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. 1), 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. 2).

Fig1. Measurement of immune-related proteins by a radial immunodiffusion. (From Peakman M, Vergani D: Basic and clinical immunol ogy, ed 2, Edinburgh, 2009, Churchill Livingstone.)

Fig2. 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. Anti serum 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.

Fig3. 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. 4). Proteins are thus differentiated by their diffusion coefficient and antibody specificity as well as electrophoretic mobility. Anti body 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 nor mal serum into 25 to 40 distinct precipitation bands. The exact number depends on the strength and specificity of the antiserum used.

Fig4. 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.)

Normal Appearance of Precipitin Bands

Immunoprecipitation bands should be of normal curvature, symmetry, length, position, intensity, and distance from the antigen well and antibody trough. In normal serum, immunoglobulin G (IgG), IgA, and IgM are present in sufficient concentrations of 10 mg/mL, 2 mg/mL, and 1 mg/mL, respectively, to produce precipitin lines. The normal concentrations of IgD and IgE are too low to be detected by IEP.

A normal IgG precipitin band is elongated, elliptical, slightly curved, and clearly visible in undiluted serum and 1:10 diluted serum. An IgG band is located cathodic to the antigen well in the alpha (α) area of the electrophoretogram. If monospecific serum is used, it is fused with a thin precipitin line positioned midway between the antigen well and antibody trough and extending into the beta (β) area. The IgM and IgA bands are visible in undiluted serum but disappear at a 1:10 dilution of serum. The IgA band is a flattened, thin arc, slightly cathodic to the well in the α-β position. The IgM line is a barely visible thin line, slightly cathodic to the antigen well.

Clinical Applications

Immunoelectrophoresis is most often used to determine qualitatively the elevation or deficiency of specific classes of immunoglobulins. Also, IEP is a reliable and accurate method for detecting structural abnormalities and concentration changes in proteins. It is possible to identify the absence of a normal serum protein (e.g., congenital deficiency of complement component) or alterations in serum proteins. This method can be used to screen for circulating immune complexes, characterize cryoglobulinemia and pyroglobulinemia, and recognize and characterize antibody syndromes and the various dysgammaglobulinemias.

The most common application of IEP is in the diagnosis of a monoclonal gammopathy, a condition in which a single clone of plasma cells produces elevated levels of a single class and type of immunoglobulin. The elevated immunoglobulin is referred to as a monoclonal protein, M protein, or paraprotein. Monoclonal gammopathies may indicate a malignancy such as multiple myeloma or macroglobulinemia. Antikappa (anti-κ) and antilambda (anti-λ) antisera are necessary for complete typing of the immunoglobulin in the evaluation of the ratio and for the diagnosis of M proteins. The class (heavy [H] chain) and type (light [L] chain) must be established because a patient’s prognosis and treatment may differ, depending on the immunoglobulin identified.

Differentiation must also be made between monoclonal and polyclonal gammopathies. A polyclonal gammopathy is a secondary condition caused by disorders such as liver disease, collagen disorders, rheumatoid arthritis, and chronic infection. It is characterized by elevation of two or more (often all) immunoglobulins by several clones of plasma cells. Polyclonal increases of proteins are usually twice the normal levels.

T he most important application of IEP of urine is the demonstration of Bence Jones (BJ) protein. IEP detects very low concentrations of BJ protein (≈1 to 2 mg/dL). If BJ pro tein is present in a urine specimen, precipitin lines will form with κ or λ anti–L chain antisera because BJ protein is composed of homogeneous L chains of a single antigen type, either κ or λ. Normal L chains are heterogeneous and include equal concentrations of κ and λ.

Sources of Error

The prozone phenomenon is an incomplete precipitin reaction caused by antigen excess (antigen-to-antibody ratio too high). Prozoning should be suspected if a precipitin arc appears to run into a trough, if an L chain appears fuzzy when an H chain is increased, or if an arc appears to be incomplete.

Abnormal Appearance of Precipitin Bands

The size and position of precipitin bands provide the same type of information regarding equivalence or antigen-antibody excess as double immunodiffusion systems. The position and shape of precipitin bands in the IEP assay of serum are relatively stable and reproducible; almost any deviation is abnormal (Fig. 5). These abnormalities can be detected by evaluating the following features of the precipitin bands:

• Position of the band in relation to electrophoretically identified protein fractions

• Position of the band between the antigen well and antibody trough

• Distortion of the curvature or arc formation

• Thickening (density) and elongation of a band

 • Shortening (inhibition), thinning, or doubling of a band

Fig5. Example of immunoglobulin (Ig) profile on immunoelectrophoresis showing abnormal Ig pattern. (Adapted from Ritzman SE, Daniels JC: Laboratory notes—serum proteins, No. 3, Somerville, NJ, 1973, Behring Diagnostics–Hoechst Pharmaceuticals.)

Position of Band

The precipitin band may be displaced compared with its normal position in the control serum because molecular charges in the abnormal protein may affect its speed of migration in the electrophoresis phase of IEP. A precipitin band may form a line of fusion or partial fusion with another protein, indicating the presence of proteins immunologically similar but electrophoretically distinct.

A distinct abnormality in the position of the band is seen in cases of monoclonal IgA gammopathy. The monoclonal IgA band is closer to the antibody trough than normal IgA.

Distortion of Curvature or Arc

An abnormal curvature of the precipitin band can be observed with M proteins because of an antigen excess. The monoclonal IgG band shows an arc of a circle rather than the elongated elliptical shape of a normal band. This distortion of IgG reflects its homogeneous nature and limited electrophoretic mobility of the abnormal protein.

Normal IgM and IgD bands are hardly visible, but the monoclonal IgM or IgD bands are skewed arcs of a circle.

Thickening and Elongation

T hickening and elongation can be seen in the presence of M proteins because excess antigen diffuses a greater distance. Monoclonal IgM, IgG, IgD, and IgA all demonstrate denser than normal bands. In addition, monoclonal IgG touches the antisera trough.

 Shortening, Thinning, or Doubling

 A band may be shortened and incomplete because of inhibition of a segment, resulting from the antibody’s reacting with only a portion of the abnormal protein. Monoclonal IgE elevation leads to a short thick arc in the antigen well area, extending to the anodal side.

Polyvalent and Monovalent Antisera

Polyvalent antiserum confirms the presence or absence of major protein fractions. Monovalent antiserum for specific individual immunoglobulins identifies only the corresponding proteins. If the nonspecific antisera have combining sites for H and L chains, the combining sites will react with L chains of other immunoglobulins or with the free L chains of BJ protein. H-chain–specific sera do not cross-react with other proteins.

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