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), B-cell non-Hodgkin lymphoma (B-NHL), and other B-cell lymphoproliferative disorders (LPD)³. A retrospective study conducted in Spain reported that among these conditions, MGUS is the most prevalent, accounting for 54.1% of cases, followed by MM (31.3%) and other malignant gammopathies (14.6%)⁴. It is also important to note that M protein may also be produced in response to certain non-neoplastic conditions, such as nephrotic syndrome⁵.

M protein testing plays a critical role in the diagnosis and monitoring of M protein-related disorders⁶. However, early diagnosis remains a clinical challenge, as patients with MM are often asymptomatic in the initial stages, leading to delayed diagnosis at a relatively advanced phase of the disease. Approximately 1% of individuals with MGUS progress to a malignant condition annually-most commonly to MM, WM, or SPC⁷. Although nearly all MM cases are believed to evolve from MGUS, fewer than 10% of MM patients have a documented history of prior MGUS diagnosis. Consequently, timely identification of M protein in serum and/or urine can provide valuable clues for early diagnosis of these disorders, which is crucial for improving patient survival and prognosis. Research indicates that patients with MGUS identified through active screening experience fewer complications than those diagnosed incidentally⁸. During therapeutic monitoring, information regarding the isotype and electrophoretic migration pattern of the M protein is essential to distinguish between recurrence of the original monoclonal protein and the emergence of oligoclonal bands or a secondary reactive M protein⁹. The appearance of a new M protein, often observed following autologous stem cell transplantation, is generally regarded as a favorable prognostic indicator for most patients¹⁰. Consistent with this observation, comparative survival data indicate that patients who develop a secondary M protein exhibit a median overall survival of 115.3 months, in contrast to 31.0 months among those who do not¹¹.

Protein electrophoresis is a highly sophisticated analytical technique. A range of methods is available for detecting serum M protein, including serum protein electrophoresis (SPE), IFE, capillary electrophoresis immunotyping (IT), free light chain (FLC) assay, heavy and light chain (HLC) detection, and mass spectrometry (MS). Each approach offers distinct advantages while also facing inherent limitations. SPE can be further subdivided into agarose gel electrophoresis (AGE) and CE. AGE, which uses an agarose-based medium, is a simple, cost-effective method suitable for rapid separation and analysis. However, it is limited by relatively low sensitivity, with a typical detection limit of 0.3-0.5 g/L¹², which may result in failure to detect small-volume or low-concentration M proteins. Furthermore, the technique is susceptible to band bending and deformation, which compromises reproducibility.

IFE is widely recognized as the gold standard for M protein typing. Following initial electrophoresis, specific antibodies (anti-IgG, anti-IgA, anti-IgM, κ, λ) are applied to form immunoprecipitated bands, enabling the identification of immunoglobulin type and light-chain subtype. IFE can detect M protein at concentrations as low as 0.05-0.15 g/L, providing a sensitivity more than 10 times that of SPE. Despite this advantage, specialized pretreatment is required for certain samples, such as those containing M protein polymers, to ensure dissociation with β-mercaptoethanol. Inadequate treatment may lead to misleading results. Additionally, patients undergoing novel combination therapies (e.g., regimens containing daratumumab, bortezomib, dexamethasone, and thalidomide/lenalidomide) may yield false‑positive IFE findings¹³. A key limitation of IFE is that it provides only qualitative results, as it cannot quantify M protein levels and is therefore unsuitable for monitoring therapeutic efficacy¹⁴. Among treated patients, IFE results may remain positive even as M protein concentration declines during successful treatment. 

In contrast, IT employs capillary buffers as the separation medium, offering higher resolution than conventional IFE. Nevertheless, the process requires laborious sample pretreatment steps, such as antiserum mixing, which prolongs the overall detection workflow. FLCs, which are immunoglobulin light chains (κ and λ) not bound to heavy chains, are directly quantified in serum using antibodies targeting their "hidden zone" epitopes, providing high sensitivity for detecting light chain-related disorders with a detection limit as low as 10–30 mg/L. In MM, elevated serum FLC levels correlate with tumor burden and allow for earlier assessment of treatment response, owing to their shorter half-life compared to intact immunoglobulins¹⁵. However, its clinical application should be combined with other diagnostic methods, such as electrophoresis, to rule out other conditions and ensure a comprehensive evaluation. For example, impaired renal function may lead to falsely elevated light-chain levels. HLC assays represent another quantitative approach that uses antibodies targeting epitopes on both the constant and light chains of immunoglobulin. This design allows for the separate quantification of isotype-specific immunoglobulin pairings (e.g., IgGκ and IgGλ), and the derived HLC ratio serves as a sensitive indicator of clonality. However, the results may be influenced by background polyclonal immunoglobulin levels, and the method may be less reliable when analyzing complex M-protein variants. MS offers exceptional sensitivity and enables in-depth proteomic characterization for M proteins. However, its application is constrained by high equipment costs, lengthy analytical procedures, and specialized expertise required for data interpretation.

