Immunoelectrophoresis is a general term describing many combinations of the principles of
electrophoresis and reaction of
antibodies, also known as immunodiffusion.
Agarose as 1% gel slabs of about 1 mm thickness buffered at high
pH (around 8.6) is traditionally preferred for electrophoresis and the reaction with antibodies. The agarose was chosen as the gel matrix because it has large pores allowing free passage and separation of proteins but provides an anchor for the immunoprecipitates of protein and specific antibodies. The high pH was chosen because antibodies are practically immobile at high pH. Electrophoresis equipment with a horizontal cooling plate was normally recommended for the electrophoresis. Immunoprecipitates are visible in the wet agarose gel, but are stained with protein stains like
Coomassie brilliant blue in the dried gel. In contrast to SDS-
gel electrophoresis, the electrophoresis in agarose allows native conditions, preserving the native structure and activities of the proteins under investigation, therefore immunoelectrophoresis allows characterization of
enzyme activities and ligand binding etc. in addition to electrophoretic separation.
Counterimmunoelectrophoresis is the combination of
immunodiffusion with electrophoresis. In essence electrophoresis speeds up the process of moving the reactants together. The '
immunoelectrophoretic analysis ad modum
Grabar' is the classical method of immunoelectrophoresis. Proteins are separated by electrophoresis, then antibodies are applied in a trough next to the separated proteins and immunoprecipitates are formed after a period of diffusion of the separated proteins and antibodies against each other. The introduction of the immunoelectrophoretic analysis gave a great boost to protein chemistry, some of the first results were the resolution of proteins in biological fluids and biological extracts. Among the important observations made were the great number of different proteins in serum, the existence of several immunoglobulin classes and their electrophoretic heterogeneity.
Crossed immunoelectrophoresis is also called two-dimensional quantitative immunoelectrophoresis
ad modum Clarke and Freeman or
ad modum Laurell. In this method the proteins are first separated during the first dimension electrophoresis, then instead of the diffusion towards the antibodies, the proteins are electrophoresed into an antibody-containing gel in the second dimension. Immunoprecipitation will take place during the second dimension electrophorsis and the immunoprecipitates have a characteristic bell-shape, each precipitate representing one antigen, the position of the precipitate being dependent on the amount of protein as well as the amount of specific antibody in the gel, so relative quantification can be performed. The sensitivity and resolving power of crossed immunoelectrophoresis is than that of the classical immunoelectrophoretic analysis and there are multiple variations of the technique useful for various purposes. Crossed immunoelectrophoresis has been used for studies of proteins in biological fluids, particularly human serum, and biological extracts.
Rocket immunoelectrophoresis is one-dimensional quantitative immunoelectrophoresis. The method has been used for quantitation of human serum proteins before automated methods became available.
Fused rocket immunoelectrophoresis is a modification of one-dimensional quantitative immunoelectrophorsis used for detailed measurement of proteins in fractions from protein separation experiments.
Affinity immunoelectrophoresis is based on changes in the electrophoretic pattern of proteins through specific interaction or
complex formation with other
macromolecules or ligands.
Affinity immunoelectrophoresis has been used for estimation of
binding constants, as for instance with
lectins or for characterization of proteins with specific features like
glycan content or
ligand binding. Some variants of affinity immunoelectrophoresis are similar to
affinity chromatography by use of immobilized
ligands.
Binding of ligands. The open structure of the immunoprecipitate in the agarose gel will allow additional binding of radioactively labeled antibodies and other ligands to reveal specific proteins. Application of this possibility has been used for instance for identification of allergens through reaction with immunoglobulin E (IgE) and for identification of glycoproteins with
lectins.
General comments. Two factors determine that immunoelectrophoretic methods are not widely used. First they are rather work intensive and require some manual expertise. Second they require rather large amounts of polyclonal antibodies. Today
gel electrophoresis followed by
electroblotting is the preferred method for protein characterization because its ease of operation, its high sensitivity, and its low requirement for specific antibodies. In addition proteins are separated by
gel electrophoresis on the basis of their apparent molecular weight, which is not accomplished by immunoelectrophoresis, but nevertheless immunoelectrophoretic methods are still useful when non-reducing conditions are needed. ==Applications==