Immunoelectrophoresis refers to a group of biological techniques for isolating and characterizing proteins using electrophoresis and antibody reactions. Immunoglobulins, antibodies that react with proteins, are required for all forms of immunoelectrophoresis.

  • Rocket immunoelectrophoresis is quantitative immunoelectrophoresis in one dimension.
  • Fused rocket immunoelectrophoresis is a modification of one-dimensional quantitative immunoelectrophoresis that is used for comprehensive protein quantification in fractions from protein separation investigations.
  • Affinity Immunoelectrophoresis is based on variations in protein electrophoretic patterns caused by particular interactions or complex formation with other macromolecules or ligands.

• Immunoelectrophoresis is a strong qualitative approach for antibody characterisation. It is first coined by Grabar and Williams in 1953.

• Immunoelectrophoresis is a method that combines immunodiffusion and electrophoresis.

• In this procedure, one antigen combination is electrophoresed in an agarose gel, which permits separation of its various components depending on charge along the gel slide, followed by lateral diffusion of the serum or monoclonal antibody within the gel.

• Antibodies specific to the antigens produce white precipitation arcs against a dark background.

Principle of Immunoelectrophoresis

The antigen mixture deposited in wells is divided into distinct antigen components according to their charge and size when an electric current is supplied to a slide covered with gel. Following electrophoresis, the antigens are reacted with specific antisera in troughs parallel to the electrophoretic migration, and diffusion is permitted to take place. In 18-24 hours, the antiserum in the trough travels toward the antigen components, forming distinct precipitin lines, each showing a response between different proteins and their antibody.

Procedure of Immunoelectrophoresis

An agarose gel is created on a glass slide that has been placed horizontally.

• Wells are carefully borne on the application zone using the sample template.

• With protein diluent solution, dilute the sample 2:3 (20μl antigen solution + 10μl diluent).

• Apply 5μl of control and sample across each matching slit using a 5μl pipette (Control slit and Sample slit).

• The samples are placed on the cathodic side of the gel in the electrophoresis chamber, and the electrophoresis runs for 20 minutes at 100 volts.

• After electrophoresis, 20μl of the matching antiserum is applied to troughs in a moist chamber and incubated in a horizontal posture for 18- 20 hours at room temperature.

• The agarose gel is arranged horizontally and blotter sheets are used to dry it.

• After soaking the gel in saline solution for 10 minutes, it is dried and washed twice more.

• The gel is dried at a temperature below 70°C and stained with protein staining solution for about 3 minutes before being decolored in distaining solution baths for 5 minutes.

• After the gel has dried, the data are analysed.

Results of Immunoelectrophoresis

• Antigen-antibody interaction is represented by the presence of elliptical precipitin arcs.

• The absence of precipitation indicates that no reaction has occurred.

• The intensity, shape, and position of the precipitation lines can be used to identify different antigens (proteins).

Applications of Immunoelectrophoresis

• The test assists in the identification and approximate quantification of certain serum proteins.

• In patients with suspected monoclonal and polyclonal gammopathies, immunoelectrophoresis is used.

• Used to detect both normal and aberrant proteins in human serum, such as myeloma proteins;

• Used to assess complicated protein mixtures including multiple antigens.

• This method can be used to check antigen purity and antigen-antibody purity, as well as to identify a single antigen in a group of antigens.

• Immunoelectrophoresis is an older method for qualitative analysis of M-proteins in serum and urine.

• Immunoelectrophoresis aids in the diagnosis and evaluation of the therapeutic response in many disease states affecting the immune system.

• Immunoelectrophoresis is a more traditional approach for analyzing M-proteins in serum and urine.

• Immunoelectrophoresis aids in the diagnosis and assessment of therapy response in a variety of immune-related diseases.

Advantages of Immunoelectrophoresis

• Immunoelectrophoresis combines antigen separation by electrophoresis with immunodiffusion against an antiserum to produce a strong analytical technique with great resolving power.

• The fundamental benefit of immunoelectrophoresis is that it can identify a variety of antigens in serum.

Limitations of Immunoelectrophoresis

• Immunoelectrophoresis is more difficult to interpret than Immunofixation electrophoresis because it is slower and, less sensitive.

• Some small monoclonal M-proteins are missed by IEP because the fastest migrating immunoglobulins present in the highest concentrations may hide the presence of small M-proteins.

• The availability of particular antibodies limits the application of immunoelectrophoresis in food analysis.

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