MALDI-TOF mass spectrometry can distinguish immunofixation bands of the same isotype as monoclonal or biclonal proteins

Erica M. Fatica, Mark Martinez, Paula M. Ladwig, Josiah D. Murray, Mindy C. Kohlhagen, Robert A. Kyle, Taxiarchis Kourelis, John A. Lust, Melissa R. Snyder, Angela Dispenzieri, David L. Murray, Maria A.V. Willrich

Research output: Contribution to journalArticlepeer-review


Background: Plasma cell disorders (PCDs) are typically characterized by excessive production of a single immunoglobulin, defined as a monoclonal protein (M−protein). Some patients have more than one identifiable M−protein, termed biclonal. Traditional immunofixation electrophoresis (IFE) cannot distinguish if two bands of the same isotype represent biclonal proteins or M−proteins with some other feature. A novel assay using immunoenrichment coupled to matrix-assisted laser desorption ionization time-of-flight mass-spectrometry (Mass-Fix) was applied to determine whether two bands of the same isotype represented (1) monomers and dimers of a single M−protein, (2) an M−protein plus a therapeutic monoclonal antibody (t-mAb), (3) an M−protein with light chain glycosylation, or (4) two distinct biclonal M−proteins. Methods: Patient samples with two bands of the same isotype identified by IFE were enriched using nanobodies against IgG, IgA, IgM, or κ and λ light chains then analyzed by Mass-Fix. Light chain masses were used to differentiate IgGκ M−proteins from t-mAbs. Mass differences between peaks were calculated to identify N-glycosylation or matrix adducts. High-resolution mass spectrometry was used as a comparator method in a subset of samples. Results: Eighty-one residual samples were collected. For IgA, 93% (n = 25) were identified as monoclonal. For IgG, 67% (n = 24) were monoclonal, and 33% (n = 12) were truly biclonal. Among the monoclonal IgGs, the second band represented a glycosylated form for 21% (n = 5), while 33% (n = 8) had masses consistent with a t-mAb. 44% (n = 8) of IgM samples were biclonal, and 56% (n = 10) were monoclonal, of which one was glycosylated. Conclusions: We demonstrate the utility of mass spectrometry in the characterization of multiple IFE bands of the same isotype. Improved reporting accuracy of M−proteins is useful for monitoring of patients with PCDs.

Original languageEnglish (US)
Pages (from-to)67-73
Number of pages7
JournalClinical Biochemistry
StatePublished - Nov 2021


  • Biclonal gammopathy
  • Immunofixation electrophoresis
  • Monoclonal gammopathy
  • Therapeutic monoclonal antibodies

ASJC Scopus subject areas

  • Clinical Biochemistry


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