Fig. 1.

Antigen recognition, C1q- and FcγR-binding of mutated MOG-specific mAbs. The MOG antibody r8-18C5 and the Fc mutant antibodies SAI and TAP show comparable binding to MOG in a cell-based flow cytometry assay (A). IgG binding to MOG was normalized to binding of the r8-18C5 and the assay was repeated two times for each antibody (delta MFI value). (B) C1q binding was analyzed with an ELISA. Specifically, ED-hMOG was coated and indicated Ab variants were added. Subsequently, C1q was added and its binding was quantified using anti-C1q Ab. Binding of the mutant Abs was normalized to the binding of the antibody r8-18C5 and the assay was repeated three times for each antibody. FcγRI, FcyRIIb, FcyRIII, and FcγRIV binding was investigated by ELISA (C–G). Anti-FLAG antibodies were coated on an ELISA plate and used to capture soluble flag-tagged FcγR (FcγRI, FcyRIIb, FcyRIII, FcγRIV) (C). Binding of antibody-MOG IgG immune complexes to the added soluble FcγRs was quantified (D-G). Binding of the mutant MOG-Abs SAI and TAP is normalized to the binding of the MOG antibody r8-18C5. The assay was repeated three times for each antibody. Data are shown as mean with SEM. Each mutant mAbs’ affinity to C1q and FcyRs was presented as log₂ fold change compared to r8-18C5 measured on the same plate and was analyzed using 2-tailed one-sample t test (parametric Welch’s t test) with Benjamini–Hochberg multiple comparison correction (n = 3).