Immunoelectrophoresis

Abstract

Immunoelectrophoresis is a two‐stage process. Electrophoresis is conducted in the first stage and immunoprecipitation using antibodies against specific proteins in the second stage without removing the proteins from the separation media (usually agarose). Four types of immunoelectrophoresis (IEP) have been used: electroimmunoassay (EIA also called ‘rocket’ or ‘Laurell rocket’), classical IEP, immunofixation electrophoresis (IFE) and immunoprecipitation of proteins after capillary electrophoresis. The procedure used in most medical laboratories is IFE where the proteins are immunoprecipitated on the gel in a pattern that is similar to their location on routine agarose gel electrophoresis. This type of immunoelectrophoreisis is easier to interpret and more sensitive than the IEP that it replaced. The principles underlying each method and the strengths and weaknesses of each is described. Example of IEP and IFE for identifying monoclonal proteins in serum and urine and the relationship of the proteins to disease is discussed.

Key concepts

  • Immunoelectrophoresis differs from blotting techniques because with IE the entire procedure is conducted in an agarose gel and blotting is not necessary.

  • These techniques are based on the concept that near equivalency an antigen–antibody complex will precipitate and become trapped in the gel.

  • Immunofixation electrophoresis (IFE) is preferred over immunoelectrophoresis because of its greater sensitivity and simpler interpretation characteristics.

  • Immunofixation electrophoresis is largely used in medical or clinical laboratories for identifying abnormal monoclonal immunoglobulins associated with diseases of lymphocytes such as multiple myeloma.

  • The abnormal protein bands identified on IFE line up with bands seen on routine agarose electrophoresis making interpretation simpler.

  • Proteins in large concentration can cause prozoning on IFE causing more difficulty in interpretation.

  • IFE is widely used in medical laboratories for identifying monoclonal free light chains in urine, called Bence Jones proteins.

  • IFE can also be used for identifying oligoclonal banding in cerebrospinal fluid.

Keywords: immunoelectrophoresis; immunofixation electrophoresis; electro immunoassay; agarose gel electrophoresis

Figure 1.

Examples of prozoning. (a) Minimal prozoning in a serum as a result of a high antigen concentration. The track on the left shows agarose gel serum electrophoresis (SPE). The bands represent various serum proteins, as indicated. The track on the right shows the same serum sample after immunofixation of IgG with antibody specific for IgG, washing and staining. Only the IgG remains in the gel (see Figure b and text for more details). The distinct band (green arrow) reflects a monoclonal IgG protein from a patient with multiple myeloma. The same band can be seen in the SPE gel before fixation (red arrow). Notice, the band looks like a donut. This is because the amount of protein antigen decreases from the centre of the band so that there is antigen excess in the centre but equivalency of antibody and antigen concentration towards the edges, causing less reaction in the centre and more towards the periphery. (b) A concentrated urine containing a large concentration of monoclonal free light chain (Bence Jones protein). On the far left, is shown agarose gel electrophoresis (Age) of the urine. The thick band just below the origin represents a highly concentrated monoclonal free light chain. The 7 tracks to the right show immunofixation with an antibody specific for κ light chain. Notice that as the urine is diluted, a thinned out donut starts to develop because the antigen concentration is too high so that little reaction occurs with the fixation antibody. The donut is seen at the 1:25 and 1:50 dilutions. With zero dilution, the reaction is so weak that nothing is seen. As the antigen becomes more dilute a band is seen at the 1:75 and 1:100 dilutions. The band begins to disappear at the 1:150 dilution as the antibody moves into the zone of excess (prezoning). See Figure a and b and the text associated with it for more details of the IFE method. The circles at the bottom of the gels represent control wells where antigen and antibody are placed to ensure there is a reaction. Electrophoresis is towards the anode or positive electrode. The arrow indicates the origin (o) – spot at which the sample is placed.

Figure 2.

