Electrophoresis Techniques

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Chapter 11

Electrophoresis Techniques

Learning Objectives

At the conclusion of this chapter, the reader should be able to:

• Define the term electrophoresis.

• Describe the electrophoresis technique.

• Identify the fractions into which serum proteins can be divided by electrophoresis.

• Describe the characteristics of immunoelectrophoresis.

• Explain the features of immunofixation electrophoresis.

• Discuss the clinical applications of immunoelectrophoresis.

• Compare immunoelectrophoresis and immunofixation electrophoresis.

• Compare capillary electrophoresis and microchip capillary electrophoresis.

• Describe two electrophoresis separation methods.

• Analyze a case study related to the results of serum protein electrophoresis.

• Correctly answer case study related multiple choice questions.

• Be prepared to participate in a discussion of critical thinking questions.

• Describe the principle of the immunofixation electrophoresis procedure.

• Correctly answer end of chapter review questions.

Electrophoresis

Electrophoresis is the migration of charged solutes or particles in an electrical field. Using this principle, charged molecules can be made to move and different molecules can be separated if they have different velocities in an electrical field.

The electrical field is applied to a solution with oppositely charged electrodes. Charged particles in this solution begin to migrate. Positively charged particles (cations) move to the negatively charged (−) electrode; negatively charged particles (anions) migrate to the positively charged (+) electrode (Fig. 11-1).

Figure 11-1 Application of electrical field to a solution of ions makes the ions move. (From Kaplan LA, Pesce AJ: Clinical chemistry: theory, analysis, correlation, ed 5, St Louis, 2010, Mosby.)

Serum proteins are often separated by electrophoresis. Serum electrophoresis results in the separation of proteins into five fractions using cellulose acetate as a support medium (Fig. 11-2). This separation is based on the rate of migration of these individual components in an electrical field.

Figure 11-2 Example of the effect of disease (hepatic cirrhosis) on serum protein electrophoresis pattern.
*Upper profile,* Distribution characteristic of healthy people. (From Kaplan LA, Pesce AJ: Clinical chemistry: theory, analysis, correlation, ed 5, St Louis, 2010, Mosby.)

Electrophoresis is a versatile analytic technique. Immunoglobulins are separated by electrophoresis using agarose as a support medium. The immunologic applications of electrophoresis include identification of monoclonal proteins in serum or urine, immunoelectrophoresis, and various blotting techniques (see Chapter 14).

Immunoelectrophoresis

Immunoelectrophoresis (IEP) involves the electrophoresis of serum or urine followed by immunodiffusion.

Passive Immunodiffusion Procedures

Immunodiffusion is a laboratory method for the quantitative study of antibodies (e.g., radial immunodiffusion [RID]) and rocket electrophoresis or for identifying antigens (e.g., Ouchterlony technique). Single diffusion preceded radial immunodiffusion. In the single diffusion procedure, antigen was layered on top of a gel medium and, as the antigen moved down into the gel, precipitation occurred and migrated down a tube in proportion to the amount of antigen present. In radial immunodiffusion (RID) (Fig. 11-3), antibody is uniformly distributed in the gel medium and antigen is added to a well cut into the gel. As the antigen diffuses from the well, the antigen-antibody combination occurs in changing proportions until the zone of equivalence is reached and a stable lattice network is formed in the gel. The area of the visible ring is compared with standard concentrations of antigens. A variation of this principle is rocket immunoelectrophoresis (Fig. 11-4).

Figure 11-3 Measurement of immune-related proteins by a radial immunodiffusion. (From Peakman M, Vergani D: Basic and clinical immunology, ed 2, Edinburgh, 2009, Churchill Livingstone.)

