To measure the binding affinities (KD equilibrium dissociation constant) of the purified antibodies (bispecific or monoclonal) to soluble antigens, the antibodies were first captured onto streptavidin SAX biosensor tips with a biotinylated (1 biotin/molecule), polyclonal capture antibody (Jackson ImmunoResearch, catalog# 109–005-098) and then incubated with a dilution series of each soluble antigen. This assay format was chosen so the bivalent antibodies would be immobilized and on the biosensor tips and tested versus the same serial dilution of each soluble antigen. The measured quantitative KD affinities thus represent monovalent 1:1 binding interaction and can be directly compared. Experiments were run in a ForteBio Octet HTX instrument using the 96-tip mode with standard 5 Hz data acquisition rate at 27°C and 1000 RPM. Using Genedata Screener V16 software, raw Octet binding data was processed with installed SPR kinetic curve fitting package and globally fit to a 1:1 binding model to determine the association rate constant (ka) and the dissociation rate constant (kd). The equilibrium dissociation constant (KD) was then calculated as a ratio of kd/ka. The KD values are average ± standard error from at least three independent experiments. The representative sensorgrams shown in Figure 6 are in each case from one of the experiments used in the analysis.

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