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. 2019 Jun 20;4(12):e127167. doi: 10.1172/jci.insight.127167

Figure 2. Characterization of human MuSK mAb–binding properties.

Figure 2

Binding properties of mAbs MuSK1A, MuSK1B, and MuSK3-28 were tested in several in vitro antibody-binding assays. (A) Representative cell-based assay (CBA) flow cytometry plots are shown for 3 MuSK mAbs and a negative control (AChR-specific mAb 637). Binding was tested at both 1.25 and 0.08 μg/mL. The x axis represents GFP fluorescence intensity and, consequently, the fraction of transfected HEK cells. The y axis represents Alexa Fluor 647 fluorescence intensity, which corresponds to secondary anti–human IgG antibody binding and, consequently, primary antibody binding to MuSK. Hence, transfected cells are located in the right quadrants and transfected cells with MuSK autoantibody binding in the upper right quadrant. (B) Binding to MuSK was tested over a wide range of mAb concentrations in the CBA. Controls included the MuSK-specific humanized mAb 4A3 and AChR-specific mAb 637 tested with MuSK mAbs MuSK1A, MuSK1B, and MuSK3-28. Each data point represents a separate replicate within the same experiment. Bars represent means and error bars SDs. (C) A solution phase radioimmunoassay was used to measure MuSK binding over a range of mAb concentrations. Each data point represents a value within the same experiment. (D) Specificity of the mAbs was evaluated using CBAs that tested binding to HEK cells transfected with MuSK, GFP alone, AChR, or MOG. Positive controls included MuSK-specific humanized mAb 4A3, AChR-specific mAb 637, and MOG-specific 8-18C5. Each data point represents a separate replicate within the same experiment. Bars represent means and error bars SDs. (E) Immunofluorescent staining of mouse NMJs. Tibialis anterior muscles were cut longitudinally in cryosections and fixed with PFA. AChRs were stained with Alexa Fluor 648 α-bungarotoxin (shown in red) and DNA with Hoechst (shown in blue in the merged panels). The first row shows staining with polyclonal IgG4 from a patient with MuSK MG. Binding of mAbs (MuSK1A, MuSK1B, MuSK3-28) against MuSK (1.6 μg/mL for 1 hour) was detected with goat anti–human IgG Alexa Fluor 488 (IgG, shown in green). In A–E the IgG4 subclass mAbs MuSK1A and MuSK3-28 were tested in their native IgG subclass unless indicated otherwise.