Goat anti human fc f ab 2 af 488

The assay, including the preparation of soluble membrane protein and ovalbumin reagents, were performed as described in our previous work.^{46 (link)} Briefly, non-specific binding measurements with soluble membrane proteins (PSP #1) and ovalbumin (PSP #2) were incubated with overnight antibody (15 μg/mL) coated Protein A magnetic beads (10002D, Invitrogen). The reagent and bead complexes were incubated at 4 °C for 20 min for soluble membrane proteins, while ovalbumin was incubated 3 h at room temperature. The beads were then washed once and incubated with 0.001x streptavidin-AF647 (S32357, Invitrogen) and 0.001x goat anti-human Fc F(ab’)2 AF-488 (H10120, Invitrogen) on ice for 4 min. As a final step, the beads were washed once more and resuspended in PBSB. The samples were analyzed for non-specific binding via flow cytometry to measure their median fluorescent intensities (MFI). Adalimumab and bococizumab (generated using the antibody variable regions from the clinical-stage antibodies on a common IgG1 framework) were analyzed in each experiment as negative (adalimumab) and positive (bococizumab) controls. Polyspecificity scores were calculated as the MFI for a given mAb minus the MFI for adalimumab divided by the difference in MFI values for bococizumab and adalimumab.