Quantifying FcRn-Ligand Interactions via ELISA

Recombinant human single-chain soluble FcRn (sFcRn), containing both β and α chains in a 1:1 molar ratio, was expressed in mammalian cells and purified as described previously (18 (link)). ELISA wells were coated with sFcRn at 50 ng per well in PBS (pH 7.4) overnight at 4°C, and blocked with protein-free blocking buffer (Thermo Scientific) at 37°C for 2 h. Twofold serially diluted protein, prepared with PBS containing 0.2% BSA, pH 6.0 or PBS containing 0.2% BSA, pH 7.4, was added and incubated at 37°C for 1.5 h. The plates were washed with PBST (PBS plus 0.05% Tween-20), pH 6.0 or pH 7.4, and horseradish peroxidase (HRP)-conjugated anti-FLAG tag antibody (Sigma-Aldrich) in PBS (pH 6.0 or 7.4) was incubated with wells for 45 min at 37°C. After extensive washes with PBST (pH 6.0 or 7.4), the binding was detected by the addition of ABTS substrate (Roche, Indianapolis, IN, USA), and monitored at 405 nm.