Human fc domain

DO24 single-chain binding affinities were determined by ELISA assay; the MET extracellular domain fused in frame with a human Fc domain (100 ng/well, R&D System) was in the solid phase and increasing concentrations of the purified DO24 antibody formats (range 0–33 nM) were in the liquid phase. Binding was revealed using horseradish peroxidase (HRP)-conjugated anti-human k light chain antibodies (Sigma-Aldrich). The colorimetric signal was quantified by the multi-label reader VICTOR X4 (Perkin Elmer Instrument INC.). To calculate Kd and Bmax, data were analyzed and fitted according to nonlinear regression, one-site binding hyperbola curve, using GraphPad Prism software (GraphPad Software).

For affinity determination, the MET extracellular domain fused in frame with a human Fc domain (R&D System) was in the solid phase and pure MvDN30 or hOA-DN30 were in the liquid phase. ELISA signal was quantified by the multi-label reader VICTOR X4 (Perkin Elmer Instrument INC.).
For hOA-DN30 quantification in mouse serum, a goat anti-human IgG antibody (Sigma Aldrich) was in the solid phase. For hOA-DN30 quantification in monkey serum samples, neutravidin-coated plates were used to immobilize biotin-labeled goat anti-human IgG monkey adsorbed antibody (Southern Biotech). In both cases, pure hOA-DN30 and serum samples were in the liquid phase. The concentration of hOA-DN30 in serum was achieved by interpolating the optical density (OD) readings of the samples with the calibration curve fitted by a weighted (1/Y) 4 parameters regression function.