Method Description

This assay is a live cell-based binding assay using flow cytometry to detect ganglionic acetylcholine receptor (AChR) binding antibodies in patient serum. Human embryonic kidney cells (HEK293) transiently expressing human alpha-3 beta-4 ganglionic AChR subunits, along with accessory proteins required for receptor expression, are used as the antigen source. Receptor expression is enhanced prior to analysis under controlled culture conditions. Cells are incubated with diluted patient serum to allow binding of AChR-specific antibodies to surface-expressed receptors. Bound human IgG is detected using a fluorophore-conjugated anti-human IgG secondary antibody, with a viability dye included to exclude non-viable cells. Fluorescence intensity is measured by flow cytometry, and antibody binding is assessed by comparing signal from AChR-expressing cells to control cells lacking receptor expression. A humanized AChR-specific monoclonal antibody is used as an assay calibrator. The IgG binding index (IBI) of patient samples is compared to the assay calibrator to calculate a cut-off Index (COI). Patient samples with a COI greater than or equal to 1.0 are reported as positive. Positive samples are further titrated to determine the end-point dilution, defined as the highest serum dilution that remains above the calibrator.(Unpublished Mayo method)