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

Young Gi Min^1,2, Paula Llarch³, Cinta Lleixà³

¹Department of Neurology, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, Republic of Korea.

Journal of the Peripheral Nervous System : JPNS

|July 12, 2026

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A new multiplex flow cytometry assay accurately detects autoimmune nodopathies (AN) antibodies, including IgG4 subclass, aiding diagnosis of these rare autoimmune neuropathies.

Area of Science:

Neuroimmunology
Autoimmune Disorders
Diagnostic Assays

Background:

Autoimmune nodopathies (AN) are rare disorders involving autoantibodies against cell-adhesion molecules at the node of Ranvier.
Accurate antibody testing is crucial for managing autoimmune neuropathies.

Purpose of the Study:

Develop and validate a multiplex flow cytometry (FCM) assay for simultaneous detection of four major AN autoantibodies.
Assess the diagnostic performance of the multiplex FCM assay through inter-laboratory comparison.

Main Methods:

Established a multiplex FCM assay using live cell lines expressing NF155, CNTN1, CASPR1, and NF186.
Tested 87 serum samples (46 AN, 39 controls) and compared results with ELISA and cell-based assays (CBA).
Analyzed IgG4 subclass positivity and autoantibody titers.

Keywords:

CIDP ELISA autoimmune nodopathy cell‐based assay multiplex flow cytometry assay

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Main Results:

The multiplex FCM assay showed high diagnostic performance: 95.1% sensitivity, 97.8% specificity, and 99.6% accuracy.
Approximately 80% of AN sera were positive for IgG4 subclass, with high concordance to ELISA.
Positive correlations were observed between ELISA titers and FCM fluorescence intensity.

Conclusions:

The multiplex FCM assay provides rapid, objective, and quantitative detection of AN autoantibodies and IgG4 subclass.
Its high sensitivity and specificity suggest utility in routine serological testing for AN.
Minimal sample volume requirement enhances its practicality for clinical use.