Patrick Vanderboom (Presenter)
Bio: Patrick Vanderboom is from Rochester Minnesota. After earning a bachelors degree in chemistry from Winona State University in 2004 he took a job in the Toxicology Lab at Mayo Clinic. In 2012 he obtained a Masters degree in Molecular Pharmacology and Experimental Therapeutics at the Mayo Clinic School of Graduate Medical Education. Since then, Patrick has been working on the Technology Development Team in Proteomics for the Center for Individualized Medicine at Mayo Clinic.
Authorship: Patrick Vanderboom, Kari Gurtner, David Barnidge, David Murray, Maria Willrich
Mayo Clinic, Rochester, MN
Isoelectric focusing followed by IgG specific immunoblotting is used to detect immunoglobulins specific to the CNS compartment (oligoclonal banding) and is routinely used as part of the diagnostic criteria for multiple sclerosis. The current isoelectric focusing based reference method is labor intensive and relies on a subjective interpretation of IgG bands from paired CSF and serum. Here, we demonstrate the advantages of a recently developed high throughput mass spectrometry based method which leverages the power of accurate molecular mass to detect and characterize oligoclonal immunoglobulins in CSF.
Background: Isoelectric focusing followed by IgG specific immunoblotting (IgG-IEF or oligoclonal banding) is used to detect immunoglobulins specific to the central nervous system (CNS) and is routinely used as part of the diagnostic criteria for multiple sclerosis. Unfortunately, this process is labor intensive and relies on a subjective interpretation of IgG bands from paired cerebrospinal fluid (CSF) and serum. Recent work has demonstrated the utility of accurate molecular mass measurements obtained by microLC-ESI-Q-TOF MS miRAMM analysis for the detection of monoclonal immunoglobulins in CSF (1). Here, we describe adding an immunoaffinity purification step before miRAMM analysis to enable lower limits of detection using a benchtop LC-ESI-TOF mass spectrometer. In this study the diagnostic performance of the IgG-IEF reference method is compared with LC-ESI-TOF mass spectrometry analysis to identify immunoglobulins in CSF.
Objective: To develop an immunoaffinity-MS method for detecting oligoclonal immunoglobulins in CSF and serum using camelid nanobodies on a benchtop LC-ESI-TOF MS platform. The diagnostic performance of this method will then be compared to the current reference test.
Methods: Fifty residual paired CSF/serum samples analyzed previously as positive (OCB+, N=30) and negative (OCBneg, N=20) were used for this analysis. Immunoglobulins were affinity purified from serum and CSF with CaptureSelect™ anti-kappa and anti-lambda camelid nanobody affinity resins in a 96 well PVDF filter plate. The captured immunoglobulins were eluted with 5% acetic acid, reduced with TCEP and separated on an Agilent Poroshell 2.1 x 75mm 300SB-C3 column before LC-ESI-TOF MS analysis on an Agilent 6224 TOF mass spectrometer. Clonal light chains unique to serum and CSF were identified using accurate molecular mass measurements with distinguishable signal to noise over the polyclonal background, and were called positive when CSF had 4 unique clones not present in serum. Readers were blinded to the original OCB results.
Results: In the OCB+ samples, the mean±SD number of IgG bands identified by IEF was 10.7±3.9 whereas in the OCBneg cohort it was 0.8±1.2. In contrast, samples identified as positive by LC-ESI-TOF MS analysis resulted in a mean of 22.8±11.5 light chain clones compared to 3.6±5.5 in those identified as negative. In the positive cohort, 26 samples were identified with kappa clones (16.3±8.8), whereas only 19 samples had lambda clones (9.4±7.1). Overall, LC-MS-TOF analysis resulted in 86% agreement with IgG-IEF. The lack of agreement resulted from four discordant samples in the OCB+ cohort, all of which were false negatives by LC-ESI-TOF MS. In two of these four samples several light chain clones were detected; however, clones with the same molecular mass were also determined to be present in serum, indicating that these clones were not unique to the CNS compartment. Of the remaining two false negative results, one was caused by a technical issue resulting in low recovery and the other had 6 clones in CSF and 9 clones in serum putting it within 1 unique clone of the cutoff.
Conclusion: Analysis of oligoclonal immunoglobulins from CSF and serum using immunoaffinity-LC-ESI-TOF MS correlates well with IgG-IEF. As indicated by these results, the accurate molecular mass measurements obtained by mass spectrometry analysis allow for unparalleled specificity when profiling immunoglobulins unique to the CNS compartment. An additional advantage of this method is the isotype specificity of the camelid nanobodies used to isolate oligoclonal immunoglobulins from CSF and serum. This improves the ability to characterize CSF oligoclonal immunoglobulins, and could potentially provide insight to the progression of multiple sclerosis.
References & Acknowledgements:
1.Barnidge, D. Kohlhagen, M. Zheng, S. Willrich, M. Katzmann, J. Pittock, S. Murray, D. Monitoring Oligoclonal Immunoglobulins in Cerebral Spinal Fluid Using microLC-ESI-Q-TOF Mass Spectrometry. J Neuro Im. 2015.09.011, In Press
IP Royalty: no
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