Figure 11-4 Configuration for immunoelectrophoresis.
Sample wells are punched in the agar-agarose, sample is applied, and electrophoresis is carried out to separate the proteins in the sample. Antiserum is loaded into the troughs and the gel is incubated in a moist chamber at 4° C (39° F) for 24 to 72 hours. Track x represents the shape of the protein zones after electrophoresis; tracks y and z show the reaction of proteins 5 and 1 with their specific antisera in troughs c and d. Antiserum against proteins 1 through 6 is present in trough b. (From Burtis CA, Ashwood ER, Bruns DB: Tietz fundamentals of clinical chemistry, ed 6, St Louis, 2008, Saunders.)

The classic Ouchterlony double diffusion technique (Fig. 11-5). performed on a gel medium is used to detect the presence of antibodies and determine their specificity by visualization of lines of identity, or precipitin lines. The reaction of antigen-antibody combination occurs by means of diffusion. The size and position of precipitin bands provide information regarding equivalence or antibody excess. Proteins are differentiated not only by their electrophoretic mobility, but also by their diffusion coefficient and antibody specificity. Although double immunodiffusion produces a separate precipitation band for each antigen-antibody system in a mixture, it is often difficult to determine all the components in a complex mixture.

Figure 11-5 Rocket immunoelectrophoresis of human serum albumin.
Patient samples were applied in duplicate. Calibrators were placed at opposite ends of the plate. (From Burtis CA, Ashwood ER, Bruns DB: Tietz fundamentals of clinical chemistry, ed 6, St Louis, 2008, Saunders.)

Principle

Immunoelectrophoresis is a combination of the techniques of electrophoresis and double immunodiffusion. IEP separates the antigen mixture by electrophoresis before performing immunodiffusion. In the first phase, electrophoresis, serum is placed in an appropriate medium (e.g., cellulose acetate or agarose) and then electrophoresed to separate its constituents according to their electrophoretic mobility—albumin; α₁-, α₂-, β-, and γ-globulin fractions

After electrophoresis, in the second phase, immunodiffusion, the fractions are allowed to act as antigens and to interact with their corresponding antibodies. Antiserum (polyvalent or monovalent) is deposited in a trough cut into the gel to one side and parallel to the line of separated proteins. Incubation allows double immunodiffusion of the antigens and antibodies. Each antiserum diffuses outward, perpendicular to the trough, and each serum protein diffuses outward from its point of electrophoresis. When a favorable antigen-to-antibody ratio exists (equivalence), the antigen-antibody complex becomes visible as precipitin lines or bands. Diffusion is halted by rinsing the plate in 0.85% saline. Unbound protein is washed from the agarose with saline and the antigen-antibody precipitin arcs are stained with a protein-sensitive stain.

Each line represents one specific protein (Fig. 11-6). Proteins are thus differentiated by their diffusion coefficient and antibody specificity as well as electrophoretic mobility. Antibody diffuses as a uniform band parallel to the antibody trough. If the proteins are homogeneous or of like composition, the antigen diffuses in a circle and the antigen-antibody precipitation line resembles a segment, or arc, of a circle. If the antigen is heterogeneous or not uniform in composition, the antigen-antibody line assumes an elliptical shape. One arc of precipitation forms for each constituent in the antigen mixture. This technique can be used to resolve the protein of normal serum into 25 to 40 distinct precipitation bands. The exact number depends on the strength and specificity of the antiserum used.

Figure 11-6 Double immunodiffusion in two dimensions by the Ouchterlony technique.
A, Reaction of identity. B, Reaction of nonidentity. C, Reaction of partial identity. D, Scheme for spur formation. *Ab,* Antibody; *Ag,* antigen. (From Burtis CA, Ashwood ER, Bruns DB: Tietz fundamentals of clinical chemistry, ed 6, St. Louis, 2008, Saunders.)

Immunofixation Electrophoresis

Immunofixation electrophoresis (IFE), or simply immunofixation, has replaced IEP in the evaluation of monoclonal gammopathies because of its rapidity and ease of interpretation. IFE is a two-stage procedure, agarose gel protein electrophoresis and immunoprecipitation. The test specimen may be serum, urine, cerebrospinal fluid (CSF), or other body fluids. The primary use of IFE in clinical laboratories is for the characterization of monoclonal immunoglobulins.

