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Fig. 1 | Acta Neuropathologica Communications

Fig. 1

From: Reemergence of pathogenic, autoantibody-producing B cell clones in myasthenia gravis following B cell depletion therapy

Fig. 1

MuSK autoantibody binding. A live cell-based assay (CBA) was used to screen B cell culture media for MuSK IgG and to validate the binding of the human MuSK specific mAbs 2E6 and 6C6. a To generate MuSK mAbs, patient-derived B cells were sorted for single cell culture, after which the secreted IgG was tested for MuSK-specificity using a CBA. The contour plots from this screening show that MuSK-specific IgG are present in the supernatant of two culture wells from which the mAbs 2E6 and 6C6 were subsequently derived. The x-axis represents GFP fluorescence intensity and, consequently, the fraction of HEK cells transfected with MuSK. The y-axis represents Alexa Fluor 647 fluorescence intensity, which corresponds to secondary anti–human IgG Fc antibody binding and, consequently, primary antibody binding to MuSK. Hence, transfected cells are located in the right quadrants and cells with MuSK antibody binding in the upper quadrants. b Binding to MuSK by mAbs 2E6 and 6C6 was tested over a series of ten two-fold dilutions ranging from 10-0.02 µg/ml. The ∆MFI was calculated by subtracting the signal acquired from non-transfected cells from the signal of transfected cells. The MuSK-specific human mAb MuSK1A was used as a positive control and the AChR-specific human mAb 637 used as a negative control. Each data point represents the mean value from three independent experiments. Bars or symbols represent means and error bars SDs. Values greater than the mean + 4SD of the negative control mAb at 1.25 µg/ml (indicated by the horizontal dotted line) were considered positive

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