A new bioluminescent cell-based assay for the detection of alpha-3 acetylcholine receptor antibodies: a multicentre comparison with current methods

Autoimmune autonomic ganglionopathy (AAG) is a disorder of the autonomic nervous system associated with autoantibodies against the α3 subunit of the ganglionic acetylcholine receptor (gnACHR). gnACHR antibodies exert their effects via receptor internalisation, but most current gnACHR diagnostic assays only detect antibody binding. Hence, we developed a new bioluminescent cell-based assay (BLIA) to measure internalising gnACHR antibodies. The BLIA was performed by incubating patient sera with live IMR-32 cells that express surface gnACHR. Remaining surface gnACHR was quantified using a novel split luciferase system with NanoBiT conjugated detecting antibodies. The percentage of receptor internalisation was calculated with comparison to cells incubated with reference sera, with > 20% receptor internalisation considered positive for gnACHR antibodies. The BLIA was validated using 46 samples from 20 AAG patients and 203 control samples. Subsequent comparisons to four existing assays were performed: (1) radioimmunoprecipitation assay (RIPA), (2) flow cytometric internalisation assay (FCIA), (3) cell-based assay (CBA-1) where HEK293T cells were transfected with α3β4 gnACHR subunits and (4) optimised CBA-2 with additional transfection of RIC3 and NACHO and incubation with 1 mM nicotine. The BLIA detected gnACHR antibodies with 100% sensitivity and 100% specificity (AUROC = 1.0) with detection of the internalising effects of antibodies to concentrations of < 0.1 pM. There was complete qualitative agreement with CBA-2 (Cohen’s kappa = 1). No statistical difference between these two assays and the RIPA, which had slightly lower sensitivity (98%) and specificity (95%), or the FCIA, which had slightly reduced specificity (95%), was found. CBA-1 had significantly lower sensitivity (69%, p = 0.0003). Overall, the BLIA had perfect clinical sensitivity and specificity in detecting gnACHR antibodies in AAG patients and detection of the functional effects of gnACHR antibodies via internalisation offers a suitable alternative to current assays. Further assessment of its possible role in monitoring disease severity or treatment response is warranted.