Nickel plates

Binding to the neonatal Fc receptor was measured as described in (24 (link)). Nickel plates (Qiagen) were coated with 2 μg/ml his-tagged FcRn/β2 (Sinobiological, Bejing) for a minimum of 1 hour, washed with PBS 0.05% Tween-20, and blocked with 5% milk/PBS. Five-fold serial dilutions starting at 5 μg/ml of bNAbs were incubated with the receptor in 100mM NaPO4, 0.05% (v/v) Tween20, pH 6.0 for 1 hour at room temperature. Following this, plates were either washed with 100 mM NaPO₄, 0.05% (v/v) Tween20, pH 6.0 or 100 mM NaPO₄, 0.05% (v/v) Tween20, pH 7.4 three times with 30 minute incubations in between washes. Residual binding of antibodies was detected by a goat Anti-Human IgG (Fab specific) antibody at 1 in 5,000 (Sigma) and were visualized with tetramethylbenzidine (TMB) at OD450nm.

Antibody binding to FcγR was measured by ELISA as described previously (55 (link)). Briefly, FcγRI, FcγRIIa, FcγRIIb and FcγRIIIa His6-tagged receptors (R&D Systems Minneapolis, MN) were coated on nickel plates (Qiagen) at 2 μg/ml or 4 μg/ml. Five-fold serial dilutions starting at 5 μg/ml of bNAbs were added. Binding was detected by a goat Anti-Human IgG (Fab specific) antibody goat at 1 in 10,000 (Sigma). Results were visualized with tetramethylbenzidine (TMB).