Supplementary Figure 3: Binding analysis of RA801 or RA802 to human C1q, FcγRs and FcRn. | Nature Immunology

Supplementary Figure 3: Binding analysis of RA801 or RA802 to human C1q, FcγRs and FcRn.

From: IgG Fc domains that bind C1q but not effector Fcγ receptors delineate the importance of complement-mediated effector functions

Supplementary Figure 3

(a) ELISA analysis of C1q-specific antibody variants binding to human FcγRs. ELISA plates were coated with aglycosylated IgG1 (negative control), glycosylated IgG1 (Rituxan, positive control), RA801 or RA802. 500 nM or 50 nM of his-tagged FcγRI, GST-tagged FcγRIIaH131, GST-tagged FcγRIIaR131, GST-tagged FcγRIIb, GST-tagged FcγRIIIaV158, or GST-tagged FcγRIIIaF158 was added and binding was detected using anti-His IgG conjugated to HRP or anti-GST IgG-HRP, accordingly. Errors bars: standard deviations from triplicate experiments. (b-c) SPR analysis of C1q-specific antibody variants. (b) SPR analysis of antibody binding to purified C1q or to effector Fcγ receptors. Antibody variants were immobilized on CM5 chips. The binding of C1q, or of GST fusion proteins high affinity FcγRI, or low affinity FcγRIIa, FcγRIIb, or FcγRIIIa, all expressed as dimers, to enhance binding, was assayed. (c) pH-dependent binding to human FcRn. In all sensorgrams, x-axis is time (sec) and y-axis is RU (response unit). Data are from one experiment representative of three experiments (a-c).

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