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~ TheTito Discuss

Immuno
A combination of the techniques of electrophoresis and immunodiffusion used to separate the components of a mixture of antigens and make them visible by reaction with specific antibodies.

Immunoelectrophoresis is a general name for a number of biochemical methods used to separate and characterize proteins based on electrophoresis and to make them visible by reacting them with antibodies. All of the variants of immunoelectrophoresis require immunoglobulins, also known as antibodies, to react with the target proteins. The methods were developed and used extensively during the second half of the 20th century. In somewhat chronological order: Immunoelectrophoretic analysis (one-dimensional immunoelectrophoresis, crossed immunoelectrophoresis (two-dimensional quantitative immunoelectrophoresis, rocket-immunoelectrophoresis (one-dimensional quantitative immunoelectrophoresis, fused rocket immunoelectrophoresis, affinity immunoelectrophoresis.

Preparation
For the electrophoresis, agarose gel slabs of about 1 mm thickness buffered at a high pH (around 8.5) are cast. At this basic pH, most proteins will have a strong charge. As an electric field is passed through the agarose, the proteins will migrate based on their acquired charge. The agarose matrix has large pores allowing free passage and separation of proteins based on thie size of the particles. Larger particles and those with less charge will move slower through the agarose than smaller, highly charge molecules. However, at a high pH antibodies are practically immobile.

Immunoprecipitates may be seen in the wet agarose gel, but protein stains like Coomassie blue are applied so that the results can be visualized in the dried gel. In contrast to SDS-gel electrophoresis where proteins end up being denatured, the electrophoresis in agarose preserves the structure and activities of the proteins under investigation; therefore, immunoelectrophoresis allows characterization of enzyme activities and ligand binding in addition to electrophoretic separation.

Immunoelectrophoretic analysis
The immunoelectrophoretic analysis is the classical method of immunoelectrophoresis. Proteins are separated by electrophoresis, then antibodies are applied in a trough next to the separated proteins. 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 very 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 and the existence of several immunoglobulin classes with their electrophoretic heterogeneity.

Crossed immunoelectrophoresis
Crossed immunoelectrophoresis, also called two-dimensional quantitative immunoelectrophoresis, is where the proteins are first separated during a 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 takes place during the second dimension electrophoresis where the immunoprecipitates form a characteristic bell-shape. Each precipitate represents one antigen, the position of the precipitate being dependent on the amount of protein and the amount of specific antibody in the gel. This allows relative quantification.

The sensitivity and resolving power of crossed immunoelectrophoresis is greater 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
Rocket immunoelectrophoresis is one-dimensional quantitative immunoelectrophoresis. The methods has been used for quantitation of human serum proteins before automated methods became available.

Fused rocket immunoelectrophoresis
Fused rocket immunoelectrophoresis is a modification of one-dimensional quantitative immunoelectrophoresis used for detailed measurement of proteins in fractions from protein separation experiments.

Affinity immunoelectrophoresis
Affinity immunoelectrophoresis is based on changes in the electrophoretic pattern of proteins through biospecific 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.

Extra
The open structure of the immunoprecipitate in the agarose gel will allow additional binding of radioactively labeled antibodies to reveal specific proteins. This variation has been used for identification of allergens through reaction with IgE.

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.