in human IgG expression vectors obtained from Dr. Michel Nussenzwieg (The Rockefeller University). Membranes were washed three times with PBST, incubated with horseradish peroxidase-conjugated anti-human IgG (Invitrogen, ThermoFisher, Waltham, MA) and anti-mouse IgG (ThermoFisher, Waltham, MA) for 1 h at room temperature and developed with SuperSignal West Pico PLUS chemiluminescent substrate (ThermoFisher Scientific, Waltham, MA).