“Background: Myasthenia gravis (MG) and neuromyelitis optica spectrum disorders (NMOSD) are neurological conditions associated respectively with Acetylcholine receptor (ACHR) and Aquaporin4 (AQP4)-antibodies. These pertain to the IgG1/3 subclass and can induce antibody-mediated-complement-activation. Other MG forms associate with MUSK-antibodies, that are IgG4 and unable to induce ACA.
Objective: to develop a screening assay to assess ACA in the serum of MG/NMOSD patients.
Methods: HEK293T cells transfected with either AQP4, ACHR, or MUSK were incubated for 2 hours with complement inactivated polyclonal serum from MG/NMOSD patients with or without healthy donors as complement source, or with healthy donor serum alone. Three independent raters scored the ACA by evaluating the reduction in secondary antibody labeling compared to the control group using a semiquantitative scale (0= no reduction, 4= strong reduction).
Results: ACA was identified by all raters in NMOSD (13/22 patients, 59%) and ACHR-MG (6/30 patients, 20%). Interrater agreement was higher for AQP4 (Fleiss’ kappa=0.804) compared to ACHR (Fleiss’ kappa=0.585). Agreement became perfect (Fleiss’ kappa=1) when considering scores >1 in NMOSD and >1.5 in ACHR-MG. Only 1/15 MUSK-MG showed low levels (score=1) of ACA.
Results from qualitative assay were further validated with a luminescent assay of cell-viability, which showed an average 40% cell-killing in two AQP4 samples with high ACA (score=4).
Conclusions: our assay allowed to detect ACA in IgG1/3, but not in IgG4 samples. The high interrater agreement for high scores suggests that this might be a reliable tool to stratify patients who could benefit from complement inhibitor drugs.”