Development and Inter-Laboratory Validation of Multiplex Flow Cytometry Assay for Autoimmune Nodopathy

Autoimmune nodopathies (AN) are a group of rare disorders caused by autoantibodies targeting cell-adhesion molecules located at the node of Ranvier. Reliable and efficient antibody testing for AN has become essential for optimal care of autoimmune neuropathies. In this study, we developed a multiplex flow cytometry (FCM) assay capable of simultaneously detecting four major AN antibodies and validated its diagnostic performance through inter-laboratory collaboration.

Multiplex FCM assay was established using live cell lines stably expressing each of the four AN targets-neurofascin-155 (NF155), contactin-1 (CNTN1), contactin-associated protein 1 (CASPR1), and NF186. Using this assay, we tested 87 serum samples (46 AN, 39 disease controls, and 2 healthy controls) and compared the results with those obtained by enzyme linked immunosorbent assay (ELISA), fixed and/or live cell-based assay (CBA) conducted at an independent laboratory. IgG4 subclass positivity and autoantibody titers were also analyzed and compared between multiplex FCM assay and ELISA.

Multiplex FCM assay demonstrated excellent diagnostic performance, with overall sensitivity of 95.1%, specificity of 97.8%, and accuracy of 99.6%. IgG4 subclass was detected in approximately 80% of AN sera, showing high concordance with ELISA. Antibody titers showed positive correlations between ELISA end-point titers and FCM-derived fluorescence intensity values.

This multiplex FCM assay enables rapid, objective, and quantitative detection of AN autoantibodies and the presence of IgG4 subclass using minimal sample volume. High sensitivity and specificity support its potential utility for routine serological testing for AN.