NanoBiT FcRn assay

NanoBiT FcRn assay involves four steps. In the first step, 25 µl (or 10 µl) of IgG-LgBiT solution was mixed with 25 µl (or 10 µl) of the IgG or Fc fusion proteins in a white, 96-well, nonbinding plate (Corning). In the second step, FcRn–biotin tag and streptavidin–SmBiT were mixed at 1:1 molar ratio to form FcRn–biotin–streptavidin–SmBiT (FcRn-SmBiT). In the third step, 50 µl (or 20 µl) of FcRn-SmBiT is added to each well containing IgG and IgG-LgBiT and plate incubated for 30–60 min. In the final step, furimazine substrate (Promega) is diluted 1:50-fold, and 25 µl (or 10 µl) is added to plate, and signal is allowed to stabilize for 3 min, and the bioluminescence signal (relative light units [RLU]) is measured in a GloMax Discover Luminometer (Promega). All the dilutions were made in PBS containing 10% superblock (pH 6). Before running the assay, pH of the Abs was adjusted to pH 6 by addition of 1/10th the volume of 0.5 M citrate buffer containing 50% superblock (pH 5.7). All the experiments were run in duplicate or triplicate. Normalized RLU data are generated by assigning 100% to the maximum bioluminescent signal obtained in absence of the IgG and then calculating percentage drop in signal in the presence of an IgG. Inhibition curves were generated by plotting normalized RLU as a function of log value of the IgG concentration (in nM) and fitted to a four-parametric equation with 1/y² weightage using GraphPad Prism. IC₅₀ values that are equivalent to apparent affinity values (K_D) are used when comparing data generated using biosensor and reported in the literature.