Immunoelectrophoresis- Principle, Procedure, Results and Uses
Immunoelectrophoresis is a technique that combines electrophoresis and immunodiffusion to separate and identify proteins based on their antigenic properties. Electrophoresis is the process of applying an electric field to a gel containing a mixture of proteins, which causes them to migrate according to their charge and size. Immunodiffusion is the process of allowing antibodies to diffuse from a trough into the gel and form visible precipitates with their corresponding antigens.
The principle of immunoelectrophoresis is based on the fact that different proteins have different charges and molecular weights, and therefore move at different speeds and distances in an electric field. Moreover, each protein has a specific antigenic structure that can be recognized by a specific antibody. By using an antiserum that contains antibodies against the proteins of interest, immunoelectrophoresis can reveal the presence and identity of these proteins in a complex mixture.
- A gel is prepared on a glass slide and wells are made on one end of the gel.
- A sample containing a mixture of proteins (such as serum) is diluted and applied to the wells.
- The gel is placed in an electrophoresis chamber and an electric current is applied for a certain period of time.
- The proteins in the sample separate into distinct bands according to their charge and size.
- After electrophoresis, troughs are made parallel to the direction of migration and filled with antiserum.
- The gel is incubated in a moist chamber for several hours or overnight, allowing the antibodies to diffuse from the troughs into the gel.
- The antibodies react with their specific antigens and form visible precipitates (also called precipitin arcs) in the gel.
- The gel is dried and stained to enhance the visibility of the precipitates.
Immunoelectrophoresis produces a pattern of precipitin arcs that corresponds to the different proteins in the sample. Each arc represents an antigen-antibody reaction between a protein and its specific antibody. The position, shape, and intensity of the arcs can provide information about the identity, quantity, and purity of the proteins. Immunoelectrophoresis can also detect abnormalities or deficiencies in protein levels or structure, such as in some diseases or genetic disorders.
Immunoelectrophoresis has various applications in medical diagnostics and protein analysis. It can be used for the identification and quantification of serum proteins, detection of monoclonal and polyclonal gammopathies, analysis of complex protein mixtures, assessment of antigen and antigen-antibody purity, and identification of a single antigen in a mixture of antigens. These applications help in diagnosing diseases, monitoring therapeutic responses, and analyzing protein mixtures.
Immunoelectrophoresis has several advantages over other methods of protein analysis. It combines the separation of antigens by electrophoresis with the specificity of immunodiffusion against an antiserum, resulting in high resolving power and sensitivity. It can detect multiple antigens in a single sample, identify normal and abnormal proteins, monitor the purity and identity of antigens and antibodies, and diagnose various diseases that affect the immune system.
Immunoelectrophoresis has some limitations that need to be considered. It is a slow and labor-intensive process, less sensitive and less quantitative than some other techniques, may fail to detect small or rare monoclonal proteins, depends on the availability and specificity of antisera, and may not resolve complex mixtures of antigens. It can be compared with other techniques such as immunofixation electrophoresis, enzyme-linked immunosorbent assay (ELISA), and Western blotting, which offer advantages in terms of sensitivity, specificity, speed, or quantification.
Immunoelectrophoresis is an important technique in immunology and protein analysis, but it has some limitations. Other techniques may offer advantages in specific situations. However, each technique has its own strengths and weaknesses, and the choice of the best technique depends on the purpose and context of the analysis.
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