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. 2017 Sep 1;6(3):12. doi: 10.3390/antib6030012

Figure 3.

Figure 3

Testing for interactions of cross-linked huIgG variants with Fcγ receptors. Binding of huIgG Ab1 (anti-TNFα) molecules to human FcγRs was assessed using AlphaScreen bead assays in a competitive format. To increase sensitivity of the assays, test samples were cross-linked to introduce binding avidity by using a goat F(ab′)2 anti-huIgG F(ab′)2-specific fragment in 1:1 molar ratio with the test antibodies. Cross-linked test antibodies at the designated concentrations were co-incubated with biotin-labeled huIgG Fc fragment (to avoid binding to cross-linker), the respective His-tagged FcγRs, nickel chelate acceptor beads, and streptavidin donor beads. Plates were read on the EnVision multi-label plate reader, and data plotted with GraphPad Prism v6.0 software. Shown are binding of cross-linked huIgG variants to: (A) huFcγRI, (B) huFcγRIIa-H131, high affinity allotype, (C) huFcγRIIa-R131, low affinity allotype, (D) huFcγRIIb, (E) huFcγRIIIa-V158, high affinity allotype. Non-binding of cross-linker alone is shown with the high affinity huFcγRI. All points represent the mean of duplicate samples ± range. The plot labels refer to IgG1 AA—human IgG1 L234A/L235A; IgG1 FES—human IgG1 L234F/L235E/P331S; and IgG1 WT—wild type human IgG1.