CE is a powerful analytical separation technique that employs elastic fused-silica capillaries as separation channels and a high-voltage direct electric field as the driving force. It delivers high-resolution separation by leveraging differences in the electrophoretic mobility and/or partitioning behavior of target analytes. Since its inception, the technology has progressed from single-capillary configurations to high-throughput capillary arrays and miniaturized microfluidic devices. Its applications now span diverse fields, including pharmaceutical analysis, biomedical and clinical diagnostics, food safety testing, and biotherapeutic characterization^16,17. CE is increasingly recognized as a crucial tool for the screening and quantitative analysis of M proteins. It determines the percentage of M protein relative to total protein, which can then be multiplied by the total protein concentration to calculate absolute M protein levels. Most notably, its minimal sample requirement makes it particularly suitable for analyzing precious or volume-limited specimens.

CE offers three key technical advantages. Firstly, its high analytical sensitivity (0.1–0.5 g/L) enables the effective detection of low-concentration M proteins, thereby reducing the risk of misdiagnosis in conditions such as MGUS and light-chain diseases. Secondly, the fully automated workflow eliminates the subjective interpretation errors inherent in traditional gel-based staining methods. Finally, the assay time is significantly reduced to 15–20 min, markedly shorter than the 60–90 min required for conventional electrophoresis. As a pivotal analytical technique between zone electrophoresis and liquid chromatography, CE performs electrophoretic separation in a free solution. Under an applied electric field, protein molecules are primarily separated by electroosmotic flow within an alkaline buffer at a specific pH. Following dilution with a dedicated buffer, samples are injected at the capillary anode under high voltage to initiate rapid, efficient protein separation, and then detected at the cathode at 200 nm. After each analytical cycle, the capillaries undergo automated cleaning and buffer replenishment to process subsequent samples. Protein separation occurs in the following order: gamma-globulin, beta-2-globulin, beta-1-globulin, alpha-2-globulin, alpha-1-globulin, and albumin, enabling the identification of abnormalities in one or more serum protein fractions. Notably, CE provides enhanced resolution within the beta region, distinctly separating it into beta-1 and beta-2 globulin components, thereby delivering more detailed clinical information¹⁸. Driven by the integration of artificial intelligence and ongoing advancements in multi-parameter detection, CE is poised to evolve into a first-line screening modality for M protein detection. Therefore, this protocol describes the use of capillary electrophoresis for the rapid detection, localization, and quantitative analysis of serum monoclonal proteins in clinical samples.

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.
   NOTE: The running buffer is ready-to-use with a pH of 9.9 ± 0.5. The reagent kit contains three 700 mL bottles of alkaline buffer used as the running buffer for serum protein separation.
2. Dilute 75 mL of concentrated rinse solution stock to 750 mL with deionized water before use. Store the diluted rinse solution at room temperature. Once opened, the solution remains stable for up to 3 months. Use this solution to clean capillary tubes after electrophoresis.
   NOTE: The rinse solution contains sodium hydroxide and is used for capillary cleaning.
   CAUTION: The rinse solution contains sodium hydroxide, a corrosive substance that can cause severe burns. In case of eye contact, immediately rinse with plenty of water and seek medical attention. If skin or clothing gets contaminated, remove the contaminated garments immediately. Always wear appropriate protective clothing and use eye and face protection.
3. Store the reagent cups in the original sealed package at 2–30 °C in a clean and dry place. Use the reagent cups for sample dilution on fully automated instruments.
4. Store the sealed filter in a dry place at room temperature (15–30 °C). Use the disposable filters to filter buffer solutions and deionized water.
5. Store the capillary cleaning solution (see Table of Materials) at 2–8 °C and avoid precipitation.
   ​NOTE: Each bottle of capillary cleaning solution concentrate (25 mL/bottle) contains proteolytic enzymes, surfactants, and other additives. Additives are non-hazardous at the application concentration and are necessary for optimal performance. The solution prevents capillary blockage and maintains optimal performance.
6. Use filtered deionized water with a pore size ≤ 0.45 µm, a conductivity below 3 µS/cm, and resistivity exceeding 0.33 MΩ·cm. Change the water daily to prevent microbial contamination. Use this water to clean the capillary tubes of the electrophoresis analyzer.
7. Prepare a sodium hypochlorite solution containing 2–3% available chlorine. Use this solution for sample-probe cleaning to remove adsorbed proteins. Store the working chlorinated solution at room temperature in a closed container. The solution remains stable for up to 3 months. Do not store near sunlight, heat, ignition sources, acids, or ammonia.

