Fig. 1
Design, production, and characterization of MOG-Fc fusion protein. a Schematic representation of MOG-Fc fusion protein. The extracellular domain of the mouse MOG protein (amino acids 1–118) is depicted in green and the Fc portion of the mouse IgG2c is depicted in blue. The mutations to abolish glycosylation of MOG protein and to disrupt complement and Fc receptor binding sites are indicated within brackets. His-Tag and AviTag sequences were introduced at the C-terminus for purification and detection. b SDS-PAGE (both reducing and non-reducing) of the purified MOG-Fc, SD-Fc proteins, and MOG-specific antibody 8.18C5. c Dose-dependent binding analysis of 8.18C5 and control IgG1. ELISA plates were coated with MOG-Fc or SD-Fc and serially diluted 8.18C5 or IgG1 were added. OD at 450 nm is shown. d Binding of MOG-Fc to serum anti-MOG antibodies. Serum from 2 individual WT SJL/J and RR mice were used (n = 3) for the detection of MOG antibodies by ELISA. OD at 450 nm is shown. e Comparison of the binding efficiencies of MOG-Fc and monomeric MOG to 8.18C5. MOG-Fc or monomeric MOG was coated and detected using serially diluted anti-MOG monoclonal antibody 8.18C5. OD at 450 nm is shown. f Comparison of the binding of MOG-Fc and monomeric MOG to serum anti-MOG antibodies from RR mice. OD at 450 nm is shown. g Detection of MOG-specific B cells by MOG-Fc and monomeric MOG. MOG-Fc and monomeric MOG were biotinylated at their AviTag and tetramerized with a streptavidin-coupled fluorochrome. IgHMOG splenocytes were mixed with wild-type mouse splenocytes at various ratios and used for the staining with tetramerized MOG-Fc or monomeric MOG proteins. Samples were analyzed by flow cytometry and the percentage of MOG binding B cells was shown. **P = 0.0078 (Wilcoxon matched-pairs signed rank test). h Residual MOG-Fc or SD-Fc in WT SJL/J mice after a single injection. 200 µg of MOG-Fc or SD-Fc was injected into the mice (n = 6 per group) and sera were collected after 4 h and on days 1, 3, 5, and 8 post-injections. Residual-MOG ELISA was performed and the OD at 405 nm is shown. All experiments were performed at least twice