MSACL 2026 Abstract

High-Resolution Mass Spectrometry Detection of CSF-Restricted Oligoclonal Immunoglobulins in Paired CSF and Serum for Multiple Sclerosis Diagnosis

Ruben Y. Luo (1,2), Danya Ortiz (1), Priscilla S.-W. Yeung (1,2)
(1) Department of Pathology, Stanford University, Stanford, CA, USA (2) Clinical Laboratories, Stanford Health Care, Palo Alto, CA, USA

Ruben Y. Luo, PhD, DABCC (Presenter) Stanford University

Presenter Bio: Ruben Y. Luo, PhD, DABCC, FADLM is an Assistant Professor of Pathology at Stanford University and an Associate Director of Clinical Chemistry Laboratory at Stanford Health Care. He received PhD in chemistry from Stanford University, worked in the clinical diagnostic industry for several years, and then completed clinical chemistry fellowship at the University of California San Francisco. Dr. Luo is dedicated to innovations in clinical diagnostics. His research focuses on (1) discovering the clinical diagnostic value of molecular characteristics of protein biomarkers, and (2) applying top-down mass spectrometry and label-free optical sensing immunoassays to characterization and accurate measurement of biomarkers. He has been an active member and conference speaker in the international clinical chemistry and mass spectrometry communities, e.g., Association for Diagnostics and Laboratory Medicine (ADLM; formerly American Association for Clinical Chemistry, AACC), American Society for Mass Spectrometry (ASMS), Mass Spectrometry & Advances in Clinical Lab (MSACL). His research awards include 2022 AACC George Grannis Award for Excellence in Research and Scientific Publication, 2020 American Society for Clinical Pathology (ASCP) “40 Under Forty” Honoree, etc. He currently serves as an associate editor of JMSACL and an editorial board member of Scientific Reports.

Relevant Financial Disclosures (within past 24 months, reported on Apr 21, 2026) Other Potential Conflicts Thermo Fisher Scientific (San Jose, CA) / Research CollaborationGator Bio (Palo Alto, CA) / 2 Seed InstrumentsInstant Nanobiosciences (New Taipei City, Taiwan) / 1 Seed InstrumentCMP Scientific (Brooklyn, NY) / 1 Seed Instrument Discussing Products? NO, presenter will NOT present educational content related to products/services of the ineligible company(ies) listed above. Conflict mitigation NOT REQUIRED.

INTRODUCTION:
Oligoclonal bands (OCB) in cerebrospinal fluid (CSF), defined as CSF-restricted immunoglobulins present in CSF but not in paired serum, are a longstanding biomarker of intrathecal humoral immune activation and are most classically used in the diagnosis of multiple sclerosis.(1,2) OCB analysis remains clinically important because CSF-specific bands can support multiple sclerosis diagnosis, while also occurring in other inflammatory and infectious disorders of the central nervous system. Conventional testing relies on isoelectric focusing (IEF) with immunoblotting, an informative but labor-intensive assay that provides limited molecular characterization. High-resolution mass spectrometry (HR-MS) offers the potential to move OCB testing from pattern recognition toward direct molecular readout of clonal immunoglobulin species. Prior reports have shown that clones of serum immunoglobulins can be profiled by long-gradient HPLC coupled with HR-MS,(3) and that OCB-related peptides can be linked to B-cell repertoires and light-chain-based MS approaches can achieve good agreement with IEF.(4,5)

METHOD:
We developed and optimized a liquid chromatography-high-resolution mass spectrometry (LC-HR-MS) workflow for characterization and detection of OCB-related immunoglobulin species in paired CSF and serum. Method development proceeded iteratively from direct protein G capture and Melon Gel enrichment to a Fab-focused strategy using IdeS digestion followed by capture on kappa and lambda light chain-selective resins. The LC-HR-MS analysis was performed on a Vanquish Neo HPLC-Orbitrap Eclipse Tribrid mass spectrometer system using a MabPac RP column (1 mm x 150 mm) in microflow setup (100 µl/min) and positive-ion mode for data acquisition. Deconvolution of raw data was performed using the Respect algorithm in Biopharma Finder software.

RESULTS:
Data interpretation emphasized detection of oligoclonal (Fab)2 species in CSF by subtraction of clones shared with matched serum using mass- and retention-time-based filters. Noise-filtering and relative-abundance thresholds were evaluated during method optimization. In positive OCB specimens, typically CSF-restricted oligoclonal (Fab)2 peaks were observed; in negative OCB specimens and paired serum specimens, typically CSF-restricted oligoclonal peaks were absent. In multiple OCB sample sets, the LC-HR-MS analysis was mostly consistent with IEF with immunoblotting, with a small number of discordant cases, indicating the potential of the LC-HR-MS method to complement or improve the conventional test.

CONCLUSION:
The LC-HR-MS method can detect and molecularly profile oligoclonal immunoglobulins in paired CSF and serum, moving beyond the band-pattern abstraction of electrophoretic assays. In this optimization study, the most effective strategy was (Fab)2-centered sample preparation with paired sample contrasting, enabling detection of CSF-restricted oligoclonal immunoglobulins and showing broad concordance with conventional IEF. The LC-HR-MS method therefore has strong potential both as a research tool for OCB study and as a foundation for next-generation clinical testing of intrathecal humoral immune responses, especially multiple sclerosis.