Recombinant human ephrin b3 fc chimera biotinylated protein

The inhibition of the binding to the ephrin receptors by the purified G proteins was measured to establish 50% inhibiting concentration (IC50) values for each serum sample. The G protein-coupled magnetic microspheres were mixed after sonication such that all assays were multiplexed. A total of 1500 microspheres was added to each well. The recombinant mouse ephrin-B2 Fc chimera biotinylated protein (R&D systems) or recombinant human ephrin-B3 Fc chimera biotinylated protein (R&D systems) were diluted in PBSA and added into the microsphere-containing wells at a final concentration of 25 ng/mL, and the serum was three-fold serially diluted from 1:20 with two replicates per dilution. The assay plate was incubated for 60 min at 24 °C on a plate shaker at 800 rpm. Streptavidin-R-phycoerythrin (Invitrogen) was diluted to a concentration of 12 μg/mL. Thereafter, 10 µL of diluted SAPE was added to each well. The assay plate was incubated for 30 min at 24 °C on a plate shaker at 800 rpm. The supernatant was removed using a magnetic plate separator (Luminex Corporation). The plate was washed three times with PBSA, and the binding was measured using a Luminex MAGPIX instrument (Luminex Corporation). The MFI was recorded for a four-parameter logistic curve fitting, and the IC50 was calculated based on this curve.

The binding between the purified G proteins and ephrinB2 and ephrinB3 was measured to determine the ligand cross-reactivity. G protein-coupled magnetic microspheres were mixed after sonication such that all assays were multiplexed. A total of 1500 mixed microspheres were added per well. The recombinant mouse ephrin-B2 Fc chimera biotinylated protein (R&D systems, Minneapolis, MN, USA) and recombinant human ephrin-B3 Fc chimera biotinylated protein (R&D systems) were diluted in PBSA and added into the microsphere-containing wells with two replicates per concentration. The 96-well assay plate (Corning Inc, Corning, NY, USA) was incubated for 60 min at 24 °C on a plate shaker at 800 rpm. Streptavidin-R-phycoerythrin (SAPE) (Invitrogen) was diluted to a concentration of 12 µg/mL. A total of 10 µL of diluted SAPE was added to each well. The assay plate was incubated for 30 min at 24 °C on a plate shaker at 800 rpm. The supernatant was removed using a magnetic plate separator (Luminex Corporation). The plate was washed three times with PBSA, and the binding was measured using Luminex MAGPIX instrument (Luminex Corporation). The net median fluorescence intensities (MFI) were recorded and used to draw the binding curve.