Capillary Zone Electrophoresis
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The Hunter Area Pathology Service has implemented the Beckman CZE 2000 Capillary Electrophoresis System, which offers automated Capillary Zone Electrophoresis (CZE) technology for the analysis of serum protein electrophoresis (SPE) and the identification and typing of serum monoclonal proteins by Immunosubtraction (IS). Serum samples are injected into a narrow-bore capillary tube and the protein fractions are separated in a liquid phase buffer by the application of high voltage across the capillary.
Serum Protein Electrophoresis
The reference ranges were established by the Hunter Immunology Unit using fresh sera from 99 normal blood bank donors. One impact of the new technology is that low levels of Alpha-1-Antitrypsin are not as obvious on the CZE as on previous methods using cellulose acetate or agarose electrophoresis. The locations of specific proteins within the CZE electrophoresis pattern are indicated in Figure 1.
Table 1. Patient Reference Ranges |
Electrophoretic Fractions |
Range (g/L) |
Total Protein |
64 - 80 |
Albumin |
38 - 50 |
Alpha-1 |
3 - 5 |
Alpha-2 |
3 - 7 |
Beta |
6 - 10 |
Gamma |
7 - 14 |
Figure 1. CZE Electrophoresis Pattern
- pre-albumin
- Albumin
- alpha 1-Acid-Glycoprotein
- alpha 1-Antitrypsin
- Haptoglobin
|
- alpha2 macroglobulin
- Hemopexin
- Transferrin
- Complement
- Gamma
|
The detection limit for monoclonal proteins on the CZE-SPE was determined by our laboratory to be approximately 0.5 g/L. The CZE technology is able to more accurately quantitate the concentration of monoclonal proteins on the SPE, as the detection does not rely on the uptake of dyes, as is the case in cellulose-acetate or agarose EPG methods.
Immunosubtraction of Monoclonal Proteins
Serum monoclonal immunoglobulins have been traditionally identified by immunofixation methods such as immunoelectrophoresis (IEP), isoelectricfocusing (IEF) or high resolution agarose electrophoresis. The CZE uses a reverse technique of immunosubtraction (IS). The sample is allowed to incubate simultaneously, in different wells, with five different solid supports (sepharose beads), to which are bound specific antisera for alpha, gamma and mu heavy chains and kappa and lambda light chains. After incubation, the treated samples are assayed simultaneously by capillary electrophoresis in adjacent capillaries. The typing of the monoclonal component is determined by overlaying SPE electropherograms from before and after incubation with antisera to visually determine which heavy and light chain antisera has removed the monoclonal protein (immunosubtraction) from the serum.
The identification of IgD and IgE monoclonal proteins is currently not available by CZE and confirmatory tests by IEP or IEF/Immunofixation will continue to be used when appropriate. The technology is currently uderevaluation for urine and is not available for CSF samples.
The IS example provided in Figure 2 shows a distinct monoclonal protein peak (1) on CZE-SPE, that is removed from the electrophoresis pattern by antisera to gamma (2) heavy chains (Anti-IgG) and kappa light chains (3). The immunosubtraction indicates the monoclonal protein is IgG Kappa.
Figure 2. Monoclonal Protein Typing by Immunosubtraction on CZE
 Anti-IgG
|
 SPE
|
 Anti-IgA
|
 Anti-Kappa
|
 Anti-IgM
|
 Anti-Lambda
|
- Monoclonal protein on CZE-SPE
- Monoclonal subtraction of heavy chain
- Monoclonal subtraction of light chain
Further Information
The CZE-SPE and CZE-IS are assayed daily (weekdays) by the Hunter Immunology Unit. Further information about the CZE or assistance with interpretation of the patterns is available at any time by contacting the laboratory or one of the on-call immunopathologists.
Written by: Christine Burns and Dr Maree Gleeson, Immunology, HAPS
Written: November 1998
Last Reviewed: 10.5.2001