Optimization of the Cell-Based Assay Methodology to Study Degradation of Acetylcholine Receptors (Antigenic Modulation) by Antibodies From Myasthenia Gravis Patients.

Abstract

Introduction/aims: Degradation of cell-surface acetylcholine receptors (AChRs) by antigenic modulation is a key pathogenic mechanism of myasthenia gravis (MG) autoantibodies, yet standard assays primarily detect antibody binding rather than functional effects. We therefore aimed to establish a standardized assay to evaluate the functional effects of MG autoantibodies on AChR modulation.

Methods: CN21 cells expressing adult and fetal-type AChR were used to measure the modulation capacity of AChR antibodies in vitro. In this cell-based assay, the remaining surface AChR levels were determined using iodine-125-conjugated α-bungarotoxin. Experimental variables including plate surface and coating, cell density, dexamethasone treatment, incubation time, and complement effect were systematically evaluated and optimized. The optimized assay was later tested and qualified using sera from AChR-MG patients and healthy individuals.

Results: CellBIND coated 48-well plates were selected because of robust cell adhesion. An increased cell density resulted in increased baseline AChR expression levels without affecting the modulation capacity. Prolonged incubation of cells with AChR antibodies for 3 h resulted in higher sensitivity compared with 1 h. Heat inactivation of complement in serum samples did not affect antigenic modulation.

Discussion: The optimized modulation assay showed strong sensitivity and specificity, and is a robust and reproducible method to quantify modulation capacity in the sera of individual AChR-MG patients. We encourage the use of this assay to characterize the relevance of modulating antibodies in clinical severity and treatment response in individual patients.