Diagrammatic illustration of IEP. To the left serum agarose protein electrophoresis (SPE) illustrates the migration of serum proteins. Polyclonal immunoglobulins are illustrated by the speckled pattern and specific protein by the solid bands. The arrow illustrates a monoclonal immunoglobulin in the gamma region. The direction of migration is down towards the positive electrode. Normal serum sample which serves as a control (C) or a sample from the patient (P) is placed in the sample well of appropriate strips, as indicated. After electrophoresis, specific antisera with activity against immunoglobulin heavy chains IgG, IgA, IgM, and light chains κ and λ are placed in troughs (T) that separate the samples. The separated proteins from the sample and antisera from the trough diffuse towards one another and form precipitation arcs. Each patient arc is compared with the control arc on the other side of the respective trough (see Figure a for an actual illustration). A disadvantage of this method is that the resultant pattern is difficult to relate to the migration of each of the proteins on SPE.

Figure 3.

Diagrammatic illustration of IFE. (a) Serum to the left shows agarose electrophoresis without fixation. Five tracks to the right show IFE with specific antisera against immunoglobulin heavy chains IgG, IgA, IgM and light chains κ and λ. The antisera are layered separately over each lane. The antisera against light chains includes antibodies that react with intact (bound to heavy chains) light chain (B) and unattached (free) light chains (F). (The black arrow identifies the IgA‐κ monoclonal on the SPE track.) The red arrow identifies an IgA monoclonal immunoglobulin and the green arrow shows it is an IgA with a κ light chain attached. The blue arrow identifies this IgA‐κ monoclonal protein on the (UPE) track. The dark bands represent specific protein fractions and the dotted areas immunoglobulins that normally migrate in diffuse patterns because of their diversity. Notice that the normal IgA migrates the closest to the positive electrode and the IgG closest to the negative electrode. (b) Illustrates IFE after urine electrophoreisis. The red arrow indicates that there is an intact IgG‐κ monoclonal protein and the green arrow points to a κ free light chain. The blue arrows indicate the monoclonal intact and monoclonal free κ light chain in agarose electrophoresis before fixation. Also, see Figure and Figure b for a reproduction of actual gel plates. The direction of migration is down towards the positive electrode. Symbols and abbreviations are the same as in Figure .

Figure 4.

Actual immunoelectrophoretic plates. (a) Classical immunoelectrophoresis (IEP). The patient (P) shows an arc that is more dense than the control on the other side of the trough containing antisera against IgG. The enlarged arc that also shows a distortion is illustrated by the red arrow. This indicates the patient's serum contains an elevated monoclonal IgG. A similar large arc is seen adjacent to the trough containing anti‐κ antisera. This indicates that the monoclonal protein is IgG‐κ. The blue arrow indicates a smaller arc that is continuous with the larger arc adjacent to the anti‐κ trough. Since the smaller arc is not seen adjacent to the trough containing IgG antisera, this indicates there is a κ‐free light chain as well. Notice that the arcs representing IgA, IgM and λ are decreased as compared to the control, indicating normal immunoglobulins are decreased in this patient. TV, represents a trough containing trivalent antisera: against IgG, IgM and IgA. (b) IFE of the same patient. A dense IgG band migrating coincidental with a κ band indicates an intact monoclonal IgG‐κ (red arrows) and a faster migrating κ band that did not stain for IgG indicates a κ‐free light chain. Both can be seen on the agarose gel electrophoresis (SPE) to the left. This greater simplicity of interpretation of IFE patterns are a major reason that IEF has replaced IPE (for additional details with diagrammatic illustration see Figure and Figure and the text).

Figure 5.

Multiple band pattern (ladder) on urine IFE after fixation with κ antisera. Migration is towards the positive electrode at the bottom. The red arrows indicate the location of the multiple bands. They do not represent monoclonal free light chains (Bence Jones proteins). For comparison, the track to the right shows a true Bence Jones protein. Other symbols and abbreviations are the same as in Figure .

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References

Bence Jones H (1847) Papers on chemical pathology: lecture III. Lancet 2: 88–92.

Cawley LP, Minard BJ, Tourtellotte WW, Ma BI and Chelle C (1976) Immunofixation electrophoretic techniques applied to identification of proteins in serum and cerebrospinal fluid. Clinical Chemistry 22: 1262–1268.