Clinical Applications

Although IFE was first described in 1964, it was introduced as a procedure for the study of immunoglobulins in 1976. IEP and IFE are complementary techniques best used in the workup of a patient with a suspected monoclonal gammopathy. The laboratory protocol for ruling out monoclonal gammopathy should include high-resolution electrophoresis, IEP of both serum and urine, and a quantitative immunoglobulin assay. These procedures are usually sufficient to detect and characterize monoclonal proteins with a serum concentration of 1 g/dL or more.

The following three protein variables can be determined using IFE:

1. Antigenic specificity

2. Electrophoretic mobility

3. Quantity or ratio of test and control proteins

Comparison of Techniques

IEP is technically simpler and less subject to antigen excess phenomenon than IFE. If high concentrations of monoclonal protein with IFE give no visible reactions, IEP is considered to be a better technique for typing large monoclonal gammopathies.

Immunofixation electrophoresis can be optimized to give greater sensitivity and resolution than IEP. IFE should be reserved for anomalous proteins, which are difficult to characterize by IEP. These include small bands, such as those exhibited in the early stages of monoclonal gammopathies or L-chain disease, and any multiple, closely spaced bands. The results of IFE are easier to interpret than those of IEP because interpretation is based on examination of a precipitate pattern directly analogous to routine electrophoresis; IFE does not depend on detecting slight deviations in the shape of a precipitin arc (Fig. 11-8; Table 11-1).

Table 11-1

Comparison of Immunoelectrophoresis and Immunofixation Electrophoresis

Feature	IEP	IFE
Ease of use	Easy	More complex
Sensitivity	Less sensitive	More sensitive
Monoclonal gammopathies	Better for typing large monoclonal gammopathies	Used for difficult to characterize anomalous proteins
Interpretation	Challenging	Easier

Figure 11-8 Comparison of immunofixation electrophoresis (IFE) and immunoelectrophoresis (IEP) for two patients with monoclonal gammopathies.
A, Patient specimen with an IgG (κ) monoclonal protein, as identified by IFE. Note the position of the monoclonal protein *(arrow).* After electrophoresis, each track except serum protein electrophoresis (SPE) is reacted with its respective antiserum; then, all tracks are stained to visualize the respective protein bands. Immunoglobulins G, A, and M (IgG, IgA, IgM); kappa (κ); and lambda (λ) indicate antiserum used on each track. B, Same specimen as in A, with proteins identified by IEP. Note the position of the monoclonal protein *(arrow).* Normal control (C) and patient sera (S) are alternated. After electrophoresis, antiserum is added to each trough, as indicated by the labels Ig, IgG, IgA, IgM, κ, and λ. The antisera react with separated proteins in the specimens to form precipitates in the shape of arcs. The IgG and κ arcs are shorter and thicker than those in the normal control, showing the presence of the IgG (κ) monoclonal protein. The concentrations of IgA, IgM, and λ light chains also are reduced. C, Patient specimen with an IgA (λ) monoclonal protein identified by the IFE procedure, as described in **A. D,** Same specimen as in C, with proteins identified by IEP, as described in B. The abnormal IgA and λ arcs for the patient specimen indicate an elevated concentration of a monoclonal IgA (λ) protein. All separations were performed with the Beckman paragon system. (From Burtis CA, Ashwood ER, Bruns DB: Tietz fundamentals of clinical chemistry, ed 6, St Louis, 2008, Saunders.)

Capillary Electrophoresis

In capillary electrophoresis (CE) the classic separation techniques of zone electrophoresis, isotachophoresis, isoelectric focusing, and gel electrophoresis are performed in small-bore (10- to 100-µm), fused silica capillary tubes, 20 to 200 cm in length (Box 11-1). The CE method is efficient, sensitive, and rapid. High electrical field strengths are used to separate molecules based on differences in charge, size, and hydrophobicity. Sample introduction is accomplished by immersing the end of the capillary into a sample vial and applying pressure, vacuum, or voltage.