2. Sample collection

1. Ensure that patients fast for a minimum of 8–12 h prior to blood collection.
2. Ask patients to rest in a seated or supine position for 10–15 min to stabilize hemodynamics.
3. Prepare the following supplies: disposable venous blood needles, vacuum blood collection tubes (serum tubes), tourniquet, sterile cotton swabs, 75% alcohol or iodophor disinfectant, medical adhesive bandage, and biohazard waste container.
   ​NOTE: The median cubital vein in the elbow fossa is the preferred puncture site.
4. Apply a tourniquet 3–5 cm above the puncture site to engorge the vein sufficiently.
5. Disinfect the skin with 75% alcohol or iodophor in a centrifugal spiral pattern from the center outward, covering an area ≥5 cm in diameter, and allow the disinfectant to air-dry naturally without touching the area again.
6. Hold the needle with the bevel upward at an angle of 15–30° to the skin and puncture the skin and vein wall smoothly.
7. Upon observing blood return, secure the needle and connect the vacuum tube. Let blood fill the tube passively under negative pressure, without manual compression, to prevent hemolysis.
8. After collection, first release the tourniquet, then pull out the needle gently, and immediately apply pressure to the puncture site with a sterile cotton swab for 3–5 min to ensure hemostasis.
9. Place the collected sample upright at room temperature and allow the blood to clot naturally.

3. Sample quality assessment

1. Centrifuge serum samples at 2,095 x g for 10 min at room temperature.
2. Subject serum samples to strict visual quality assessment under adequate natural light against a plain white background.
3. Evaluate hemolysis by examining the color and clarity of the supernatant. Grade samples as non-hemolyzed if the supernatant appears clear and yellow, while any pink or red discoloration is indicative of slight to severe hemolysis.
   NOTE: Hemolysis may produce a double alpha-2 zone. Beta-2 fraction may decrease in aged or improperly stored serum samples.
4. Inspect for fibrinogen interference by checking for visible fibrin strands, clots, or gelatinous networks within the supernatant, as well as incomplete phase separation, cloudiness, or inconsistent analytical signals.
   NOTE: Fibrinogen migrates in the beta-2 position (shoulder on beta-2 or superimposed with the beta-2 zone, with possibly an increase of this fraction). In some samples (plasma, serum not fully defibrinated, or patients on anticoagulant treatment), fibrinogen may interfere with analysis and lead to inaccurate interpretation, such as the suspicion of a monoclonal band or an increase in the beta-2 fraction.
5. Exclude samples showing hemolysis, fibrinogen interference, or other pre-analytical abnormalities once detected from analysis.
6. Collect a new blood specimen following standardized phlebotomy and processing protocols, ensuring appropriate clotting time and standardized centrifugation parameters. Use only samples with satisfactory appearance—clear, free of hemolysis, fibrin, lipemia, or contamination for subsequent testing.
7. Test the separated serum samples as soon as possible at room temperature. If immediate testing is not possible, store the samples at 2–8 °C for up to 10 days and at -20 °C for up to 30 days. Do not repeatedly freeze and thaw samples.
   NOTE: Avoid plasma samples. When analyzing aged plasma samples (which is not recommended), labile components such as C3 complement degrade over time, so the beta-2 zone essentially corresponds to fibrinogen.
8. Prior to testing, restore the samples to room temperature and mix them thoroughly. Ensure the frozen samples are completely thawed before use.
   NOTE: Use undiluted serum protein samples. Some refrigerated sera may become viscous or turbid, particularly those containing cryoglobulins or cryogel. Use samples containing polyimmunoglobulins directly without additional pre-treatment.

