Figure 1.

UCAs of MuSK mAbs bind to the MuSK autoantigen. MuSK-specific mAbs and their UCAs were tested for surface binding to MuSK on MuSK-GFP–transfected human embryonic kidney (HEK) cells. (A) Illustration of the differences in amino acid sequence between the mature mutated heavy and light chains compared with the UCA sequences. The different amino acids are shown in white letters. The number indicates the position of the amino acid within the variable region. The glycosylation sites are indicated with * if present and ^ if not present. (B) Representative CBA contour plots are shown for the three MuSK mAbs and their UCA. 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. The plots show testing with a mAb concentration of 1.25 µg/ml. (C) Binding to MuSK was tested over a series of 10 twofold dilutions of each mAb ranging from 10 to 0.02 µg/ml. Humanized MuSK mAb 4A3 was used as the positive control and AChR-specific mAb 637 as the negative control. The ΔMFI was calculated by subtracting the signal from nontransfected cells from the signal of transfected cells. Each data point represents the mean value from three independent experiments, and error bars represent SDs. Values greater than the mean + four SDs of the negative control mAb at 1.25 µg/ml (indicated by the horizontal dotted line) were considered positive.