Edelman GM and Gally JA (1962) The nature of Bence‐Jones proteins. Chemical similarities to polypetide chains of myeloma globulins and normal gamma‐globulins. Journal of Experimental Medicine 116: 207–227.

Falk RH, Comenzo RL and Skinner M (1997) The systemic amyloidoses. New England Journal of Medicine 337: 898–909.

Grabar P and Williams CA (1953) Method permitting the combined study of the electrophoretic and the immunochemical properties of protein mixtures; application to blood serum. Biochimica et Biophysica Acta 10: 193–194.

Graziani M, Merlini G and Petrini C (2003) Guidelines for the analysis of Bence Jones protein. Clinical Chemistry and Laboratory Medicine 41: 338–346.

Harrison HH (1991) The “ladder light chain” or “pseudo‐oligoclonal” pattern in urinary immunofixation electrophoresis (IFE) studies: a distinctive IFE pattern and an explanatory hypothesis relating it to free polyclonal light chains. Clinical Chemistry 37: 1559–1564.

Hess PP, Mastropaolo W, Thompson GD and Levinson SS (1993) Interference of polyclonal free light chains with identification of Bence Jones proteins. Clinical Chemistry 39: 1734–1738.

Keren DF, Alexanian R, Goeken JA et al. (1999) Guidelines for clinical and laboratory evaluation patients with monoclonal gammopathies. Archives of Pathology & Laboratory Medicine 123: 106–107.

Kyle RA, Remstein ED, Therneau TM et al. (2007) Clinical course and prognosis of smoldering (asymptomatic) multiple myeloma. New England Journal of Medicine 356: 2582–2590.

Laurell CB (1966) Quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies. Analytical Biochemistry 15: 45–52.

Levinson SS and Keren DF (1994) Free light chains of immunoglobulins: clinical laboratory analysis. Clinical Chemistry 40: 1869–1878.

MacNamara EM, Aguzzi F, Petrini C et al. (1991) Restricted electrophoretic heterogeneity of immunoglobulin light chains in urine: a cause for confusion with Bence Jones protein. Clinical Chemistry 37: 1570–1574.

Ritchie RF and Smith R (1976) Immunofixation. III. Application to the study of monoclonal proteins. Clinical Chemistry 22: 1982–1985.

Shaheen SP, Talwalkar SS and Medeiros LJ (2008) Multiple myeloma and immunosecretory disorders: an update. Advances in Anatomic Pathology 15: 196–210.

Sheldon J and Riches P (2004) Capillary electrophoresis for investigation of proteins in biological fluids. Journal of Clinical Ligand Assay 27: 227–233 (www.ingentaconnect.com).

Sølling K (1981) Free light chains of immunoglobulins: studies of radioimmunoassay of normal values, polymerism, mechanisms of renal handling and clinical significance. Scandinavian Journal of Clinical and Laboratory Investigation 41(suppl. 157): 15–83.

Towbin H, Staehalin T and Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets, procedures and some applications. Proceedings of National Academy of Sciences of the USA 76: 4350–4357.

Further Reading

Hamilton RG and Keren DF (eds) (2006) Manual of Molecular and Clinical Laboratory Immunology, 7th edn, chaps 6–10. Philadelphia, PA: ASM Press.

Kaplan LA and Pesce AJ (1989) Clinical Chemistry, Chap. 63 (a new 5th Edition of Kaplan and Pesce's Clinical Chemistry is due in 2010, ed: Hickman PE and Koerbin, G). St. Louis, MO: CV Mosby Company.

Karcher R and Landers JP (2006) Electrophoresis. In: Burtis CA, Ashwood ER and Bruns DE (eds) Tietz Textbook of Clinical Chemistry and Molecular Diagnostics, 4th edn, Chap. 5. St. Louis, MO: Elsevier‐Saunders.

McPherson RA and Pincus MR (eds) (2007) Henry's Clinical diagnosis and Laboratory Management, 21st edition, chaps 19 and 45. Philadelphia, PA: Saunders–Elsevier.

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Levinson, Stanley S(Sep 2009) Immunoelectrophoresis. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0001136.pub2]