Immunoelectrophoresis
Encyclopedia
Immunoelectrophoresis is a general name for a number of biochemical methods for separation and characterization of protein
s based on electrophoresis
and reaction with antibodies. All variants of immunoelectrophoresis require immunoglobulins, also known as antibodies reacting with the proteins to be separated or characterized. The methods were developed and used extensively during the second half of the 20th century. In somewhat chronological order: Immunoelectrophoretic analysis (one-dimensional immunoelectrophoresis ad modum Grabar), crossed immunoelectrophoresis (two-dimensional quantitative immunoelectrophoresis ad modum Clarke and Freeman or ad modum Laurell), rocket-immunoelectrophoresis (one-dimensional quantitative immunoelectrophoresis ad modum Laurell), fused rocket immunoelectrophoresis ad modum Svendsen and Harboe, affinity immunoelectrophoresis
ad modum Bøg-Hansen.
Agarose as 1 % gel slabs of about 1 mm thickness buffered at high pH
(around 8.6) is traditionally preferred for the electrophoresis as well as 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. An electrophoresis equipment with a horizontal cooling plate was normally recommended for the electrophoresis.
Immunoprecipitates may be seen 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.
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 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, 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 methods 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 biospecific interaction or complex formation with other macromolecules or ligands. Affinity immunoelectrophoresis
has been used for estimation of binding constants, as for instance with lectin
s 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.
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.
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
s based on electrophoresis
Electrophoresis
Electrophoresis, also called cataphoresis, is the motion of dispersed particles relative to a fluid under the influence of a spatially uniform electric field. This electrokinetic phenomenon was observed for the first time in 1807 by Reuss , who noticed that the application of a constant electric...
and reaction with antibodies. All variants of immunoelectrophoresis require immunoglobulins, also known as antibodies reacting with the proteins to be separated or characterized. The methods were developed and used extensively during the second half of the 20th century. In somewhat chronological order: Immunoelectrophoretic analysis (one-dimensional immunoelectrophoresis ad modum Grabar), crossed immunoelectrophoresis (two-dimensional quantitative immunoelectrophoresis ad modum Clarke and Freeman or ad modum Laurell), rocket-immunoelectrophoresis (one-dimensional quantitative immunoelectrophoresis ad modum Laurell), fused rocket immunoelectrophoresis ad modum Svendsen and Harboe, affinity immunoelectrophoresis
Affinity electrophoresis
Affinity electrophoresis is a general name for many analytical methods used in biochemistry and biotechnology. Both qualitative and quantitative information may be obtained through affinity electrophoresis. The methods include the so-called mobility shift electrophoresis, charge shift...
ad modum Bøg-Hansen.
Agarose as 1 % gel slabs of about 1 mm thickness buffered at high pH
PH
In chemistry, pH is a measure of the acidity or basicity of an aqueous solution. Pure water is said to be neutral, with a pH close to 7.0 at . Solutions with a pH less than 7 are said to be acidic and solutions with a pH greater than 7 are basic or alkaline...
(around 8.6) is traditionally preferred for the electrophoresis as well as 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. An electrophoresis equipment with a horizontal cooling plate was normally recommended for the electrophoresis.
Immunoprecipitates may be seen 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
Gel electrophoresis
Gel electrophoresis is a method used in clinical chemistry to separate proteins by charge and or size and in biochemistry and molecular biology to separate a mixed population of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to separate proteins by charge...
, the electrophoresis in agarose allows native conditions, preserving the native structure and activities of the proteins under investigation, therefore immunoelectrophoresis allows characterization of enzyme
Enzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...
activities and ligand binding etc in addition to electrophoretic separation.
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 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, 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 methods 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 biospecific interaction or complex formation with other macromolecules or ligands. Affinity immunoelectrophoresis
Affinity electrophoresis
Affinity electrophoresis is a general name for many analytical methods used in biochemistry and biotechnology. Both qualitative and quantitative information may be obtained through affinity electrophoresis. The methods include the so-called mobility shift electrophoresis, charge shift...
has been used for estimation of binding constants, as for instance with lectin
Lectin
Lectins are sugar-binding proteins that are highly specific for their sugar moieties. They play a role in biological recognition phenomena involving cells and proteins. For example, some viruses use lectins to attach themselves to the cells of the host organism during infection...
s or for characterization of proteins with specific features like glycan content or ligand
Ligand
In coordination chemistry, a ligand is an ion or molecule that binds to a central metal atom to form a coordination complex. The bonding between metal and ligand generally involves formal donation of one or more of the ligand's electron pairs. The nature of metal-ligand bonding can range from...
binding. Some variants of affinity immunoelectrophoresis are similar to affinity chromatography
Affinity chromatography
Affinity chromatography is a method of separating biochemical mixtures and based on a highly specific interaction such as that between antigen and antibody, enzyme and substrate, or receptor and ligand.-Uses:Affinity chromatography can be used to:...
by use of immobilized ligands.
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
Gel electrophoresis
Gel electrophoresis is a method used in clinical chemistry to separate proteins by charge and or size and in biochemistry and molecular biology to separate a mixed population of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to separate proteins by charge...
followed by electroblotting
Electroblotting
Electroblotting is a method in molecular biology/biochemistry/immunogenetics to transfer proteins or nucleic acids onto a membrane by using PVDF or nitrocellulose, after gel electrophoresis. The protein or nucleic acid can then be further analyzed using probes such as specific antibodies, ligands...
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
Gel electrophoresis
Gel electrophoresis is a method used in clinical chemistry to separate proteins by charge and or size and in biochemistry and molecular biology to separate a mixed population of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to separate proteins by charge...
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.
Further reading
- A manual of quantitative immuno-electrophoresis: Methods and applications (Scandinavian journal of immunology) J. Kroll, B. Weeke N. H. Axelsen (Editor) Published 1977 by Blackwell Scientific Publications
External links
- Comprehensive text edited by Niels H. Axelsen in Scandinavian Journal of Immunology, 1975 Volume 4 Supplement
- http://www.lib.mcg.edu/edu/esimmuno/ch4/immelec.htm
- Immuno-Electrophoresis. Immuno-Diffusion