Hrp conjugated anti human k light chain antibodies

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 analysis of the interaction between the antibody domain of the fusion proteins and MET, wild type decoyMET (100 ng/well), produced by U-Protein Express BV, was immobilized on enzyme-linked immunosorbent assay (ELISA) plates and increasing concentrations (0–1 μM) of the fusion proteins were added in liquid phase. Binding was revealed using horseradish peroxidase (HRP)-conjugated anti-human k light chain antibodies (Sigma-Aldrich). For the analysis of the interaction between the decoy domain of the fusion proteins and HGF, DO24 anti-MET antibody [24 (link)] (100 ng/well) was immobilized on ELISA plates to capture the fusion proteins (5 nM) in solid phase; then, increasing concentrations (0–10 nM) of HGF (R&D Systems, Minneapolis, MN, USA) were added in liquid phase. Binding was detected using the anti-HGF biotinylated antibody BAF294 (R&D Systems) and revealed with HRP-conjugated streptavidin (GE Healthcare, Chicago, IL, USA). Colorimetric assay was quantified by the multi-label plate reader VICTOR-X4 (Perkin Elmer Instruments INC., Whaltman, MA, USA).

The extracellular region of MET fused to the Fc portion of a human IgG (MET-Fc chimera, R&D Systems, Minneapolis, MN, USA) was immobilized on ELISA plates, and increasing concentrations (0–20 nM) of short-sc25, long-sc60, or MvDN30 were added in liquid phase. Binding was revealed using horseradish peroxidase (HRP)-conjugated anti-human K light chain antibodies (Sigma-Aldrich, St. Louis, MO, USA). Colorimetric assay was quantified by the multi-label plate reader VICTOR-X4 (Perkin Elmer Instruments INC., Waltham, MA, USA).