Box 11-1 Separation Techniques Used in Capillary Electrophoresis

Capillary Zone Electrophoresis

Capillary zone electrophoresis (CZE) is the most widely used type of CE because of its simplicity and versatility. As long as a molecule is charged, it can be separated by CZE. Also, CZE is simple to perform because the capillary is only filled with buffer. Separation occurs as solutes migrate at different velocities through the capillary. Another advantage of CZE is that it separates anions and cations in the same run, which is not done in other CE methods. However, CZE cannot separate neutral molecules.

Isotachophoresis

Isotachophoresis (ITP) is a focusing technique based on the migration of the sample components between the leading and terminating electrolytes. Solutes with mobilities intermediate to those of the leading and terminating electrolytes stack into sharp focused zones. Although used as a mode of separation, transient ITP has been used primarily as a sample concentration technique.

Capillary Isoelectric Focusing

Capillary isoelectric focusing (CIEF) is a separation method that allows amphoteric molecules, such as proteins, to be separated by electrophoresis in a pH gradient generated between the cathode and anode. A solute will migrate to a point at which its net charge is zero. At the solute’s isoelectric point (pI), migration stops, and the sample is focused into a tight zone. In CIEF, once a solute has focused at its pI, the zone is mobilized past the detector by either pressure or chemical means. CIEF is often employed in protein characterization as a mechanism to determine a protein’s pI.

Modified from www.chemsoc.org and www.beckmancoulter.com, November 2007.

Microchip CE was developed in the early 1990s. The advantages of microchip CE include high speed, reduced reagent consumption, integration analysis, and miniaturization. The applications of microchip CE are diverse and include immune disorders.

Conventional CE revolutionized DNA analysis and was vital to the Human Genome Project. Microchip CE is still in the early stages of development but has demonstrated distinct advantages compared with traditional CE (Table 11-2).

Table 11-2

Comparison of Traditional Capillary Electrophoresis and Microchip Capillary Electrophoresis

Feature	Conventional CE	Microchip CE
Separation channels	Mainly silica, single capillary or capillary array	Glass or polymer
Separation media	Buffers, gels, sieving polymers, microparticles	Buffers, sieving polymers, microparticles
Speed of analysis	Fast (typically minutes)	Very fast (typically seconds)
Integration	Difficult to connect capillaries	Easy to integrate multiple functions (e.g., PCR CE)
Potential for growth	Relatively mature	Emerging technology with potential for new designs and applications

PCR, Polymerase chain reaction.

Adapted from Li SFY, Kricka LJ: Clinical analysis by microchip capillary electrophoresis, Clin Chem 52:42, 2006.

CASE STUDY

History and Physical Examination

MA is a 40-year-old woman with a long history of alcohol abuse. She came to the emergency department complaining of difficulty breathing.

Physical examination revealed a slightly jaundiced appearance, icteric sclera, hepatomegaly, and splenomegaly. She had decreased breathing sounds and swollen legs (edema). Laboratory tests were ordered.

Case Study Laboratory Data

Hematology	Patient’s Results	Reference Range
Hemoglobin	12.5 g/dL	12-16.0 g/dL
Hematocrit	42%	36%-45%
Mean corpuscular volume	100 fL	80-96 fL
Total leukocyte count	13.5 × 10⁹/L	4.5-11.0 × 10⁹/L
Platelets	95.0 × 10⁹/L	150-450 × 10⁹/L
Coagulation—Prothrombin Time	17 sec	10-14 sec
Urinalysis
Occult blood	1+	Negative
Bilirubin	Moderate	Negative
Clinical Chemistry
Bilirubin	2.5 mg/dL	0.3-1.2 mg/dL
Liver enzymes (alanine aminotransferase [ALT])	55 IU/L	10-35 IU/L
Total protein	5.5 g/dL	6.4-8.3 g/dL
Albumin	2.5 g/dL	3.9-5.g/dL