Advanced Application of Capillary Electrophoresis in High-resolution Serum Monoclonal Protein Detection

Summary

Here we present a protocol for high-resolution serum protein analysis using capillary electrophoresis. Under a high-voltage electric field, serum proteins migrate through a capillary based on charge and electrophoretic mobility. The resulting electropherogram enables detection, localization, and quantification of monoclonal protein peaks.

Abstract

Monoclonal immunoglobulin, also known as M protein or paraprotein, is produced by clonal proliferation of plasma cells or B lymphocytes and serves as a key biomarker for monoclonal gammopathies. Accurate detection and quantification of serum M protein are essential for early disease diagnosis, clinical classification, and therapeutic monitoring. This protocol describes a method using capillary electrophoresis (CE) for high-resolution separation of serum proteins. Proteins are separated in an alkaline buffer under high-voltage electrophoresis and detected by ultraviolet absorbance, generating electropherograms that allow clear visualization and quantification of abnormal monoclonal protein peaks. Compared to traditional methods, CE provides improved resolution of the beta region and enables automated, rapid analysis with minimal sample handling requirements. Five representative electropherograms demonstrated M protein migration in the β1, β2, and γ regions, with M protein levels of 0, 44.1% (45.8 g/L), 10.5% (8.6 g/L), 35.5% (28.4 g/L), and dual peaks of 41.3% (46.0 g/L) and 12.4% (13.8 g/L). In a retrospective study of 700 clinical serum samples, CE achieved a positive detection rate of 51.86%, compared with 52.86% for immunofixation electrophoresis (IFE). Using IFE as the reference method, CE demonstrated a sensitivity of 92.70% and specificity of 93.94%. These findings confirmed CE as a rapid, automated approach for screening and quantifying serum M protein in routine clinical laboratories.

Introduction

M protein-related disorders are characterized by the presence of a monoclonal immunoglobulin (M protein) in the serum and/or urine. These proteins are typically secreted by clonal plasma cells or B lymphocytes and are primarily associated with malignant plasma cell dyscrasias or benign monoclonal gammopathies^1,2. The clinical spectrum of these conditions includes monoclonal gammopathy of undetermined significance (MGUS), solitary plasmacytoma (SPC), smoldering multiple myeloma (SMM), multiple myeloma (MM), plasma cell leukemia (PCL), Waldenström macroglobulinemia (WM), systemic light-chain amyloidosis (AL....

Protocol

The study protocol was reviewed and approved by the Ethics Review Committee of Guangdong Provincial People’s Hospital (Approval Number: KY2025-176-01). All participants provided written informed consent prior to sample collection.

1. Material preparation

NOTE: The reagent kit should be stored at 2–30 °C and has a shelf life of up to 3 years.

1. Store the running buffer at 2–8 °C and allow it to reach room temperature before use. Once opened, the solution remains stable for up to 3 months. Use this solution as the running buffer for serum protein separation in capillary electrophoresis.

Results

The migration positions, peak characteristics, and relative percentages of each protein band were quantitatively analyzed by detecting the absorbance of serum protein. Clinical interpretation was subsequently performed based on the presence and pattern of abnormal bands observed in the electropherogram. A representative capillary electropherogram showing the normal migration pattern of serum protein fractions is shown in Figure 1. Variations in peak migration times are primarily attributed t.......

Discussion

SPE is a fundamental diagnostic technique in clinical laboratories, separating serum proteins based on differences in their isoelectric points, molecular weights, and conformations¹⁹. This study systematically outlines a protocol for the rapid detection and quantification of serum M protein using CE. The protocol highlights the method's excellent analytical performance, with its core advantages being high sensitivity, high throughput, and full automation²⁰. Due to its m.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
CAPI 3 PROTEIN (E) 6 buffer	SEBIA	2503	Running buffer used for serum protein separation in capillary electrophoresis (referred to as the running buffer in the manuscript).
De lavage wash solution	SEBIA	2062	Diluted rinse solution used for cleaning capillary tubes after electrophoresis.
Reagent cups	SEBIA	2582	For sample dilution on fully automatic instruments.
FILTER CAPILLARYS	SEBIA		Disposable filter used for filtering buffer solutions and deionized water during capillary cleaning.
CAPICLEAN	SEBIA	2060	For cleaning capillary tubes of CAPILLARYS 3 OCTA instruments (referred to as the capillary cleaning solution in the manuscript)
Sodium hypochlorite solution	Guangzhou Chemical Reagents Factory	1703079	For sample probe cleaning.
Deionized water	Watsons	/	Used for capillary rinsing in the automated capillary electrophoresis analyzer.
Randox quality control	Randox	HN1530	For quality control.
Fully automated capillary electrophoresis analyzer	SEBIA	CAPILLARYS 3 OCTA	Instrument used for high-resolution and high-throughput serum protein analysis.
PHORESIS 9.30 software	SEBIA	/	Software used for automated acquisition and analysis of electrophoresis patterns.