Immunoelectrophoresis

Immunoelectrophoresis was first coined by Grabar and Williams in 1953. 

*Principle :-
Immunoelectrophoresis created a important development in protein identification and in immunology.
After electrophoretic separation of serum proteins in an agar gel, the serum proteins were allowed to diffuse against antibodies, leading to a pattern of precipitation arcs in the agar representing the major constituents among the serum proteins.
immunoelectrophoresis is also known as electro-immunodiffusion; it is a simple and rapid method to quantify the amount of either a protein or a protein mixture. 
Immunoelectrophoresis refers to precipitation in agar under an electric field. It is a process of a combination of immuno-diffusion and electrophoresis. An antigen mixture is first separated into its component parts by electrophoresis and then tested by double immuno-diffusion.

*Protocol :-
Agarose gel is prepared on a glass slide put in a horizontal position.
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Using the sample template, wells are borne on the application zone carefully.
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The sample is diluted 2:3 with protein diluent solution (20μl antigen solution +10 μl diluent). 
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Using a 5 μl pipette, 5 μl of control and sample is applied across each corresponding slit (Control slit and Sample slit). 
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The gel is placed into the electrophoresis chamber with the samples on the cathodic side, and electrophoresis runs for 20 mins/ 100 volts.
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After electrophoresis completes, 20 μl of the corresponding antiserum is added to troughs in a moist chamber and incubated for 18- 20 hours at room temperature in a horizontal position.
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The agarose gel is placed on a horizontal position and dried with blotter sheets.
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The gel in saline solution is soaked for 10 minutes and the drying and washing repeated twice again.
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The gel is dried at a temperature less than 70°C and may be stained with protein staining solution for about 3 minutes followed by decolorizing the gel for 5 minutes in distaining solution baths.   
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The gel is dried and results evaluated

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