Immunoelectrophoresis: Principle, Procedure, Results, Applications, Advantages, And Limitations

Immunoelectrophoresis Overview

  • Immunoelectrophoresis is the process of precipitation in an electric field on agar.
  • It is a procedure that combines electrophoresis with immuno-diffusion.
  • First, electrophoresis is utilised to separate an antigen mixture into its constituent parts, and second, double immuno-diffusion is utilised to analyse the mixture.
  • Without the use of an antibody, inserting antigens into gel-cut wells and electrophoresis. The gel is then sliced into a trough, which is subsequently filled with antibodies.
  • When antibodies come into contact with antigens that are diffusing, lattice formation and precipitation take place, allowing the antigens’ nature to be identified.
  • Grabar and Williams were the ones who originally used the phrase “immunoelectrophoresis” in 1953.      

Principles of Immunoelectrophoresis

While a gel-coated slide is subjected to an electric current, the antigen mixture placed in wells is separated into discrete antigen components based on their charge and size. After electrophoresis, specific antisera positioned in troughs parallel to the electrophoretic migration and diffusion are permitted to react with the separated antigens. In the course of 18 to 24 hours, the antiserum in the trough travels in the direction of the antigen components, causing the creation of distinct precipitin lines, each of which represents the interaction of a particular protein with its corresponding antibody.

Procedure for Immunoelectrophoresis

  1. On a glass slide that is horizontal, agarose gel is formed.
  2. Using the sample template, the application zone is meticulously drilled.
  3. Protein diluent solution (20 l of antigen solution plus 10 l of diluent) is used to dilute the sample 2:3.
  4. Using a 5 l pipette, 5 l of control and sample are administered across each matching slit (Control slit and Sample slit).
  5. The samples are put on the cathodic side of the gel in the electrophoresis chamber, and electrophoresis is conducted for 20 minutes at 100 volts.
  6. After electrophoresis is complete, 20 l of the appropriate antiserum is poured into troughs in a humid chamber and incubated horizontally for 18–20 hours at room temperature.
  7. The agarose gel is placed in a horizontal posture and blotter sheets are utilised to dry it.
  8. Ten minutes are spent immersing the gel in a saline solution before it is dried and rinsed twice more.
  9. The gel could be coloured for three minutes using a protein staining solution before being decolored for five minutes in discolouring solution baths. The gel is then left to dry at temperatures below 70 degrees Celsius.
  10. The gel is dried, and the outcomes are assessed.

Results of Immunoelectrophoresis

  • Ellipsoidal precipitin arcs are an indication of antigen-antibody interaction.
  • Precipitate formation not occurring indicates no response.
  • The strength, form, and location of the precipitation lines may be used to distinguish between various antigens (proteins).

Applications of Immunoelectrophoresis

  • The analysis facilitates the detection and approximate measurement of serum proteins. Immunoelectrophoresis made a turning point in immunology as well as protein identification possible.
  • Patients suspected of having monoclonal or polyclonal gammopathies are subjected to immunoelectrophoresis.
  • The technique can identify both normal and aberrant proteins, including myeloma proteins, in human blood.
  • It is used to evaluate complicated protein mixtures, including several antigens.
  • When certain proteins are anticipated to be deficient (as in hypogammaglobulinemia) or mass-produced, the diagnostic use is advantageous. (e.g., multiple myeloma).
  • This technique may be used to monitor antigen purity in order to identify a particular antigen in a combination of antigens and assess antigen-antibody purity.
  • An earlier technique for qualitatively analysing M-proteins in serum and also urine is immunoelectrophoresis.
  • In many immune system-related illness situations, immunoelectrophoresis helps with diagnosis and assessment of the therapy response.

Advantages of Immunoelectrophoresis

  • As it combines electrophoresis-based antigen separation with immunodiffusion using an antiserum, immunoelectrophoresis is a highly effective analytical technique with exceptional resolution.
  • The key benefit of immunoelectrophoresis is the capacity to recognise a range of antigens in serum.

Limitations of Immunoelectroctrophoresis

  • Compared to immunofixation electrophoresis, immunoelectrophoresis is more difficult to interpret, slower, and less sensitive.
  • Because the greatest quantities of the fastest moving immunoglobulins might mask the existence of tiny M-proteins, IEP is unable to identify certain small monoclonal M-proteins.
  • The use of immunoelectrophoresis during food testing is hampered by the dearth of widely accessible specific antibodies.


  1. Lydyard, P.M., Whelan,A.,& Fanger,M.W. (2005).Immunology (2 ed.).London: BIOS Scientific Publishers.
  2. Parija S.C. (2012). Textbook of Microbiology & Immunology.(2 ed.). India: Elsevier India.
  3. http://www.hellabio.com/E89B86BA.en.aspx
  4. Actor, J.K. (2014). Assessment of Immune Parameters and Immunodiagnostics. Introductory Immunology. Pages 135-152
  5. Sastry A.S. & Bhat S.K. (2016). Essentials of Medical Microbiology. New Delhi : Jaypee Brothers Medical Publishers.
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