MSACL 2023 Abstract

Comparison of Three IgG Purification Methods for M-protein Detection Using MALDI-TOF Mass Spectrometry Jikyo Lee (1, 2), Seunghwan Kim (1, 2), Seojin Yang (3), Jung Hoon Choi (4), Heeyoun Hwang (4, 5), Joon Hee Lee (6), KyungHoon Lee (6), JungHan Song (6), Seungman Park (7), Sang Hoon Song (1, 2) (1) Department of Laboratory Medicine, Seoul National University College of Medicine, Seoul, Korea(2) Department of Laboratory Medicine, Seoul National University Hospital, Seoul, Korea(3) Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology, Seoul National University, Seoul, Korea(4) Research Center for Bioconvergence Analysis, Korea Basic Science Institute, Chungbuk, Korea(5) Critical Diseases Diagnostics Convergence Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea(6) Department of Laboratory Medicine, Seoul National University Bundang Hospital, Seongnam, Korea(7) Department of Laboratory Medicine, National Cancer Center, Seoul, Korea. Jikyo Lee, M.D. (Presenter) Seoul National University Hospital >> POSTER (PDF)

Presenter Bio: 2016 Grauate of Yonsei University Wonju College of Medicine,
2019~2021 Resident training in Seoul Nationl University Hospital (SNUH),
2022~2023 Fellow in SNUH as a specailist in Labroatory Medicine.

Introduction: Recently, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has emerged as a sensitive method to detect small amounts of M-protein. Dominantly, IgG is the most common type of monoclonal protein (M-protein) observed in patients with plasma cell disorders (PCDs). There are various techniques for IgG purification, such as Melon™ gel IgG spin purification kit, magnetic beads consist of protein G, and IgG affinity beads. We compared those three methods to find the most effective IgG purification approach for detecting M protein.

Objectives: We sought to find out the most effective method to purify IgG for detecting M-protein by MALDI-TOF MS.

Methods: A normal and an abnormal patient’s serum samples were selected from residual samples from immunofixation electrophoresis (IFE) tests on HYDRASIS 2 (Sebia, Fulda, German). In the IFE result, a patient serum showed IgG and kappa type of monoclonal antibody. To observe analytical sensitivity, daratumuamb was serially diluted into a normal serum at concentration of 0, 0.05, 0.1, 0.2, 0.5, 1.0 g/dL. C4 ZIPTIP was used for desalting after Melon™ Gel IgG Spin Purification Kit (Thermo Fisher Scientific Inc., MA, USA). For beads based methods, Dynabeads™ magnetic beads (Thermo Fisher Scientific Inc., MA, USA) and CaptureSelect™ affinity beads (Thermo Fisher Scientific Inc., MA, USA) were used. For each purification method, samples were spotted five times onto a 96-well plate. Each set of replicates was measured by MALDI-TOF MS (Bruker Daltonics, Bremen, Germany) for five days. We used α-Cyano-4-hydroxycinnamic acid (CHCA) matrix, and 500 shots were summed up.

Results: Peaks indicating reduced light chains with single and double charge of purified IgG were observed in mass spectrum within the 22000-23000 and 11000-12500 range of mass-to-charge ratio (m/z), respectively. Polyclonal peak consisting of kappa light chain and lambda light chain was observed in normal patient, while monoclonal peak was observed with higher intensity in abnormal patient. Coefficients of variation of m/z values for all the methods were in less than 20%. Limit of detection of IgG affinity beads was 0.1 g/dL, which was the lowest among three methods. Melon kit with C4 ZIPTIP was 0.2 g/dL, and magnetic beads showed 0.5 g/dL. IgG purification using CaptureSelect™ affinity beads showed a superior outcome compared to Melon kit or magnetic beads for detection of monoclonal protein. Melon kit might be considered as an alternative method for IgG Purification.

Conclusions: Among three methods, IgG affinity beads showed the best outcome to purify IgG for identifying M-protein using MALDI-TOF MS.