Immunoelectrophoresis Test – Principle (Steps), Uses, Limitations and Facts

What is Immunoelectrophoresis?

It is agar’s precipitation under the electric field. As the name suggests, it is a combination of electrophoresis and immune-diffusion. Immunoelectrophoresis is an umbrella term for a number of biochemical procedures to separate and characterize proteins according to electrophoresis and reaction with antibodies.

Immunoelectrophoresis variants need immunoglobulins/antibodies to react to proteins to be characterized or separated. The process of Immunoelectrophoresis is begun by separating the antigen mixture into component parts through the process of electrophoresis and double immuno-diffusion. (1, 2, 3, and 4)

immunoelectrophoresis procedure is performed using the machine as illustrated in the image

Diagram 1: An immunoelectrophoresis procedure is performed using the machine as illustrated in the image above.

 

image above shows the schematic presentation of immunoelectrophoresis

Image 2: The image above shows the schematic presentation of immunoelectrophoresis.
Picture Source:
researchgate.net

The antigens are put into the wells divided into a gel and electrophoresed. The antibodies are placed in a trough that is cut in the gel. They are diffused in a lateral motion for the diffusing antigens to meet allowing the formation of lattice and precipitation, which aids in identifying antigens’ nature. (3, 5, and 7)

It was Grabar and Williams who first coined the term immunoelectrophoresis in 1953.

During such time, they applied a current to a slide containing a layer of a gel. The mixture of antigen is placed in wells and separated to form a single component of antigen based on their size and charge.

Once the antigens are successfully separated, they are reacted with particular antiserum placed in troughs with the same distance to the electrophoretic migration and allowed to diffuse.

The trough contains antiserum and migrates toward the components of antigen leading in the formation of precipitin lines within 18 to 24 hours. (4, 5, and 6)

 

image above shows the step by step guide for conducting immunoelectrophoresis

Image 3: The image above shows the step by step guide for conducting immunoelectrophoresis.
Picture Source: slideplayer.com

 

Identifying a female specific protein with the aid of immunoelectrophoresis

Image 4: Identifying a female specific protein with the aid of immunoelectrophoresis.
Picture Source:
encrypted-tbn0.gstatic.com

 

The steps for immunoelectrophoresis are as follows:

  • The process starts with placing an agarose gel on the glass slide and position horizontally.
  • Wells are carefully carried on the application zone.
  • Dilute the sample in a ratio of 2:3 using a protein diluent solution.
  • A sample is applied across each corresponding slit using a 5 μl pipette and 5 μl of control.
  • The gel is then placed in the electrophoresis chamber. The sample is placed on the side of cathode and run for a timeframe of 20 minutes in 100 volts.(2, 5, and 8)
  • Once the electrophoresis process is completed, the next step is to add the equivalent antiserum (20 μl) to troughs in a moist chamber. It is positioned horizontally and incubated at a room temperature for about 18 hours to 20 hours.
  • The agarose gel is positioned horizontally and dried using blotter sheets.
  • The gel is soaked in a saline solution for about 10 minutes and dried and washed two times.
  • When drying the gel, the ideal temperature should be less than 70-degree Celsius and can be stained using a protein staining solution for 3 minutes. The next step is to decolorize the gel for five minutes in a distaining solution bath.
  • The final step is drying the gel and interpreting the results.(3, 6, 7, and 8)

 

Interpreting the result of a sample subjected to immunoelectrophoresis

Image 5:Interpreting the result of a sample subjected to immunoelectrophoresis.
Picture Source: springernature.com

 

Interpreting results

  • Watch for any signs of elliptical precipitin arcs as they indicate antigen-antibody interaction.
  • There is no reaction if no precipitation is formed.
  • By checking the result, you can identify different antigens according to the shape, intensity, and the precipitation line placement. (8, 9, and 10)

Uses of immunoelectrophoresis

  • It helps identify and approximate the quantity of some types of proteins present in the serum. It paves the way in identifying protein and immunology.
  • It is helpful in people suspected with monoclonal and polyclonal gammopathies.
  • It helps in identifying normal and abnormal proteins like myeloma proteins that might be present in human serum.
  • It is used complex protein mixture analysis, especially the ones containing various types of antigens.
  • It is one of the traditional ways to analyze qualitatively M-proteins in both serum and urine. (1, 5, and 9)
  • It is used in analyzing the number of proteins whether proteins are overproduced or absent at all.
  • It is useful in diagnosing and evaluating the therapeutic responses of many types of illnesses that affect the body’s immune system.
  • It is used for antigen monitoring as well as antigen-antibody purity.
  • It can identify a particular antigen in an antigen mixture. (2, 5, and 9)

APPLICATIONS OF Immunoelectrophoresis

Image 6: Immunoelectrophoresis is used in a variety of industries, especially in identifying a particular or a number of antigen in a given serum.
Picture Source:
slidesharecdn.com

 

What are the advantages of performing immunoelectrophoresis?

  1. One of the primary advantages of immunoelectrophoresis is its ability to identify a number of antigen in a serum.
  2.  It is an analytical tool with high resolving capability as it can separate antigens by electrophoresis with immune-diffusion against an antiserum. (1, 6, and 7)

Are there any limitations?

Although immunoelectrophoresis does have a lot of advantages, it does have its shares of limitations such as:

  • The process is slower and less sensitive when compared to other types of electrophoresis such as immunofixation.
  • Interpreting results can be quite challenging.
  • It might not be able to fully detect some small monoclonal M-proteins.
  • Its use in food analysis is limited by specific antibody availability. (3, 5, 9, and 10)

A rocket immunoelectrophoresis is a sub-type of immunoelectrophoresis, which is an adaptation of a radial immunodiffusion

Image 7: A rocket immunoelectrophoresis is a sub-type of immunoelectrophoresis, which is an adaptation of a radial immunodiffusion.
Picture Source:
163602-560839-raikfcquaxqncofqfm.stackpathdns.com

 

Immunoelectrophoresis facts

  • Immunoelectrophoresis is simply agar’s precipiration in an electric field.
  • It is a combination of two processes – electrophoresis and immuno-diffusion.
  • Grabar and Williams first coined the term immunoelectrophoresis in 1953.
  • It needs antibodies, also known as immunoglobulins to react with proteins for characterization and separation.
  • A classic immunoelectrophoresis method is called immunoelectrophoretic analysis ad modum Grabar.
  • There are other methods or sub-types of immunoelectrophoresis such as crossed immunoelectrophoresis, rocket immunoelectrophoresis, fused rocket immunoelectrophoresis, and affinity immunoelectrophoresis. (2, 5, and 10)

 

References

  1. https://microbenotes.com/immunoelectrophoresis-principle-procedure-results-and-applications-advantages-and-limitations/
  2. https://www.sciencedirect.com/topics/neuroscience/immunoelectrophoresis
  3. https://medlineplus.gov/ency/article/003541.htm
  4. https://www.ncbi.nlm.nih.gov/pubmed/22585499
  5. https://iai.asm.org/content/iai/6/4/564.full.pdf
  6. https://link.springer.com/chapter/10.1007/978-3-642-67356-6_72
  7. https://www.healthline.com/health/immunoelectrophoresis-serum
  8. http://lvts.fr/Pages_html/Encyclopedies/FundamentalImmunology/ramiCOMMAND=applyStylesheet(interface.xsl,pau@CH004S0503.pub)&p_userid=pau.html
  9. https://www.slideshare.net/SaajidaSultaana/immunoelectrophoresis-169939054
  10. http://pennstatehershey.adam.com/content.aspx?productid=117&pid=1&gid=003541

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