GABA_AR subunit epitopes and mAb binding properties. (A) Immunofluorescence stainings as CBAs using COS7 cells overexpressing individual or multiple GABA_AR subunits (as illustrated in left column) to evaluate subunit-specific binding of patient’s polyclonal samples and derived recombinant mAbs (red; nuclei in blue). Negative controls #mGO53 and #113-109 showed no binding. Underlined subunits were stained with subunit-specific commercial antibodies (shown in green in image inserts). Note that β3 alone is expressed on the cell surface, but α1 alone is not (Ebert et al., 1999). (B) For relative quantification of human GABA_AR mAb binding to natively expressed receptors, the MFI was measured from binding in the granule cell layer of the murine cerebellum. Representative images from #113-115 are shown in top row with IgG concentrations as indicated. Per condition, MFIs from 15 ROIs from three independent experiments were used to fit nonlinear regression models of specific binding. Bars indicate mean ± SEM. (C) For analysis of competitive binding, fluorophore-coupled GABA_AR mAbs (detection mAbs) were stained on murine brain tissue in combination with an uncoupled mAb (competing mAb) in excess. The degree of detection mAb labeling was between 7.2 (#113-115) and 17.5 (#113-198). Exemplary images from coupled #113-101 are shown in top row. Quantified mean MFIs as relative values to noncompeting condition of the respective coupled mAb are shown as a heat map, each derived from 45 ROIs from three independent experiments. Receptor binding competition is visualized in black and signal enhancement in yellow. Representative scale bars indicate 20 µm in A and 100 µm in B and C.