B-179 Development of a Reflex Mass Spectrometric Workflow for Confirmation of IgE and IgD Specificity Paraproteins Utilising the EXENT® Platform
Authors: Wright N , Bermingham W , Rollins A , Berlanga O , Pasha S , North S , Wallis G , Harding S 
Published: September 2023

Abstract

Background
The EXENT® (GAM) assay is a MALDI-TOF (Matrix Assisted Laser Desorption Ionisation Time of Flight) mass spectrometry-based solution which utilises IgG, IgA, IgM, total kappa and total lambda specific paramagnetic beads, which could be used in the investigation of monoclonal proteins in multiple myeloma and other plasma cell disorders (in development by The Binding Site (TBS), part of Thermo Fisher Scientific). Rare cases of IgD and IgE myelomas will be partially typed with the current iteration of the EXENT (GAM) assay. We present here a workflow to confirm the heavy chain specificity of such paraproteins as IgD or IgE using reflex LC-MS analysis of EXENT eluates.

Methods
Samples known to contain IgD (2) or IgE (3) paraproteins were provided from either routine workload at the immunology laboratory at Birmingham Heartlands Hospital, UK or an internal library of historical myeloma samples. Capillary zone electrophoresis (CZE) and immunofixation (IFE) were performed on Capillarys2™ and Hydrasys2 Scan™ platforms, respectively (Sebia, France). Serum free light chain (sFLC) measurement was performed using Freelite® reagents (The Binding Site, UK) on a Cobas c501 turbidimeter (Roche, Switzerland). Samples were prepared via EXENT (GAM) assay, by separately incubating diluted serum with antisera specific for IgG, IgA, IgM, total kappa and total lambda conjugated to paramagnetic beads. After washes, purified immunoglobulins were treated to reduce and elute the light chain component. Samples were mixed with matrix, spotted onto a MALDI target plate and acquired by MALDI-TOF MS. Once light-chain specificity of the suspected IgD or IgE paraprotein was determined, repeat immuno-precipitation with the same light-chain bead was carried out. Eluted samples were neutralised, transferred to fresh tubes, reduced, alkylated and digested with trypsin for 3 h. After acidification digested samples were analysed by C18 reverse phase UPLC on a BioAccord LC-MS system (Waters, UK). Data was then screened against a database of known immunoglobulin heavy chain peptides to assess the presence of heavy chain peptides specific for each immunoglobulin isotype.

Results
In each sample tested, the EXENT solution identified at least one monoclonal light chain that was only seen in the total kappa or total lambda immunoprecipitations with no corresponding monoclonal peak(s) at the same mass(es) in the IgG, IgA or IgM specificities. This finding would be consistent with the presence of clonal plasma cells producing either monoclonal free light chains only, or IgD and IgE paraproteins. Subsequent LC-MS and LC-MSe (tandem MS) analysis of reduced, alkylated and tryptically digested eluates corresponding to these suspected IgD and IgE samples produced high quality peptide sequencing data. The data identified multiple IgD or IgE specific peptides in each sample and showed between 40% and 66% sequence coverage for IgD or IgE heavy-chain constant regions. Thus, confirming the presence of IgD or IgE in these samples which had been partially typed by the EXENT solution.

Conclusion
This data demonstrates that the EXENT (GAM) assay could potentially aid in detection of IgD and IgE paraproteins when combined with LC-MS proteomic techniques, as it allows efficient and facile confirmation of these rare heavy chain isotypes.