4. Operational procedures

1. Power on the automated capillary electrophoresis analyzer (see Table of Materials) and the connected computer system. Wait until the instrument is fully initialized.
   NOTE: The capillary length and inner diameter are 17.5 cm and 25 µm, respectively. The separation voltage ranges from 8,000–10,000 V. Electrokinetic injection is used, and the separation temperature is maintained at approximately 25 °C. The total run time is approximately 10 min per sample.
2. Launch the PHORESIS software (see Table of Materials) installed on the computer for data processing.
3. From the software interface, select the PROTEIN (E) 6 program and then insert the running buffer bottle into the instrument.
   NOTE: This program is a factory-calibrated preset protocol specifically designed for serum protein fractionation. All parameters are pre-configured in the instrument’s PHORESIS software with no need for manual modification.
4. If necessary, place the reagent bottle containing resolubilized cleaning solution within the instrument.
5. Mount a new reagent cup onto the automatic loading platform of the capillary electrophoresis analyzer.
6. Install a new waste box at the standard waste disposal position of the capillary electrophoresis analyzer for used reagent cups.
7. Load the sample racks into the analyzer through the opening on the right side (maximum capacity: 15 racks).
   NOTE: Each rack can accommodate up to 8 sample tubes. The bar code on each test tube must be visible through the rack opening.
8. Use sample rack No. 0 for the control serum. Use third-party quality control based on the cumulative mean and coefficient of variation calculated from 20 test data points.
9. Remove the completed sample rack from the unloading tray on the left side. Remove and discard the waste box containing the used reagent cups if necessary.
   CAUTION: Handle used reagent cups with caution, as they may contain biological samples.
10. Place capillary cleaning solution at position S2 in the secondary reagent chamber. Click Maintenance and Cleaning on the instrument screen, then select Start Capiclean.
11. Place 2-3% sodium hypochlorite solution in the secondary reagent chamber (T1 position). Click Maintenance and Cleaning on the instrument screen, and select Start Decontamination.
    ​NOTE: The entire capillary cleaning process automatically runs for 45 min.
12. Initiate the cleaning sequence at least once per week.

5. Results analysis

1. After completing each analysis run, transmit raw data from the instrument to the software for processing. The software generates an electropherogram that is displayed on the computer screen.
2. Analyze the electropherogram and quantify the relative absorbance intensities of each protein band at a wavelength of 200 nm using the software.
   NOTE: Based on total protein concentrations measured by the fully automated biochemical analyzer, the software calculates the concentration of each protein band.
3. Use the generated electropherograms for visual assessment and identification of abnormal bands.
   NOTE: By default, the system displays electropherograms in redraw mode.
4. Use a standard mode to present initial electropherograms derived from raw data if necessary.
   ​NOTE: The reference ranges of the capillary electrophoretic analyzer were established by statistical analysis of 246 healthy adult participants (both males and females) with normal triglyceride levels (Table 1). It is recommended that each laboratory establish its own standards.

6. Instrument shutdown

1. At the end of each analysis sequence, initiate the shutdown procedure of the capillary electrophoresis analyzer to store the capillary under ideal conditions.
   1. To initiate the shutdown procedure, select “Shutdown” from the main menu or use the shutdown rack provided with the analyzer.
   2. Press “Shutdown” in the main menu. In the window displayed on the screen, press  to initiate the procedure. The shutdown process will commence automatically upon completion of the ongoing analysis program.
   3. The shutdown rack is labeled "EXTINCTION/SHUTDOWN CAPILLARYS 3 & MC". Insert this rack into the end of the sample rack queue, and the instrument will automatically initiate the shutdown procedure.
      NOTE: When the shutdown procedure is complete, the instrument is powered off, and the screen will turn off.
2. Use the physical power switch on the back of the instrument to disconnect the analyzer from mains power.
3. Remove all reagent bottles from the two reagent chambers and store them under recommended storage conditions. Common faults, possible causes, and solutions of the fully automated electrophoresis analyzer are provided in Supplementary Table 1.

7. Performance parameters

1. To determine the analytical sensitivity of the capillary electrophoresis procedure, prepare and analyze serial dilutions of a serum sample containing an abnormal protein in the gamma zone at 1.198 g/dL. The highest dilution with a discernible abnormality corresponds to a concentration of 19 mg/dL of the abnormal protein.
   NOTE: Depending on the position of the abnormal protein and polyclonal background in the gamma zone, the detection limit may vary.
2. Accuracy assessment – Internal correlation
   1. To ensure sample integrity, process all the serum samples (including 18 normal samples and 100 pathological samples) identically and handle them according to the same guidelines.
   2. Measure the levels of each protein fraction both by electrophoretic separations obtained with the capillary electrophoretic procedure on the analyzer and another commercially available capillary electrophoresis technique for protein analysis.
   3. Analyze the measured values from both procedures by a linear regression statistical procedure. The obtained results demonstrated a perfect correlation between both procedures. 

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.