Method Description

This method uses flow cytometry to measure the loss of acetylcholine receptors (AChR) expressed on the surface of live cells. The cell line used is an immortalized human rhabdomyosarcoma cell line that expresses endogenous muscle-type nicotinic AChR on its surface. Cells are plated in a 96-well plate and cultured prior to the addition of patient serum, which is then incubated on the cells to enable antibody-mediated internalization of AChR (modulation).

Subsequently, modulation is then stopped and the amount of AChR on the cell surface is measured by flow cytometry. The detection of receptors is performed using a recombinant rat monoclonal antibody specific for the human alpha-subunit of the AChR followed by a secondary goat anti-rat IgG antibody conjugated with allophycocyanin (APC). The amount of AChR on the cell surface is proportional to the median fluorescence intensity (MFI) of APC. To calculate the amount of modulation (ie, % loss of AChR), the APC MFI is compared between cells treated with patient serum and cells treated with serum lacking AChR modulating antibodies. Background signal is established in each experiment utilizing cells stained with secondary antibody alone (no patient sera). The percent loss of AChR is calculated as 1-([Patient MFI-Background MFI]/[Negative calibrator MFI-Background MFI]*100%).(Unpublished Mayo method)