The technique exploits the specificity of reaction between an antigen and antibody and molecular sieving of the gel in which this reaction is taking place for analysis of components of a given sample. The technique was developed by Grabar and Williams and is actually a modification of Ouchterlony double diffusion technique. To understand the technique better a brief discussion of Ouchterlony double diffusion technique is given below.

The Ouchterlony method depends on the formation of a micellar structure during gelatin of agar or sucrose. Molecular aggregates with molecular weights not exceeding 200,000 Daltons can diffuse freely through micellar channels. The aggregates with higher molecular weights are, however, retarded. Thus, the individual antigen and antibody molecules filled in adjacent wells cut into the gel can diffuse freely till they come into contact. On contact, if the antibody is specific for the antigen, they form complexes with larger molecular weight; these complexes are immobile. If the concentrations of corresponding antigen and antibody are sufficient to form a visible aggregate, a precipitin band appears.

Immunoelectrophoresis, a powerful modification of Ouchterlony method, is performed using a double diffusion chamber. The chamber may either be purchased from the market or prepared readily by coating a glass microscope slide with agarose/purified agar and punching appropriate holes in the supporting medium. The antigen is filled in the small round well (1-100mg). The current is switched on (8 mA, 4-8 volts/cm) and the electrophoresis is allowed to continue for 1-2 hours. Immediately, after disconnecting the voltage supply, the rectangular well is filled with appropriate antisera and the gel incubated overnight at room temperature in a humid chamber to permit diffusion of antigen and antibody toward each other. This leads to the formation of precipitin bands at the site lateral to the position where the desired components has separated during electrophoresis from the rest of the components. The major advantage of this method is its increased resolving ability due to the combination of electrophoresis with immune specificity. The technique can be made quantitative and has been used to detect particular antigens in sera, tissue or cell extracts, and culture filtrates. It has also been used to determine the purity of a given antigen.